Eliana Lucchinetti

François Boucher

Andreas Daiber

Merci À Sarah

Guillaume La Force Tranquille

Lolo Le Bô Gosse

Claire L'exubérante

Rita La Sage

Nellie Alexandre

Cindy Et J'en

Séverine Gratia

Laurence Kay

Sylvie Michelland

Michel Sève

Uwe Schlattner

Malgorzata Tokarska-Schlattner

Michael Zaugg

Rita Guzun

Valdur Saks

Cardiac phosphoproteome reveals cell signaling events involved in doxorubicin cardiotoxicity

Keywords: anthracyclines, cardiac cell signaling, cardiotoxicity, phosphoproteome anthracyclines, cardiotoxicity, doxorubicin, gene expression, microarrays, mitochondrial function, perfused heart, skinned fibers, transcriptome

also for their interests in this work, and for having accepted to evaluate this study. Mon directeur de thèse, Uwe Schlattner pour m'avoir accepté dans son équipe, m'avoir supervisé pendant ces quatre années de thèse ainsi que pour sa disponibilité, sa confiance et son aide si précieuse tout au long de ce travail et plus particulièrement à la fin. Ma directrice de thèse, Malgorzata Tokarska-Schlattner, pour avoir également été très présente tout au long de ce travail, m'encadrant de ses conseils et de son aide tant technique que scientifique. Votre confiance et votre soutien m'ont beaucoup apporté

Introduction

Conventionally, all new anthracyclines are named with the suffix "-rubicin" (reviewed in 1 ).

This molecule was the precursor of all anthracyclines existing today.

DNR showed a great efficiency against leukaemia and lymphomas, but also induced fatal cardiotoxicity 2 . Until today, cardiotoxicity remains a major problem for the clinical use of anthracyclines (for details see chapter 1.2). Since their discovery, scientists have tried to synthesize new analogues of DNR with persisting antitumor activity but less cardiotoxicic side-effects. In 1969, Arcamone et al. 3 isolated from a mutant strain of Streptomyces another anthracycline, a reddish substance they named adriamycin, now also called doxorubicin (DXR) by INN convention. DXR was more efficient against certain types of cancer and had compared to DNR a better therapeutic index, i.e. ratio of the toxic dose to the therapeutic dose. Until today, it remains the chemotherapeutic drug which is used to treat the largest number of different types of cancer, including acute leukemia, lymphomas, soft tissue and osteogenic sarcomas, pediatric malignancies and solid tumors (particularly lung and breast cancer). Indeed, there are only a few cancers for which DXR is not effective: colon cancer, melanoma, chronic leukaemia and renal cancer (reviewed in 4 ). 1.1.2. Molecular structure DXR is a tetracycle bearing an aminoglycoside moiety. The aglyconic part is composed of four rings with adjacent quinone-hydroquinone (rings C and B). The sugar, daunosamine, is linked to ring A by a glycosidic bond (Figure 1-1). The divergent properties of DXR and DNR are caused by a small structural difference: bound to C9 of ring A, there is a hydroxyoxoethyl in DXR and an oxoethyl in DNR (reviewed in 4 ).

Anticancer action

Although DXR has been studied intensively for decades, its anti-cancer mechanisms are not fully understood. Studies used various cancer cells from mice and humans with different concentrations of DXR, thus complicating comparative analysis of the resulting data. In the clinical context, during a bolus administration varying from 15 to 90 mg/m 2 , the patient's initial plasma concentration varies from 0,3-5 µM, then falling to 25-250 nM within one hour. Thus, studies using higher doses to study the drug effect in intact cancer cell are unlikely to reflect the anti-cancer mechanisms associated with clinical utilization (reviewed in 5 ). Some molecular mechanisms of DXR have been validated in cancer cells by clinical doses. For example, DXR acts as a topoisomerase II inhibitor. Topoisomerase II activity involves an intermediary complex during which DNA is cut and linked to the enzyme. DXR stabilizes this complex which leads to cell growth arrest and apoptosis. DXR is also known to induce apoptosis in cancer cells via p53 (reviewed in 4 ) or to intercalate into nuclear and mitochondrial DNA 6 . Other mechanisms remain controversial because they cannot be confirmed with clinically relevant DXR doses, such as free radical mediated toxicity and DNA cross-linking (reviewed in 4 ).

Tumors have the capacity to adapt to diverse stresses, which also includes chemotherapeutic interventions like with DXR. Cancer cells can establish adaptive mechanisms to avoid DXR toxicity (reviewed in 7 ). The major mechanism of drug resistance is the increase of drug-transporting P-glycoproteins, such that resistant cancer cells can eliminate DXR more rapidly. This decreases the cellular concentration of the drug, which will diminish or even prevent its toxic effects (reviewed in 7,8 ). Another resistance mechanism is the modification of topoisomerase II. As DXR targets this enzyme to trigger cell death, cancer cells expressing a mutated topoisomerase II, which is functional but no longer interacting with DXR, will largely prevent DXR-induced apoptosis 9 . 1.1.4. "Improved" doxorubicin analogues Since DXR has been discovered, researchers have pursued the quest to find a DXR analogue with improved properties, i.e. higher anticancer efficiency and lower toxic sideeffects. More than hundreds of molecules were synthesized in order to find a less cardiotoxic drug with a better therapeutic index and more active in DXR resistant cancer cells. Some of these compounds like epirubicin were shown to be efficient and are now commercialized. But if epirubicin is less cardiotoxic than DXR, it is also less efficient to treat cancer: the maximum cumulative dose delivered before symptoms of cardiotoxicity appear is 900 mg/m 2 for epirubicin and 500 mg/m 2 for DXR, but a 25-50% higher dose of epirubicin is necessary to achieve the same efficiency as DXR in the treatment of cancer 10,11 . So far, no analogue was identified with a better therapeutic index than DXR.

A second approach to generate an "improved" DXR is to develop some tumor-targeted formulations. These drugs would allow decreasing the DXR concentration in heart and in the whole body, but increasing it in tumors. A novel such drug, Doxil, consists of DXR encapsulated in a novel pegylated liposomal formulation The pharmacokinetics of this polyethylene-glycol-coated liposome are characterized by a reduced volume of distribution, a long intravascular circulating half-life and slow plasma clearance compared with free doxorubicin. This, coupled with a small vesicular size, uniquely promotes the localization of Doxil at tumor sites. Doxil has already shown impressive effects on metastatic breast carcinoma, lymphomas, sarcoma and ovarian cancer (reviewed in 12 ). For example, in brain cancer therapy, its use led to a ten-fold increase in the DXR concentration around the tumor 13 .

However, Doxil is presently used only in the treatment of few types of cancer as compared to DXR because of a lack of clinical studies. Further research is needed to analyze its efficiency in different tumor types. Despite the lower DXR concentration received in this therapy by the heart, this formulation does not avoid but only reduce the risk of cardiotoxicity: 2,4% of patients treated with Doxil for metastatic breast cancer still showed signs of cardiac toxicity versus 14,1% for DXR-treated patients 14 . 1.1.5. Improved administration protocols Another approach to reduce cardiotoxicity is to find appropriate administration protocols for the DXR treatment. Some precautions can be taken to reduce the risks. Patients have a probability of 0,14% to develop a congestive heart failure if they received a cumulative total dose of DXR < 400 mg/m 2 body surface area, 7% if they received a dose > 550 mg/m 2 and 18% at a dose of 700 mg/m 2 . Thus, the cumulative dose of DXR has been limited to mg/m 2 . The administration schedule has also been optimized: the cardiotoxicity is decreased when DXR is administrated over more than 48 to 96 hours, or as weekly injection instead of a unique bolus 15 . Thus, the maximal dose that a patient receives as bolus injection is around 3 mg/kg. The maximal cumulative dose generally accepted for a patient is 550 mg/m 2 or approximately 14 mg/kg.

In general, one has to be more cautious with younger patients. For example, Pein et al. have shown that 39% of children had severe cardiac dysfunction or major ventricular overload, even 15 years or more after being treated with DXR. Hequet et al. [START_REF] Gewirtz | A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin[END_REF] have shown that even with a moderate dose of DXR (≈ 300 mg/m 2 ), patients could develop cardiac abnormalities.

Taken together, risk factors that emerged in these studies include: high cumulative dose, drug administration with single bolus injection, advanced and younger age, pre-existing heart disease and hypertension 15 . Further progress in the field will need a better molecular understanding of the etiology of the disease.

Doxorubicin cardiotoxicity

DXR therapy represents a "double edge sword" for patients: its anti-cancer action is associated with a cardiotoxic effect, which can lead to congestive heart failure or chronic cardiomyopathy [START_REF] Rabilloud | Two-dimensional gel electrophoresis in proteomics: Past, present and future[END_REF] .

1.2.1. Physiological/cellular effect DXR cardiotoxicity occurs in two phases: acute toxicity, which develops directly after injection, and chronic toxicity, due to cumulative dose of the drug, which occurs months to years after the treatment. The acute cardiotoxicity provokes tachycardia, hypotension, and pericarditis. These effects mostly disappear shortly after injection or are clinically manageable 4 . The more serious chronic cardiotoxicity leads to DXR-induced cardiomyopathy with clinical characteristics, including decreased blood pressure, elevated heart rate, decreased ejection fraction, and ventricular failure [START_REF] Schulenberg | Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye[END_REF] . These effects are also present in animal models (mice, rats, or rabbits), suggesting species-independent mechanisms (reviewed in 4 ).

They lead to serious congestive heart failure, which can appear even years after chemotherapy. The pathology is characterized by a decrease of myofibril number and cellular changes that lead to apoptosis, including mitochondrial swelling, altered cristae structure, nuclear fragmentation and increased number of lysosomes (reviewed in [START_REF] De Souza | Preparation of human heart for laser microdissection and proteomics[END_REF] ). Indeed, heart damage induced by DXR is refractory to most conventional heart failure treatments (reviewed in [START_REF] De Souza | Preparation of human heart for laser microdissection and proteomics[END_REF][START_REF] Li | Evaluation of several two-dimensional gel electrophoresis techniques in cardiac proteomics[END_REF] ) and is worse in children than in adults, suggesting that the disease develops differently with age [START_REF] Mcgregor | Proteomics of the heart: unraveling disease[END_REF] . These facts emphasize the importance of a better understanding of the molecular mechanisms leading to DXR-induced cardiotoxicity for developing more efficient cardioprotective therapies.

Mechanisms

DXR-induced cardiotoxicity is a multi-factorial process involving different mechanisms that lead to cardiac dysfunction and heart failure (reviewed in 5,[START_REF] Mesnard-Rouiller | Troponin T mRNA and protein isoforms in the human left ventricle: pattern of expression in failing and control hearts[END_REF] ). It has long been thought that DXR cardiotoxicity involves mechanisms that differ from those responsible for its anticancer action (reviewed in 4 ), a hypothesis that is questionable today. This raised some promises for developing cardioprotective strategies without affecting the anti-cancer action of DXR (reviewed in [START_REF] Tokarska-Schlattner | New insights into doxorubicin-induced cardiotoxicity: the critical role of cellular energetics[END_REF].

Oxidative stress

Most studies on DXR cardiotoxicity support an important role of oxidative stress induced by DXR, which has been shown by an increase of free radicals and lipid peroxidation, as well as by a decrease of antioxidants and sulfhydryl groups (reviewed in [START_REF] De Souza | Preparation of human heart for laser microdissection and proteomics[END_REF] ). Generation of oxidative stress by DXR can be explained on the basis of its chemical structure. The quinone containing tetracyclic moiety of DXR can be reduced into a semiquinone by addition of a single electron. This can be transferred from the semiquinone to an oxygen molecule (O 2 ), leading to the formation of a superoxide radical (O 2  -). The latter is instable and rapidly generates reactive oxygen (ROS) or nitrogen species (RNS), which will lead to molecular damage that finally appears in form of cardiomyocyte injury and cardiomyopathy (reviewed in [START_REF] Wallace | Doxorubicin-induced cardiac mitochondrionopathy[END_REF] ).

Iron is a crucial element indispensable for many cellular redox processes. However, free iron is usually toxic for the cell because of its ability to catalyze the reaction of transforming O 2  -and hydrogen peroxide (H 2 O 2 ) into hydroxyl radical (HO -). The latter is one of the most toxic radicals, since it is not detoxified by enzymes, but only by some non-enzymatic reactions as e.g. different types of vitamins (reviewed in [START_REF] Ventura-Clapier | Bioenergetics of the failing heart[END_REF] ). Iron and DXR can form a DXR-Fe complex, which generates H 2 O 2 or HO -. This provokes oxidative damage of biomembranes [START_REF] Feng | Phosphoproteome analysis of isoflurane-protected heart mitochondria: phosphorylation of adenine nucleotide translocator-1 on Tyr194 regulates mitochondrial function[END_REF] , leading among others to a decrease of cell contractility (reviewed in [START_REF] Mesnard-Rouiller | Troponin T mRNA and protein isoforms in the human left ventricle: pattern of expression in failing and control hearts[END_REF] ).

Since free iron is toxic for cells, different systems evolved to confine iron, which would also make it unavailable to form DXR-Fe complexes. However, there are contradictory data concerning DXR effects on iron bioavailability. In vitro studies showed that DXR provokes a release of iron by acting on ferritin, a cytoplasmatic protein involved in storage and confinement of iron [START_REF] Wang | Metabolic profiling of hearts exposed to sevoflurane and propofol reveals distinct regulation of fatty acid and glucose oxidation: CD36 and pyruvate dehydrogenase as key regulators in anestheticinduced fuel shift[END_REF] . However, studies on cardiomyocytes showed a decrease of free iron [START_REF] Sugden | Mechanisms underlying regulation of the expression and activities of the mammalian pyruvate dehydrogenase kinases[END_REF] , which would be a protective mechanism by diminishing iron-catalyzed ROS reaction, but would also inhibit important processes requiring iron such as DNA and cytochrome c biosynthesis [START_REF] Holness | Regulation of pyruvate dehydrogenase complex activity by reversible phosphorylation[END_REF] . Studies with cardiomyocytes have also shown another mechanism, where the DXR metabolite doxorubicinol, a secondary alcohol, acts on the [4Fe-4S] cluster of cytoplasmic aconitase to release the iron, increasing free iron level and inhibiting the aconitase 4,[START_REF] Carvalho | Metabolic remodeling associated with subchronic doxorubicin cardiomyopathy[END_REF] Another way to induce redox cycling of DXR is activation through oxido-reductive enzymes (Fig. 12) like NADPH-dependent cytochrome c P450 reductase of the endoplasmic reticulum or the nuclear membrane [START_REF] Lewandowski | Pyruvate dehydrogenase influences postischemic heart function[END_REF][START_REF] Wakasugi | Myocardial substrate utilization and left ventricular function in adriamycin cardiomyopathy[END_REF] , mitochondrial NADH dehydrogenase (complex I) [START_REF] Solaro | Protein phosphorylation and signal transduction in cardiac thin filaments[END_REF] , and cytosolic xanthine oxidase [START_REF] Zaugg | Cellular mechanisms in sympatho-modulation of the heart[END_REF] . Yet, DXR has a high affinity for cardiolipin, an anionic phospholipid largely specific for the mitochondrial inner membrane [START_REF] Huang | Protein kinase C-mediated desmin phosphorylation is related to myofibril disarray in cardiomyopathic hamster heart[END_REF] . Mitochondria are in fact the most important sites of DXR accumulation in cell, in addition to nuclei (reviewed in [START_REF] Geisler | Phosphorylation of desmin in vitro inhibits formation of intermediate filaments; identification of three kinase A sites in the aminoterminal head domain[END_REF] ). DXR thus co-localizes with NADH dehydrogenase also localized at the inner membrane of mitochondria and involved in the electron transport chain. This creates ideal conditions for DXR-induced free radical generation, even at low DXR concentrations.

Heart is a prominent consumer of ATP, which is primarily generated to about 90% by oxidative metabolism (reviewed in [START_REF] Timolati | The role of cell death and myofibrillar damage in contractile dysfunction of long-term cultured adult cardiomyocytes exposed to doxorubicin[END_REF] ). As a consequence, more than in other organs like liver or kidney, mitochondria make up 25-35% of cardiomyocyte volume. Thus, the heart represents a highly respiratory tissue (reviewed in [START_REF] Timolati | The role of cell death and myofibrillar damage in contractile dysfunction of long-term cultured adult cardiomyocytes exposed to doxorubicin[END_REF] ). This fact already makes the heart a prime target for DXR-induced ROS generation. In presence of DXR, mitochondria become uncoupled, as described with isolated mitochondria and in vivo, both with high and clinically relevant DXR dose (reviewed in [START_REF] Geisler | Phosphorylation of desmin in vitro inhibits formation of intermediate filaments; identification of three kinase A sites in the aminoterminal head domain[END_REF] ). The binding of DXR to cardiolipin would also modify membrane lipid properties and thus could impair functions of some important enzymes, which either need cardiolipin to operate correctly, like cytochrome c oxidase, or use cardiolipin as a membrane receptor to interact with inner membrane processes like cytochrome c or mitochondrial isoforms of creatine kinase (MtCK) 39 . Such a disturbance could lead to energy dysbalance and favor apoptosis e.g. by facilitating release of cytochrome c (reviewed in [START_REF] Wallace | Doxorubicin-induced cardiac mitochondrionopathy[END_REF].

Energy dysbalance

Heart relies on rather constant, high rates of ATP generation to sustain energy homeostasis for its contractile function (reviewed in [START_REF] Timolati | The role of cell death and myofibrillar damage in contractile dysfunction of long-term cultured adult cardiomyocytes exposed to doxorubicin[END_REF]40 ). As described above, mitochondria are a prime site of DXR accumulation and DXR action. It is thus not astonishing, that many mitochondrial functions are affected, such as respiration and oxidative phosphorylation, ROS production or regulation of apoptosis. Many studies have investigated the effect of DXR on respiratory chain complexes of mitochondria. It has been shown that DXR inhibits complexes I, III and IV [41][42][43] , uncouples respiration, and decreases ATP concentration in cardiac Chapter 1. mitochondria, cardiomyocytes and the entire heart, in both acute and chronic DXR toxicity (reviewed in 44 ).

To maintain ATP homeostasis, the heart and other tissues with high and fluctuating energy demands rely on a back-up system to store and transfer energy. They use isoforms of creatine kinase (CK) together with creatine (Cr) to reversibly store free energy of ATP in form of phosphocreatine (PCr) by the reversible reaction ATP + Cr ↔ ADP + PCr (reviewed in 45,46 ). PCr accumulates within the cell up to 20 mM, thus representing a reserve of "highenergy" phosphates. It has also a higher diffusability as ATP, and can thus more rapidly bridge sites of ATP/PCr generation and ATP/PCr consumption that are both often associated with specific CK isoforms (see below) 45,46 . DXR is known to decrease PCr in heart in both types of toxicity, as was observed in cells, and in ex vivo or in vivo models (reviewed in 44 ). Eidenschink et al. 47 have shown diminished PCr/ATP ratios in children 4 years after the end of a DXR treatment, even in patients treated with lower DXR concentrations (270 ± 137 mg/m 2 ) and in the absence of cardiomyopathy. DXR is known to inactivate all CK isoforms in vitro and in vivo by oxidation of their active site cysteine 39 . Dimeric, cytosolic CK isoforms are localized near sites of cytosolic ATP production, such as glycolytic enzymes, or sites of cytosolic ATP consumption like myofibrils (reviewed in 45 ). Inactivation of the cytosolic muscle isoform (MCK) that is partially associated with the sarcoplasmic reticulum (SR) will impair the function of the energy-demanding SR Ca 2+ pump, leading to Ca 2+ imbalance and thus reduce heart muscle contraction and relaxation (reviewed in 45,48 ). The octameric mitochondrial CK isoform (MtCK) is located in the mitochondrial intermembrane space, bound to both mitochondrial membranes. It occurs in proteolipid complexes with adenosine nucleotide translocator (ANT) and voltage-dependent anion channel (VDAC), located in the inner and outer mitochondrial membrane, respectively. MtCK activity is coupled to oxidative phosphorylation 46,49 and "channels" mitochondrially generated ATP into PCr for export into the cytosol (or details see 46 ). DXR was shown to impair three MtCK properties that are essential for its proper functioning 39 . In addition to enzymatic inactivation, DXR dissociates MtCK octamers from mitocondrial membranes, in particular by competitive binding to cardiolipin, the MtCK membrane receptor. Further, DXR induces dissociation of MtCK octamers into dimers. These modifications will disrupt the MtCK "channeling" complex and diminish PCr export into the cytosol 39 . In addition, DXR also alters CK protein levels; it decreases MCK and increases the brain isoform (BCK), both at the mRNA and at total protein levels 50,51 . The latter mechanism may be compensatory, since BCK has higher affinity to ADP and a higher specific activity, possibly leading to more efficient ATP regeneration. DXR also impairs substrate utilization of cardiomyocytes. The main substrate used by heart is fatty-acids (FA). But DXR increases free FA in serum and perturbs FA use in cardiac cells 44,[START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF] . Palmitate, a long chain FA, is less oxidized in acute and chronic toxicity. Glycolysis is also impaired, but at higher DXR concentration than FA oxidation [START_REF] Schlattner | Creatine kinase and creatine transporter in normal, wounded, and diseased skin[END_REF] , possibly due to inhibition of phosphofructokinase (PFK), a rate limitating enzyme of glycolysis 54 . Some cell signaling pathways are important regulators of energy homeostasis. One of those is the AMP-activated protein kinase (AMPK) signaling. AMPK is activated by a high AMP/ATP ratio, e.g. by energetic stress, but also by oxidative stress and some extracellular stimuli [START_REF] Kawajiri | Functional significance of the specific sites phosphorylated in desmin at cleavage furrow: Aurora-B may phosphorylate and regulate type III intermediate filaments during cytokinesis coordinatedly with Rho-kinase[END_REF][START_REF] Inada | Rho-associated kinase phosphorylates desmin, the myogenic intermediate filament protein, at unique amino-terminal sites[END_REF] . It is a cellular energy sensor and a key enzyme in regulating energy metabolism.

Once activated, it inhibits ATP consuming pathways and activates ATP producing pathways in order to maintain energy homeostasis [START_REF] Kawajiri | Functional significance of the specific sites phosphorylated in desmin at cleavage furrow: Aurora-B may phosphorylate and regulate type III intermediate filaments during cytokinesis coordinatedly with Rho-kinase[END_REF] . In DXR-treated heart, our group has reported the inhibition of AMPK and decreased phosphorylation of its target, acetyl CoA carboxylase (ACC) 50 . This could contribute to the DXR-induced change in substrate utilization, since less phosphorylated ACC is more active and would inhibit mitochondrial FA import by wellknown mechanisms [START_REF] Kitamura | Protein kinase C phosphorylation of desmin at four serine residues within the non-alphahelical head domain[END_REF] , thus decreasing FA oxidation. AMPK inhibition would also limit PFK activity and slow down glycolysis, while increasing the activity of ATP-consuming pathways, thus aggravating the energy deficit of the heart 50 .

Taken together, DXR impairs heart substrate utilization, high-energy phosphate production, storage and transfer, and at the same time the AMPK signaling pathway involved in energy homeostasis that could compensate for these impairments. Hearts treated by DXR are thus likely to develop a critical state of energetic stress.

Cell death

Cell death is a major issue in different cardiomyopathies, including those induced by DXR. However, whether apoptotic or necrotic cell death are predominant is not fully understood [START_REF] Huang | A systematic MS-based approach for identifying in vitro substrates of PKA and PKG in rat uteri[END_REF] . Apoptosis is programmed cell death, tightly regulated and energy-dependent, and characterized by cell shrinking and DNA strand breaks. The cell forms apoptotic bodies eliminated by macrophages (reviewed in [START_REF] Inagaki | Intermediate filament reconstitution in vitro. The role of phosphorylation on the assemblydisassembly of desmin[END_REF] ). Necrosis lacks this sort of control and is mostly energy-independent, although there are forms that also show regulatory mechanisms as e.g. triggered by oxidative stress [START_REF] He | Phosphorylation of mitochondrial elongation factor Tu in ischemic myocardium: basis for chloramphenicol-mediated cardioprotection[END_REF][START_REF] Ecroyd | Mimicking phosphorylation of alphaB-crystallin affects its chaperone activity[END_REF] . Autophagy is again a highly regulated process that is involved in renewing cellular compounds and is in general significantly enhanced in pathologies [START_REF] Huang | A systematic MS-based approach for identifying in vitro substrates of PKA and PKG in rat uteri[END_REF] . It can be considered as another form of non-apoptotic death, but can also represent a survival strategy in times of stress. Some studies have shown apoptosis in cardiomyocytes treated with DXR, involving DNA strand breaks and caspase activation [START_REF] Hoover | alpha B-crystallin gene induction and phosphorylation by MKK6-activated p38. A potential role for alpha B-crystallin as a target of the p38 branch of the cardiac stress response[END_REF] . H9C2 cells, a cell line derived from embryogenic rat hearts, showed apoptosis when treated with DXR [START_REF] Kato | Phosphorylation of alphaB-crystallin in mitotic cells and identification of enzymatic activities responsible for phosphorylation[END_REF] . DXR-treated cardiomyocytes have shown degradation of titin, a protein involved in myofibrillogenesis.

This would lead to disarray of myofibrils and sarcomeres that triggers apoptosis [START_REF] Ande | Insulin induced phosphorylation of prohibitin at tyrosine 114 recruits Shp1[END_REF] . DXR is also known to activate autophagy of mitochondria and endoplasmic reticulum, leading to cell death [START_REF] He | Phosphorylation of mitochondrial elongation factor Tu in ischemic myocardium: basis for chloramphenicol-mediated cardioprotection[END_REF] . Timolati and al. [START_REF] Kwon | Cell cycle-dependent phosphorylation of mammalian protein phosphatase 1 by cdc2 kinase[END_REF] have shown an increase of autophagosomes in DXR-treated cardiomyocytes, even at low DXR concentration. This mechanism would contribute to DXR cardiotoxicity.

The three mentioned types of cell death, apoptosis, necrosis and autophagy, are interconnected, and mitochondria seem to be at the crossroads: apoptosis involves mitochondrial signaling cascades, mitochondrial transition pore opening, cytochrome c release, and further ATP consuming reactions; necrosis may be triggered at any stage because of ATP shortage, while excessive autophagy is triggered by damaged organelles such as mitochondria and release of lysosomal enzymes. All three cellular processes converge to cell death, and all three are induced by DXR [START_REF] He | Phosphorylation of mitochondrial elongation factor Tu in ischemic myocardium: basis for chloramphenicol-mediated cardioprotection[END_REF] . DXR also induces apoptosis in cancer cells (reviewed in 4 ). Thus, protective strategies should not interfere specifically with cell death mechanisms, since this could also diminish the anti-cancer action of DXR.

Calcium handling

The fluxes of calcium, a second messenger that triggers muscle contraction, are tightly controlled in the heart (reviewed in [START_REF] Wu | PP1-mediated dephosphorylation of phosphoproteins at mitotic exit is controlled by inhibitor-1 and PP1 phosphorylation[END_REF] ). In calcium handling, once again, mitochondria are involved in mediating DXR-induced impairments. In isolated mitochondria from treated rats, DXR induced a lower calcium loading capacity, which persisted five weeks after treatment in a dose-dependent and irreversible manner. Another important organelle in energy-dependent regulation of calcium flux is the SR (reviewed in [START_REF] Wu | PMP24, a gene identified by MSRF, undergoes DNA hypermethylation-associated gene silencing during cancer progression in an LNCaP model[END_REF] ). As DXR is known to decrease the energy state in the heart, it affects the energy-dependent capacity of sarcoplasmic reticulum (SR) to sequester calcium, thus perturbing contraction/relaxation mechanisms [START_REF] Xiao | Inducible activation of c-Myc in adult myocardium in vivo provokes cardiac myocyte hypertrophy and reactivation of DNA synthesis[END_REF] . DXR affects Ca 2+ pumps such as the Ca 2+ /Mg 2+ ATPase and the Na + /Ca 2+ exchanger of the SR, thus disturbing calcium movement and impairing cardiac contraction and relaxation [START_REF] Yano | Identification of genes linked to gefitinib treatment in prostate cancer cell lines with or without resistance to androgen: a clue to application of gefitinib to hormone-resistant prostate cancer[END_REF] . DXR can lead to cytosolic calcium overload, as shown in perfused hearts, possibly involving via inhibition of MCK that provides ATP to the SR calcium pump. In addition, inhibition of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme, prevents calcium overload and disturbance of the contractile apparatus, suggesting its implication in these mechanisms [START_REF] Malgorzata | tokarska-schlattner@ujf-grenoble[END_REF] . It is activated by DNA strand breaks and initiates an energy consuming cycle in order to induce DNA repair [START_REF] Malgorzata | tokarska-schlattner@ujf-grenoble[END_REF] , decreasing energy needed for calcium re-sequestration. Thus, PARP indirectly triggers calcium overload. This multi-functional protein is also a main substrate of caspases, indicating a link to apoptosis, and a main trigger of necrosis by consuming massively NAD + and ATP 71 .

Gene transcription

There is experimental evidence that DXR inhibits the transcription of specific genes, not just transcription in general. In cultured cardiomyocytes, DXR decreases the mRNA levels of some cardiac specific proteins: α-actin, myosin light chain 2, troponin I and MCK, while some non-cardiac-specific protein transcripts are unaffected like the cytoskeleton protein βactin, ferritin heavy chain involved in iron storage, and glycolytic pyruvate kinase 51 . Similar data were found in hearts of DXR-treated animals 51 . This could represent an early stage in DXR cardiomyopathy, with myofibril loss and alterations of structural integrity 51 . The mRNA levels of further key enzymes that decrease in DXR-treated cardiomyocytes include: ANT1, Reiske iron-sulfur protein (RISP, a protein involved in electron transport chain), and the PFK muscle isoform (the most abundant in cardiomyocytes and the rate-limiting enzyme of glycolysis) 54 . The same group has discovered the cardiac adriamycin-responsive protein (CARP), a nuclear protein expressed predominately in the heart. CARP mRNA levels decrease more rapidly than those of the other genes known to be down-regulated by DXR, and this occurs before any cardiac dysfunction is detectable [START_REF] Zhu | MAPK superfamily plays an important role in daunomycin-induced apoptosis of cardiac myocytes[END_REF] . CARP seems to regulate some nuclear functions, particularly involving mRNA. This could explain at least in part why DXR leads to a decrease of specific cardiac protein transcripts potentially linked to heart failure [START_REF] Zhu | MAPK superfamily plays an important role in daunomycin-induced apoptosis of cardiac myocytes[END_REF] .

Protective strategies

In order to diminish or even prevent the cardiotoxic side-effects of DXR, different strategies have been applied: design of DXR analogues with a better therapeutic index (chapter 1.1.4), improvement of administration protocols (chapter 1.1.5) and finally development of protective strategies that counteract DXR cardiotoxicity. As the molecular mechanisms by which DXR acts on cancer and heart tissue are not completely clear, any preventive strategy has to take into account not to compromise the DXR anti-cancer action.

In the early days of clinical DXR application, DXR cardiotoxicity was thought to be mediated exclusively by free radicals. Therefore, many antioxidants, like metal ion chelators, vitamins etc. have been tested and proved effective in animal models like rats [START_REF] Ventura-Clapier | Bioenergetics of the failing heart[END_REF] . However, in humans, the doses necessary for cardiac protection provoked hypervitaminosis [START_REF] Yano | Identification of genes linked to gefitinib treatment in prostate cancer cell lines with or without resistance to androgen: a clue to application of gefitinib to hormone-resistant prostate cancer[END_REF]73 , while at lower doses there was no or only very limited protective effect on the heart (reviewed in 4 ).

Another approach to prevent DXR cardiotoxicity used Dexrazoxane (DXZ), an ion chelator that could remove the critical iron (reviewed in 1 ). This drug is so far the only one allowed in some countries like the USA to be administrated together with DXR (reviewed in 74 ). A recent study has published results of DXZ effects in children who received DXR to treat acute lymphoblastic leukaemia. The control group was treated with ten injections of 30 mg/m 2 of DXR, while a second group received in addition 300 mg/m 2 DXZ prior to each anthracycline bolus. Five years later, the results of the DXZ treatment revealed a cardioprotective effect on the left ventricular structure and function, without affecting DXR effects on cancer 75 . Nevertheless, some studies have shown that DXZ could induce other sideeffects like severe myelosuppression, which is even amplified by DXR co-treatment (reviewed in [START_REF] Li | Evaluation of several two-dimensional gel electrophoresis techniques in cardiac proteomics[END_REF] ). Thus DXZ has to be used with caution and is so far limited to patients who have received a DXR cumulative dose higher than 300 mg/m 2 in USA 74 .

A different strategy used carvedilol, a β-blocker drug preventing calcium entry into cells, stabilizing mitochondrial membrane, and protecting against free radicals. Combined with DXR, it reduces cell vacuolization, mitochondrial respiration inhibition and protects against lipid peroxidation and the fall of systolic blood pressure. It has been shown to even enhance DXR anti-tumor action by reducing drug resistance of cancer cells (reviewed in 76 ). The first clinical trial in 2006 showed a significant improvement of the left ventricular systolic function. But mortality rate due to heart failure was not significantly reduced in patients treated with carvedilol as compared to the control group 77 . This has been the only clinical study so far showing beneficial effects of carvedilol against DXR-induced cardiotoxicity.

Although it may raise some hope for the future, similar expectations were also raised with earlier protective strategies and could not be confirmed later. Some other drugs have been investigated in animal models for their application together with DXR but are not applicable to humans. For example, neuregulin or growth factors (insulin-like growth factor, cardiotrophin 1) have shown to lower DXR cardiotoxicity in murine models, but were suspected to promote cancer growth (reviewed in 74 ). Nutritional status was investigated by Mitra et al. 78 , showing a protective effect of diet-restricted rats versus non-restricted rats.

Taken together, despite the efforts to find efficient cardioprotective strategies to prevent DXR cardiotoxicity, none of them is fully satisfying. It seems that some mechanisms are in common between cardiotoxicity and anti-cancer action of DXR, which complicates the search for a specific cardioprotective strategy. Possibly, DXR effects seem to be phenotypically different only because tumor and heart cells are very dissimilar. For example the mitochondria, a main target of DXR, are abundant in cardiomyocytes but not in cancer cells (reviewed in [START_REF] Timolati | The role of cell death and myofibrillar damage in contractile dysfunction of long-term cultured adult cardiomyocytes exposed to doxorubicin[END_REF]40 ). They are essential for the oxidative metabolism of the heart, while they are of minor importance for glycolytic cancer cells (Warburg effect, reviewed in 79,80 ). Since mitochondria are also the main site of DXR-induced free radical generation (reviewed in 4,[START_REF] Geisler | Phosphorylation of desmin in vitro inhibits formation of intermediate filaments; identification of three kinase A sites in the aminoterminal head domain[END_REF] ), oxidative stress generated by DXR will again be more important in heart as compared to cancer cells. Thus, functional alterations of mitochondria might be fatal to cardiomyocytes, but not for cancer. By contrast, induction of apoptosis by DXR is shared by both cell types.

Studying DXR toxicity mechanisms in heart remains a crucial issue to find an effective cardioprotective drug. It should be emphasized again that caution is needed when targeting a cellular mechanism to prevent or treat DXR cardiotoxicity, not to change the anti-tumor effect of the drug.

Doxorubicin and non-biased, screening approaches

Transcriptomics

Transcriptomics is the study of all mRNA molecules synthesized in a given cell. The cellular transcriptome varies depending on internal and external stimuli, as compared to the rather stable genome. It reflects the active genes in a cell at a given time. Transcriptomics is a powerful tool to explore how cells respond to various stimuli like DXR in heart.

DNA microarrays are the mostly applied tool for a non-biased and genome-wide screening of the transcriptome. A DNA microarray holds thousands of DNA probes or reporters that are spotted on a chip support. These probes are DNA sequences, each corresponding to a specific gene. The technique allows a high throughput analysis of genes transcripts. mRNAs are extracted from cells or organs, transformed into cDNA thanks to reverse transcriptase, labeled with a fluorescent dye and hybridized on the chip. Then positive spots are detected by the fluorescence signal, allowing the comparison between two or more different conditions or cell types. To confirm microarray results, real-time polymerase chain reaction (RT-PCR) can be used. This technique allows the quantification of a known transcript by using specific primers for PCR, followed by a DNA polymerase-catalyzed amplification in real time. Depending on the number of cDNA molecules present, PCR amplification will progress more or less rapidly, such that the more rapid the amplification is, the more abundant the transcript has been. RT-PCR thus allows a relative quantification of mRNA to compare transcripts in a sample as compared to a control after normalization with a non-regulated gene. In the context of DXR-induced cardiotoxicity, these techniques are valuable tools to detect drug effects on cardiac gene transcription for genome-wide, nonbiased analysis. Several studies have applied these techniques to both acute and chronic models of DXR cardiotoxicity (Table 1-1).

To study DXR cardiotoxicity, Yi et al. 81 have used hearts of mice having received an acute treatment (15 mg/kg of a unique injection of DXR) or a chronic treatment (cumulative dose of 36 mg/kg of DXR). A higher number of mRNA species was affected by DXR in the acute model as compared to the chronic model. The authors assumed that the effects of DXR on the transcriptome were decreased in the chronic model since the drug was metabolized and excreted. However, similar results were found in both toxicity models: changes were detected in mRNAs coding for proteins with roles in metabolism, oxidative stress response, signal transduction, apoptosis and myofibril structure. Acute DXR treatment up-regulated genes coding for proteins in antioxidant pathways, apoptosis induction and transcription. Chronic DXR treatment provoked an increase of transcripts coding for proteins in energy homeostasis, transcription, cell signaling (e.g. SNF1--like kinase), tumor suppression, apoptosis (proapoptotic factors), and cell cycle (BTG, responsible of cell growth arrest). Many of these cellular functions are related to the development of cardiomyopathy, suggesting that the changes in the identified transcripts may translate to the functional level and contribute to DXR-induced cardiotoxicity. Thompson et al. 82 have studied the effect of DXR either in acute or in chronic treatment.

Hypertensive rats received a cumulative dose of 3, 6 or 9 mg/kg DXR intravenously. Rats having received a 3 mg/kg DXR dose showed an increase of gene transcripts involved in antioxidative defense, xenobiotic metabolism and mitochondrial genes known to be stressinduced. Transcripts were lower for genes associated with mitochondria, ion transport, lipid metabolism, muscle contraction, and antigen proceeding. These results revealed a great diversity of genes affected with only one injection of DXR and confirmed some known effects, such as a decrease of transcripts in lipid metabolism. Rats having received 6 mg/kg of DXR showed an up-regulation of genes involved in antioxidative response and other molecular damage responses, and rats treated with DXR 9 mg/kg had higher copy number of transcripts in apoptosis, inflammatory processes, muscle contraction, ion homeostasis, glycolysis and gluconeogenesis. mRNA coding for mitochondrial proteins and for RyR2 calcium release channel in SR were decreased, but not for MtCK and adenylate kinase. Thus, again, nuclear transcripts for mitochondrial proteins were affected preferentially.

Berthiaume and Wallace 83 have performed a transcriptomic study with rats treated chronically by DXR (12 mg/kg). They found a down-regulation of genes involved in FA oxidation (except genes for mitochondrial FA transport) and a modification in expression of genes coding for glycolysis enzymes (genes coding for PFK and LDH were up-regulated, while those coding for fructose 1,6 bisphosphatase and enolase are down-regulated). These data may support a switch in heart substrate preference from FA to glucose. Transcripts of Krebs cycle and proteins in oxidative phosphorylation were also decreased, consistent with decreased oxidative glucose utilization and ATP generation, leading to DXR-induced energy deficit 84 . Transcripts coding for amino acid metabolism were increased, and those related to mitochondrial morphology and function were mostly down-regulated, suggesting a risk for mitochondrial dysfunction in long term. Changes in transcript levels related to oxidative stress showed no clear common tendency. This lack of a clear antioxidant response in this case may reflect the inability of heart to respond to DXR-induced ROS 83 .

Comparison of the different transcriptomic studies on DXR effects in heart obtained in different toxicity models reveals considerable variability. Since the studies differ in multiple parameters, including type of the experimental model, DXR dose, time of sacrifice after the last DXR injection, animal species, and others, it is difficult to establish general conclusions.

However, energy metabolism is clearly involved in DXR cardiotoxicity, since mitochondrial protein transcripts were largely affected in several studies. This supports the existence of a feedback control from functionally impaired mitochondria that down-regulates expression of nuclear-encoded mitochondrial genes. Taken together, the transcriptomic studies give some important clues to understand DXR effects in heart, but are on their own not sufficient to fully explain the functional changes observed in DXR cardiotoxicity. ). Whether conventional 2-DE or DIGE are used, the gels can be analyzed with powerful software for gel matching, quantification of each protein spot, and statistical analysis in order to find significant differences between two samples.

Protein identification is usually performed by protease digestion of the proteins in an identified spot, followed by mass spectrometry (MS) using peptide mass fingerprint (PMF)

algorithms. The PMF is characteristic of one unique protein and the used protease, commonly trypsin. Protein identification is based on a comparison between experimental PMF and theoretical PMF in a database (reviewed in 88 ).

Proteomic analysis can be performed also mainly based on MS techniques in combination with liquid chromatography (LC). Proteins are digested with a protease and peptides are separated using LC and analyzed with MS. This is a very sensitive and high throughput method to identify the protein species, but which is not sufficiently quantitative to deduce protein abundance. To have a relative quantification between control and treated samples, isotopic labeling can be used, where the two peptide samples are each linked to a different isotopic label, which introduces slight differences in mass without changing other properties.

This mass shift then allows distinguishing between the same peptide coming from the two different samples, which finally allows a relative quantification (reviewed in 89 ).

Non-biased studies may identify new mechanisms or pathways responsible of DXR toxicity. Three proteomics studies have been performed with different DXR toxicity models:

hearts from DXR-injected mice and rats, and cultured cardiomyocytes (Table 12 The most recent study of Kumar et al. 91 analyzed two different models: cardiomyocytes incubated with 0.5 µM of DXR during 0, 24 and 48h, and rats which have received intraperitoneally a DXR cumulative dose of 20 mg/kg. In cardiomyocytes, different protein chaperone levels were found increased, suggesting a protective function to counteract stressinduced protein denaturation. Other protein levels that were increased included intermediary filaments (lamin C2), signaling pathways (MAPK) and energy metabolism (e.g. F1 ATPase β subunit, α-enolase). Some proteins have been found decreased, including ATP synthase, electron transfer flavoprotein β subunit and VDAC, key proteins involved in mitochondrial energy transformation and transfer. The pattern of changes in protein levels was somewhat different in DXR-treated rats. Increased amounts were found for proteins involved in stress defense (e.g. chaperones), glucose and FA metabolism (β-enolase, apolipoprotein A-I precursor), proteolysis (proteasome subunit-α type 3), cytoskeleton (neurofilament triplet L protein) and contraction (actin, troponin T2). Two proteins were down-regulated (HSP 27, a small heat shock protein, and phosphocholine cytidylyltransferase A), while three proteins were up-regulated in both models: β-enolase, troponin, and the α crytallin B chain CRYAB, a small heat-shock protein. The latter is known to avoid protein aggregation and to be cardioprotective, once activated by phosphorylation. Despite differences in both models, some cellular functions seem to be preferentially affected by DXR, including again energy metabolism.

Another study reported effects of DXR on the cardiac proteome in an acute toxicity model where 15 mg/kg DXR were injected intraperitoneally into mice 92 . The treatment increased the level of proteins involved in different cellular functions, including oxidative stress defense (superoxide dismutase 2 and peroxidoxin, a peroxisomal membrane protein), contractile functions/cytoskeleton, FA oxidation and glucose metabolism. Finally, increased levels were found for proteins in mitochondrial energy transformation (cytochrome c reductase, cytochrome c oxidase) and CRYAB.

Taken together, proteomics studies have added some new insight into DXR action in the heart at the molecular level. Proteomics has contributed to understand some known molecular phenotypes of DXR cardiotoxicity like e.g. the impairment of the mitochondrial ATP generating system, the enhanced use of glucose as energy source, the cardiac remodeling, the establishing of heart protective mechanisms such as CRYAB, or molecular damage of the myofibrillar contraction/relaxation system. However, even if proteomics can provide information on enzyme levels, it will not tell us about changes in their activity. Further investigations are required to know their activation state, and the analysis of the protein phosphorylation pattern is one of the approaches that can be envisaged.

   Glucose metabolism     Mitochondrial respiratory chain    Others     Defense against stress Oxidative stress   DNA damage Chaperone    Stress-induced MAPK  Cell Structure Cytoskeleton     Contraction     Others Ion handling   Proteasome  
Protein synthesis/transport    : Level of most proteins in this category is increased : Level of most proteins in this category is increased, while a minority is decreased : Level of most proteins in this category is decreased : Level of most proteins in this category is decreased, while a minority is increased 1.3.3. Phosphoproteomics Post-translational modification of proteins plays a major role in cellular regulation, with phosphorylation being the most important one. Reversible phosphorylation is a major mechanism in cell signaling, catalyzed by the delicate equilibrium between kinase and phosphatase activities 94 .

Different methods are available to enrich phosphoproteins, which is necessary since many phospo-species have a rather low abundance. These include immunoprecipitation with antibodies specific of phosphoresidues (see below) and different types of chromatography columns like immobilized metal ion affinity columns or titanium dioxide columns (reviewed in 95 ). For separation, detection and identification of proteins, the methods described before for standard proteomics can be adapted. 2-DE can be used for separation, followed by phosphospecific detection for which different methods are available. First, phosphorylation may be visible from a simply protein-stained gel, since it adds a negative charge to the protein and provokes a shift of pI towards acid pH. This is evident for proteins with multiple phosphorylation states, giving the aspect of a "pearl necklace" to the spots. However, as a more specific and more sensitive approach, phosphoprotein-specific dyes are used such as e.g. ProQ-Diamond. This stain allows sensitive detection of phosphoamino acids in 2-DE gels. It has been shown to discriminate between phosphorylated and non-phosphorylated proteins with a high ratio of specific to non-specific staining 96 . This dye is also compatible with MS analysis and thus allows identification of the detected phosphoproteins 96,97 . Alternatively, phosphoproteins in 2-DE can be detected with phosphoresidue-specific antibodies on immunoblots. However, detection with antibodies often turned out to be less sensitive and less specific as compared to staining procedures 95 . Finally, 32 P-or 33 P-isotopes can be used to label phosphoproteins in vitro and in vivo, and to detect them by autoradiography, which is the most sensitive method available. However, for in vivo studies, the isotopes can have toxic effects independent of DXR which complicates analysis Like in standard proteomics, MS can be used as an alternative technique, which currently becomes very popular. However, phosphopeptides are rather difficult to analyze by MS because of their often low propensity to be ionized by the laser or the risk of phosphate loss upon ionization. Thus, enrichment techniques are important to achieve good detection. As in proteomics, quantification may be performed by introducing a protein tag, which will allow comparison of two samples (reviewed in 95 ).

DXR may alter cell signaling pathways, as it has been shown in a few cases such as AMPK signaling 50 . However, the effect of DXR on the cardiac phosphoproteome has not yet been investigated. 1.3.4. Redoxproteomics Oxidative stress, a well-known effect of DXR in the heart, leads to oxidative modification of many proteins 4 . Amino-acid residues can be directly modified by free radicals, and proteins can be altered by adduct formation with products of glycoxidation or lipid peroxidation.

Oxidized proteins can lose their activity and other functional properties. Depending on the dose of ROS (and RNS), this leads to severe disruption of cellular functions, finally triggering cell death, in particular if the oxidative stress response is unable to detoxify the reactive species and to repair molecular damage 88 .

Like in phosphoproteomics, oxidative modifications can be studied by specific antibodies in immunoblots of 2-DE. In the field of DXR cardiotoxicity, only one study has used this approach to analyze the acute effect of DXR on the formation of dinitrophenylhydrazone adducts 98 . Mice were injected with 20 mg/kg of DXR and sacrificed 72 h thereafter. 2-DE-MS identified only three proteins carrying this oxidative modification after DXR treatment, all involved in energy metabolism: β-enolase, triose phosphate isomerase and electrotransfer flavoprotein-ubiquinone oxidoreductase. Only enzyme activity of glycolytic β-enolase was decreased by 25%, possibly contributing to energy depletion.

Studying the abundant protein oxidation caused by DXR 4 could indeed improve our knowledge of these oxidative modifications. However, other more abundant oxidative protein modifications should be selected and analyzed, e.g. by high affinity antibodies specific for these modifications.

Metabolomics

Metabolomics is the study of complete sets of low-molecular-weight metabolites present in a given cell. Mostly MS or NMR spectroscopy are used to detect, identify and quantify these molecules. Metabolomic data, together with the knowledge on metabolic pathways and proteomics, allow to generate models of e.g. metabolic pathways and fluxes, substrate utilization, or metabolic switches 99 .

Two studies have been published so far using metabolomics approaches to study DXR effects in heart. The first study involved rats treated by a unique intraperitoneal injection of 20 mg/kg DXR. NMR analysis showed in DXR treated hearts an increase of acetate and succinate, which are both generated via non-enzymatic decarboxylation of pyruvate and αketoglutarate under oxidative stress conditions 100 . The study further revealed an unchanged lactate/pyruvate ratio, indicating that lactate did not accumulate in heart as predicted by inhibition of oxidative metabolism, probably because of the important nonenzymatic conversion of pyruvate into acetate. Finally, the data showed that branched chain amino acids like valine, leucine and isoleucine were reduced. These amino acids may be mobilized from muscle proteins in order to be used as an alternative energy substrate to compensate the energy loss. Thus, this study gave first indications how metabolic pathways are disturbed by DXR and revealed associated biomarkers such as succinate and acetate 101 . Cavalho et al. 102 have conducted a metabolomic study of DXR cardiotoxicity using rats injected weekly with 2 mg/kg DXR during 6 weeks (cumulative dose: 12mg/kg). Animals were sacrificed one week after the last injection and hearts were excised and perfused with 13 C-enriched substrates. This study confirmed a switch form long-chain fatty-acid oxidation to glucose oxidation. To a minor degree, lactate plus pyruvate oxidation was also decreased.

The change in substrate utilization did not occur via inhibition of FA transport but via decreased activity of β-oxidation enzymes. The authors suggested that inhibition of FA oxidation was compensated by glycolysis and conversion of pyruvate into lactate, which was found increased.

Both metabolomic studies agree on decreased FA oxidation, providing evidence for a switch in oxidative substrate utilization for ATP generation, while only the second study suggests a redirection of glucose metabolites towards lactate production.

1.3.6. Summary : "omics" studies on doxorubicin cardiotoxicity

The different "omics" methods described above provide more or less complete inventories of cellular levels of mRNAs, proteins, protein modifications and metabolites. The results are not always consistent, given the very different toxicity models that use of various DXR concentrations and injection protocols. However, some inventory data can be turned into insight.

First, energy metabolism is highly affected by the drug, confirming earlier studies on serious cardiac energy deficits induced by DXR (reviewed in [START_REF] Wallace | Doxorubicin-induced cardiac mitochondrionopathy[END_REF]44 ). Transcriptomic studies reveal an up-regulation of genes coding for glycolysis, while genes in FA oxidation, mitochondrial electron transport chain and Krebs cycle are down-regulated. Proteomics studies show a mixed response of proteins in FA oxidation and mitochondrial respiratory chain, but a consistent increase of proteins involved in glucose metabolism. In addition, metabolomics confirm decreased FA oxidation and, in part, an increase of glucose utilization for lactate production. As a consequence, while normal heart uses preferentially FA as energy source, DXR-treated heart seems to prefer glucose, thus confirming a switch in substrate utilization already suggested by early transcriptomic studies (see chapter 1.2.2).

Second, some protective pathways are activated in response to DXR. Genes coding for oxidative stress defense, DNA damage repair, inflammation and some others stresses are often up-regulated. Also protein levels of those enzymes are mostly higher, in particular some anti-oxidative enzymes and chaperones. Apparently, these are compensatory mechanisms.

Third, structural proteins are often affected, mostly increased, possibly to compensate the loss of cardiac performance. Indeed, changes in mRNAs coding for proteins involved in cardiac remodeling and increased levels of cytoskeletal proteins may be linked to the cardiac hypertrophy occurring in DXR-treated heart 2 . This hypertrophy is supposed to occur in order to compensate the decrease of ejection fraction [START_REF] Schulenberg | Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye[END_REF] . For similar reasons, as shown by proteomic studies, proteins involved in muscle contraction become more abundant [START_REF] Schulenberg | Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye[END_REF] .

Despite these correlations, one has to be careful with the interpretation of transcriptomic and proteomics data. Changes in transcript or protein abundance do not necessarily translate into changes in the activity of the corresponding pathway.

Doxorubicin and cardiac cell signaling

Cellular processes are controlled, among others, by cell signaling pathways that inhibit or activate proteins, often by reversible phosphorylation of serine, threonine or tyrosine residues 94 . These signaling pathways respond to different stimuli, which can be extra-or intracellular. As DXR induces various kinds of cellular stress, it can be assumed that signaling pathways are also affected by the drug and that their deregulation contributes to DXR cardiotoxicity. Here we briefly introduce some major protein kinases (Table 1-3) and their (potential) links to DXR cardiotoxicity. 1.4.1. AMP-activated protein kinase AMP-activated protein kinase (AMPK), a Ser/Thr kinase, is an energy sensor and a key regulator of cellular and whole body energy homeostasis [START_REF] Kawajiri | Functional significance of the specific sites phosphorylated in desmin at cleavage furrow: Aurora-B may phosphorylate and regulate type III intermediate filaments during cytokinesis coordinatedly with Rho-kinase[END_REF] . When energy expenditure exceeds ATP regeneration, the AMP/ATP ratio increases via the adenylate kinase reaction. High AMP levels then activate AMPK, which inhibits anabolic pathways and stimulates catabolic pathways in order to generate more ATP and to maintain energy homeostasis. Moreover, AMPK is also activated by phosphorylation at Thr172, catalyzed by different upstream kinases, including the calmodulin-dependent kinase kinase (CamKK) and the serine/threonine protein kinase 11, LKB1 [START_REF] Kawajiri | Functional significance of the specific sites phosphorylated in desmin at cleavage furrow: Aurora-B may phosphorylate and regulate type III intermediate filaments during cytokinesis coordinatedly with Rho-kinase[END_REF] .

Our group was the first to report effects of DXR on AMPK phosphorylation and activity in rat heart, perfused with 2 and 20 µM of DXR 50 . In this model, two conditions known to activate AMPK were present: free radicals and a high AMP/ATP ratio. However, the AMPK signaling pathway was down-regulated at different levels: P-AMPK, AMPK protein, and AMPK activity as assayed with the substrate ACC, a well-known target of AMPK 50 . These results suggested a paradox inhibition of AMPK in a context which should activate the kinase.

A more recent study 103 confirmed a decrease in P-AMPK by 45% in hearts of rats treated with a unique injection of DXR 20 mg/kg. More recently, Konishi et al. 104 have used an adiponectin (APN) transgenic murine model treated with a cumulative dose of 24 mg/kg DXR. They also observed a decrease of P-AMPK relative to total AMPK in wild-type animals, and also in APN depleted transgenic animals, but an opposite result in APN overexpressing animals. These data suggest that APN can overcome DXR-inhibition of AMPK, which would finally inhibit apoptosis as suggest by experiments with cultured cardiomyocytes. Thus, AMPK inhibition and its rescue may be a crucial element in DXRmediated cardiotoxicity.

Mitogen activated protein kinases

Oxidative stress implies the generation of free radicals, which are known to activate the mitogen-activated protein kinases (MAPK). Their signaling pathways involve extracellular signal-regulated protein kinase (ERK1/2), c-Jun-N-terminal kinase (JNK) and p38 (reviewed in 105 ). These kinases play a role in apoptosis and cell proliferation, and are activated by phosphorylation (reviewed in 105 ). Inhibition of ERK as well as activation of JNK and p38 triggers apoptotic events. As DXR is known to generate free radicals and to induce apoptosis, MAPK signaling in the heart has already been analyzed. In a study on chronic toxicity in rat heart induced by a cumulative dose of 15 mg/kg DXR, mRNAs of ERK and a kinase upstream of ERK, MEKK, were decreased, and ERK phosphorylation, after a transient increase, also decreased, in parallel with an increase in phosphorylation of p38 and JNK.

These changes correlated with an activation of caspase 3, a central execution element in apoptosis. Thus, DXR may trigger apoptosis by acting on MAPKs 106 . In another study on H9C2 cells, also the MAPK p38 was phosphorylated and activated at 5 and 10 µM DXR 107 . It seems thus that DXR can affect several MAPK family members to affect apoptosis.

Akt / protein kinase B

The protein kinase B (PKB), also known as Akt, a Ser/Thr kinase, is a pro-survival kinase and a critical signaling node regulating many processes, including cell growth, proliferation and survival 108,109 . Akt is fully activated when phosphorylated at two sites: Thr308 in the catalytic domain and Ser473 in its hydrophobic part. This key regulator of cell signaling is an interesting putative target of DXR in cardiotoxicity and has been studied in this context in more detail; however, some controversial results have been obtained. In cardiomyocytes treated with 1 µM DXR for 6 hours, phosphorylation and activity of Akt were decreased. This inhibition of the Akt pro-survival pathway correlated with an increase of some pro-apoptotic factors and led to apoptotic death of cardiomyocytes 110 . In an animal model, where rats received a unique injection of DXR 20 mg/kg, cardiac levels of P-Akt and Akt were also decreased after the treatment, although the P-Akt/Akt ratio remained unchanged. Another decrease in P-Akt (and also P-ERK) was seen in hearts of rat intravenously injected with 2,5 mg/kg DXR per week for six weeks 111 . These results are in contrast to several other studies showing a DXR-induced increase in P-Akt 112-114 . 1.4.4. Protein kinase C

The protein kinase C (PKC) family of Ser/Thr kinases is involved in many cellular processes like cell growth and differentiation (reviewed in 115 ). This family comprises about ten different isoforms, classified by their structure or their activation mode. PKCs are activated by growth factors via the intermediate generation of the second messenger diacylglycerol, by protein/protein interaction or by phosphorylation (reviewed in 116 ).

Activation of these kinases has been already studied in the context of DXR-induced cardiotoxicity. In vitro assays were performed using DXR-Fe complexes and PKC. DXR-Fe inhibited PKC activity in a reversible way. In fact, DXR-Fe and Mg-ATP competed for binding to PKC 117 . However, an in vivo study showed an opposite effect of DXR: PKC was activated in cultured cardiomyocytes treated with 4 µM DXR for 30 minutes. This correlated with the destabilization and disassembly of myofilaments 118 . 1.4.5. Calcium/calmodulin-dependent protein kinases Calcium/calmodulin-dependent protein kinases (CamK) are Ser/Thr kinases regulated by calcium. They are involved in cellular processes such as gene expression and pathological developments like cardiac diseases (reviewed in 119 ). In a chronic rat model, Little et al. 120 have demonstrated that DXR down-regulates genes coding for CamKIIδB, the predominant CamK form in heart. They confirmed in cardiomyocytes a decrease of CamKIIδB transcripts and protein levels already in an early stage of DXR treatment. Here, they also observed an alteration of cell integrity and a decrease of anti-apoptotic factor Bcl2, linking downregulation of nuclear CamKIIδB to induction of cardiomyocyte apoptosis.

Introduction

Anthracyclines, in particular doxorubicin (DXR), are among the most effective and commonly used anticancer drugs. However, a serious side effect of anthracycline therapy is severe cardiotoxicity. This represents a burden to the public health system, given the constantly increasing number of cancer patients and cancer survivors suffering from cardiovascular side effects of chemotherapy or pathological (ischemia) situations, leading to a rise in AMP/ATP ratios, intracellular calcium, and reactive oxygen and nitrogen species. In addition, it is also activated by extracellular signals like orexigenic/anorexigenic hormones or cytokines 5,6 . The activation mechanism consists of an allosteric component (AMP or ATP binding to the gamma subunit) and a covalent component (phosphorylation of Thr172 in the alpha subunit by the upstream kinases LKB1 and/or CamKKβ). Once activated, AMPK induces adaptation of energy metabolism through regulation of cellular signaling cascades, activity of metabolic key enzymes, and gene transcription. For example, activated AMPK stimulates ATP-generating processes, including uptake and oxidation of glucose and fatty acids, and suppresses a number of ATP-consuming processes such as protein synthesis, cellular growth and proliferation. The latter is mainly mediated by inhibition of mammalian target of rapamycin (mTOR) within the mTORC1 complex 7,8 .

Several most recent studies indicate a more general relationship between a reduced LKB1-AMPK signaling and cardiac disease 7,[START_REF] Tokarska-Schlattner | Early effects of doxorubicin in perfused heart: transcriptional profiling reveals inhibition of cellular stress response genes[END_REF]10 . In this context, the aim of the present study was to analyze the effect of DXR (i) on cardiac signaling up-and downstream of AMPK, in particular mTOR, and (ii) on Akt/PKB and mitogen activated protein kinase (MAPK) signaling, both reported to cross-talk with AMPK and to mediate also upstream signals for mTOR. In two different rat cardiotoxicity models, the isolated perfused heart and a more chronic in vivo model, we show that DXR induces combined energy, oxidative and genotoxic stress. This combined stress leads to inhibition of AMPK without negatively affecting different parameters of AMPK upstream signaling. It also activates MAPK and Akt signaling, e.g. via DNA-damage sensing DNA-PK, and this in turn would negatively impact on AMPK activation. Downstream, this would result in continued ATP consumption without compensating ATP generation, and most importantly in continuous activation of growthstimulating mTOR, both potentially contributing to the cardiotoxic phenotype.

Methods

Materials

Doxorubicin hydrochloride (DXR) was purchased from Fluka (Buchs, Switzerland). A stock solution (10 mM) was prepared in water and kept frozen until use. Further dilutions were prepared in Krebs-Henseleit buffer just before heart perfusion or in saline before injection to rats. ATP, ADP and AMP were purchased from Sigma (Saint Louis, MO), protease inhibitor cocktail tablets from Roche (Mannheim, Germany) and phosphatase inhibitor cocktail from Pierce (Rockford, IL, USA).

Rat heart perfusion and animal treatment

Perfusion experiments were essentially performed according to the protocol described earlier. 2 Briefly, hearts of male Wistar rats (∼300 g) anaesthetized with sodium pentobatbital (50 mg/kg ip) and heparinized (1500 IU/kg iv) were quickly removed after checking the depth of anaesthesia (pedal reflex, muscle relaxation) and perfused at constant pressure in a noncirculating Langendorff apparatus without (control) or with 5 µM or 25 µM DXR during 80 min (after initial 30 min period of stabilization with Krebs-Henseleit buffer) and functional parameters were recorded. At the end of perfusion, hearts were rapidly freeze-clamped in liquid nitrogen and stored at -80°C. For the animal cardiotoxicity model, 6 week old male Wistar rats (∼175 g) were anaesthetized with an intramuscular injection of ketamine (50 mg/kg)/xylazine (10 mg/kg) and catheterized via jugular vein 11 . Adequacy of anaesthesia was carefully monitored by regular checking depth of anaesthesia (pedal reflex, muscle relaxation) and physiological parameters (heart and respiratory rate). Anaesthetic support such as fluid supplementation and external heating as well as monitoring and support during anaesthetic recovery was provided. DXR (2 mg/kg) or equivalent volume of saline were administered to animals via the implanted catheter three times a week during two weeks for a total dose of 12 mg/kg. The first injection was realized on the day of catheterization. Four weeks after the last injection, rats were anesthetized again and left ventricular function was measured with a Millar pressure-volume system 12 . Subsequently, the hearts were rapidly removed, freeze-clamped in liquid nitrogen and stored at -80°C. All procedures involving animals conform to Directive 2010/63/EU and were approved by the Grenoble Ethics Committee for Animal Experimentation (Com-Eth 15_LBFA-U884-HD-01).

Quantification of nucleotides, DNA damage and protein oxidation

Freeze-clamped hearts were homogenized in 0.6 N perchloric acid in liquid nitrogen, centrifuged (12 min, 3000 g, 4°C), neutralized by addition of 5 M potassium carbonate and centrifuged again (12 min, 3000 g, 4°C). Protein-free extract (30 µl) was separated on a C18

HPLC column (Polaris 5C18-A, S250*4.6 Repl, Varian, France) in pyrophosphate buffer (28 mM, pH 5.75) at 1 ml/min flow rate and 30°C. ATP, ADP and AMP eluted at 3, 5 and 9 min, respectively. Elution peaks were integrated with STAR software (Varian, France).

Total genomic DNA was isolated with the QIAamp DNA mini kit (Qiagen). Nuclear and mitochondrial DNA damage were evaluated using a two-step strategy based on a Long PCR and a Real-Time PCR 13 . A detailed description of reagents, primers and PCR conditions is available as Supplementary Material online. Oxidative modification of proteins was probed by immunodetection of carbonyl-groups in the protein side-chains with the OxyBlot TM Kit (Millipore, Temecula, CA).

Immunoblotting and immunoprecipitation

SDS-PAGE separation of heart homogenates and immunoblotting were performed according to standard procedures 14 . Detailed descriptions including sources and dilutions of antibodies as well as the immunoprecipitation protocol are available as Supplementary Material online.

Statistics

If not stated otherwise, data are presented as means ± SD. Statistical significance was evaluated by two-tailed Student's t-test using Sigma Plot. * P < 0.05, ** P < 0.01, *** P < 0.001.

Results

Impaired cardiac function associated with energetic, oxidative and genotoxic stress

Two different models of DXR cardiotoxicity have been used throughout this study, representative for acute and chronic toxicity. Acute effects were analyzed in isolated rat hearts perfused during 80 min with 5 or 25 µM DXR, the lower concentration corresponding to DXR concentrations found in plasma of patients after bolus injection 15 . Chronic effects were analyzed in hearts of rats that received 6 intravenous injections of DXR (2 mg/kg) over a 2 week period and were sacrificed 4 weeks after the end of the treatment. The latter model has been shown to adequately mimic chronic myocardial alterations in patients 15 . Both models were first analyzed for cardiac function and markers of different forms of stress and apoptotic S2-1 ). This complete characterization of our models for acute and chronic DXR cardiotoxicity is consistent with earlier reports 2,[START_REF] Gewirtz | A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin[END_REF][START_REF] Rabilloud | Two-dimensional gel electrophoresis in proteomics: Past, present and future[END_REF] . and2-2F). On the contrary, basal AMPK phosphorylation at Thr172 was even diminished by DXR. This decrease in AMPK activity was also observed at the level of one of its important downstream targets, acetyl-CoAcarboxylase (ACC), providing an independent readout for full AMPK activity (Figure 2-3A,B). These data clearly confirm and expand our initial observation on an inhibitory effect of DXR on cardiac AMPK activity 2 that later has been reproduced by others 3,4 . Only CamKKα was reduced at 25 µM DXR, but this isoform is not considered as bona fide upstream kinase in vivo 5 . We further examined whether LKB1 regulation was altered.

status (Figures 2-1, 2-2, Table
Although this kinase is mostly considered to be constitutively active 5,[START_REF] Schulenberg | Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye[END_REF] , some studies correlated LKB1 phosphorylation at Ser428 20 or deacetylation at Lys48 21 with its downstream activity. In particular SIRT1 was proposed to deacetylate nuclear LKB1, resulting in LKB1 export into the cytosol and activation of cytosolic AMPK [START_REF] Li | Evaluation of several two-dimensional gel electrophoresis techniques in cardiac proteomics[END_REF] . Our data reveal no DXR-induced changes in SIRT1 levels, but an increase of both phosphorylation (at 5 µM DXR) and deacetylation of LKB1 (Figure 2-3C,D), which should both rather favor cytosolic LKB1 activity. Another mechanism for LKB1 inhibition suggested recently is adduct formation with HNE (4-hydroxy-2-nonenal), a byproduct of lipid peroxidation during oxidative stress in the heart 10 . However, the amount of HNE-protein adducts was not increased by DXR (Figure S2- 3). Finally, phosphatases are emerging as important players in controlling the AMPK activation state. We examined the activity of phosphatase PP2C, which can dephosphorylate AMPK in vitro 5,[START_REF] Schulenberg | Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye[END_REF] . This activity was unaltered by DXR in perfused heart (Figure S2-4).

Collectively, we could not find evidence for an altered upstream signaling to be responsible for the AMPK inhibition by DXR.

Doxorubicin activates the Akt pathway

An alternative mechanism to down-regulate AMPK activity may be a cross-talk with other signaling pathways. One of the most relevant candidate in heart is Akt, as AMPK inhibition by the Akt pathway has been reported by us and others [START_REF] Mcgregor | Proteomics of the heart: unraveling disease[END_REF][START_REF] Mesnard-Rouiller | Troponin T mRNA and protein isoforms in the human left ventricle: pattern of expression in failing and control hearts[END_REF][START_REF] Tokarska-Schlattner | New insights into doxorubicin-induced cardiotoxicity: the critical role of cellular energetics[END_REF][START_REF] Wallace | Doxorubicin-induced cardiac mitochondrionopathy[END_REF] and was detected in heart pathologies. [START_REF] Ventura-Clapier | Bioenergetics of the failing heart[END_REF] Most recent data indicate that Akt may be also activated by genotoxic stress as a substrate of DNA-dependent protein kinase (DNA-PK). The DNA-PK is itself activated by DNA double-strand breaks [START_REF] Feng | Phosphoproteome analysis of isoflurane-protected heart mitochondria: phosphorylation of adenine nucleotide translocator-1 on Tyr194 regulates mitochondrial function[END_REF][START_REF] Wang | Metabolic profiling of hearts exposed to sevoflurane and propofol reveals distinct regulation of fatty acid and glucose oxidation: CD36 and pyruvate dehydrogenase as key regulators in anestheticinduced fuel shift[END_REF] , a typical consequence of DXR action. In line with the genotoxic stress detected ( Consistently, DXR increased Akt signaling as observed by specific phosphorylation of Akt downstream targets, including GSK3 (Figures 2-4A,B, S5). GSK3 is a prominent downstream effector of the DNA-PK-Akt pathway [START_REF] Wang | Metabolic profiling of hearts exposed to sevoflurane and propofol reveals distinct regulation of fatty acid and glucose oxidation: CD36 and pyruvate dehydrogenase as key regulators in anestheticinduced fuel shift[END_REF] and essential to promote cell survival and inhibit apoptosis after DNA damage. Consistent with earlier studies [START_REF] Sugden | Mechanisms underlying regulation of the expression and activities of the mammalian pyruvate dehydrogenase kinases[END_REF][START_REF] Holness | Regulation of pyruvate dehydrogenase complex activity by reversible phosphorylation[END_REF] , an increased activating phosphorylation of Akt itself was clearly detectable at Ser473 in the animal model, but below significance in perfused heart at both Ser473 and Thr308, despite strong phosphorylation of DNA-PK and GSK3. This may relate to negative feedback loops by which activated downstream elements of the Akt-mTOR pathway limit the extent of upstream signaling 8 or by activation of local pools of Akt 28 .

Doxorubicin activates MAPKs

Another kinase family reported to cross-talk with AMPK are mitogen-activated protein kinases (MAPKs [START_REF] Carvalho | Metabolic remodeling associated with subchronic doxorubicin cardiomyopathy[END_REF][START_REF] Lewandowski | Pyruvate dehydrogenase influences postischemic heart function[END_REF] ). MAPKs respond to a variety of stresses like UV irradiation or reactive oxygen species in addition to growth factors and stretch stimuli. In particular the extracellular signal-regulated protein kinase (ERK), a MAPK family member with pro-survival functions, is activated in heart in response to practically every stress [START_REF] Wakasugi | Myocardial substrate utilization and left ventricular function in adriamycin cardiomyopathy[END_REF] . In line with these characteristics and previous reports 14,[START_REF] Sugden | Mechanisms underlying regulation of the expression and activities of the mammalian pyruvate dehydrogenase kinases[END_REF][START_REF] Solaro | Protein phosphorylation and signal transduction in cardiac thin filaments[END_REF] , an activation of ERK by DXR was observed in isolated heart Since AMPK is unable to fulfill its compensatory function in cellular energy homeostasis, we examined the status of another key factor for cardiac energy homeostasis, the creatine kinase energy buffer and transfer system [START_REF] Huang | Protein kinase C-mediated desmin phosphorylation is related to myofibril disarray in cardiomyopathic hamster heart[END_REF] . Up-regulation of the cytosolic brain isoform of creatine kinase is one of the redundant regulatory and compensatory mechanisms observed in cardiac pathologies [START_REF] Huang | Protein kinase C-mediated desmin phosphorylation is related to myofibril disarray in cardiomyopathic hamster heart[END_REF] . Consistently with our previous findings, increased BCK protein was detectable in perfused heart, already at the low DXR concentration of 5 µM, and also in the animal model of chronic cardiotoxicity (Figure S2-6).

Discussion

This study is a first analysis of the LKB1-AMPK-mTOR signaling axis and its crosstalking signaling pathways as a potential mediator of DXR-induced cardiotoxicity. Two different models were chosen to represent acute and chronic toxicity: the DXR-perfused isolated rat heart and DXR-treated rats probed four weeks after the last injection, respectively.

Both models mimic DXR plasma concentrations and myocardial alterations that are comparable to those in patients 15 . The response of cell signaling to DXR was determined by the combined induction of at least three forms of stress: energetic, oxidative and most of all genotoxic stress, as shown for the toxicity models in this and other studies 15,16,[START_REF] Rabilloud | Two-dimensional gel electrophoresis in proteomics: Past, present and future[END_REF][START_REF] Geisler | Phosphorylation of desmin in vitro inhibits formation of intermediate filaments; identification of three kinase A sites in the aminoterminal head domain[END_REF] . We show that under these conditions, the fuel-sensing AMPK signaling is not activated but instead paradoxically suppressed, whereas survival signaling by DNA-PK-Akt-GSK3, as well as stress signaling by MAPK, is up-regulated. As a consequence, mTOR, the main regulator of protein synthesis and cell growth, which integrates signals from the several protein kinase pathways, becomes activated. This signaling pattern contains some cytoprotective elements, such as pro-survival Akt-GSK3 signaling triggered by DNA damage via DNA-PK, which counteracts apoptosis due to genotoxic stress, or transient anti-apoptotic ERK activation.

However, there are several elements that likely contribute to cardiotoxicity. First, inhibition of compensating, pleiotropic AMPK downstream signaling exacerbates energy stress. This will limit the capacity for ATP regeneration, e.g. via fatty acid oxidation, and maintain or reinforce numerous energy-consuming biosynthetic processes, like via the relieved inhibition of mTOR. This will also make cardiomyocytes less resistant to additional physiological and pathological stressors like strenuous exercise, hypoxia or ischemia.

DXR induces not only energetic imbalance, a direct AMPK activator, but also reactive oxygen and nitrogen species and genotoxic stress, which should stimulate AMPK activity by more indirect mechanisms 8 . In both analyzed models, at least two of these forms of DXRinduced stress are present at a level that activates AMPK in different cellular systems. [START_REF] Tokarska-Schlattner | New insights into doxorubicin-induced cardiotoxicity: the critical role of cellular energetics[END_REF] The lack of AMPK activation by DXR, and even more the diminished basal phosphorylation by DXR is therefore unexpected. However, it is consistent with some other reported DXR effects: (i) a change in the profile of cardiac energy substrate utilization with a decrease in oxidation of both fatty acids and glucose 38 , (ii) reduced exercise tolerance affecting up to 70% of cancer patients during and after therapy 39 , (iii) protection against DXR cardiotoxicity by compounds and regimens activating AMPK like resveratrol, adiponectin, caloric restriction, and physical exercise 4,[40][41][42] , and aggravation by conditions suppressing AMPK signaling like obesity and high fat diet 43 .

The cardiac metabolic network is characterized by an unusual stability. Energy homeostasis is maintained by multiple regulatory mechanisms, including allosteric regulations and feedback loops, micro-compartmentation or metabolic channeling 44 .

Compensatory mechanisms within the cardiac energy network occur early under DXR treatment and include e.g. up-regulation of brain-type creatine kinase and increased expression of other transcripts involved in ATP generation 2,14 . AMPK activation in heart, in contrast to most other tissues, seems rather to act as a last safeguard during severe energy deprivation and pathological situations such as cardiac ischemia 44 . Thus, DXR-induced inhibition of AMPK is expected to generate a negative feedback loop that could not only contribute to the etiology of the pathological phenotype, but also aggravate it in advanced stages of DXR-induced cardiomyopathy 15 or during additional energy stress as observed with patients 39 .

In search of mechanisms responsible for AMPK inhibition, we have analyzed most known pathways upstream of or cross-talking with AMPK. The by far most important AMPK kinase in heart, the tumor suppressor LKB1 which is considered to be constitutively active 5,[START_REF] Schulenberg | Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye[END_REF] , was not affected in terms of expression level. It even showed increased deacetylation and phosphorylation, reported to favor cytosolic localization and downstream signaling. Protein adducts with HNE, a byproduct of lipid peroxidation reported to inhibit LKB1-AMPK signaling in heart upon oxidative insult 10 , were neither found increased. Levels of the second AMPK kinase, CamKKβ, were also unchanged. Phosphatases regulating AMPK activity are less defined, but at least PP2C activity was not altered. Thus, no evidence for inhibited AMPK upstream signaling could be found, though we cannot definitely reject decreased activities of LKB1 and CamKKβ due to other, unknown mechanisms. More likely, inhibitory cross-talk with other signaling pathways can play an important role. Akt, and as suggested more recently also ERK can cross-talk with and inhibit AMPK [START_REF] Mcgregor | Proteomics of the heart: unraveling disease[END_REF][START_REF] Mesnard-Rouiller | Troponin T mRNA and protein isoforms in the human left ventricle: pattern of expression in failing and control hearts[END_REF][START_REF] Tokarska-Schlattner | New insights into doxorubicin-induced cardiotoxicity: the critical role of cellular energetics[END_REF][START_REF] Wallace | Doxorubicin-induced cardiac mitochondrionopathy[END_REF][START_REF] Carvalho | Metabolic remodeling associated with subchronic doxorubicin cardiomyopathy[END_REF][START_REF] Lewandowski | Pyruvate dehydrogenase influences postischemic heart function[END_REF] . Both pathways were strongly activated by DXR in both examined models.

The interplay of AMPK with Akt is especially pronounced in heart [START_REF] Ventura-Clapier | Bioenergetics of the failing heart[END_REF] . We show here that DNA-PK, recently located upstream of Akt [START_REF] Wang | Metabolic profiling of hearts exposed to sevoflurane and propofol reveals distinct regulation of fatty acid and glucose oxidation: CD36 and pyruvate dehydrogenase as key regulators in anestheticinduced fuel shift[END_REF]45 , represents an important and novel element in the cardiac response to DXR. DNA-PK, a crucial component of the DNA repair machinery for double-strand breaks, shows rapid and strong phosphorylation and activation by DXR. It thus connects DNA-damage signaling to activation of Akt, inhibition of AMPK, and even more pronounced phosphorylation of further downstream elements such as GSK3 that promote cell survival after genotoxic stress [START_REF] Wang | Metabolic profiling of hearts exposed to sevoflurane and propofol reveals distinct regulation of fatty acid and glucose oxidation: CD36 and pyruvate dehydrogenase as key regulators in anestheticinduced fuel shift[END_REF]45 . While activation of the DNA-PK-Akt-GSK3 pathway was comparable in both models examined here, apoptosis was lower in perfused heart as compared to the chronic model, correlating with the activation pattern of MAPKs.

While phosphorylation of pro-survival ERK was prominent in the former model, proapoptotic JNK became activated in the latter, in line with previous reports of us and others 14,[START_REF] Sugden | Mechanisms underlying regulation of the expression and activities of the mammalian pyruvate dehydrogenase kinases[END_REF][START_REF] Solaro | Protein phosphorylation and signal transduction in cardiac thin filaments[END_REF] . Apoptosis, for which cardiomyocytes are relatively resistant, heavily contributes to heart failure when occurring 46 . Pathways counteracting apoptosis, as DNA-PK-Akt-GSK3

and ERK, may be a crucial to preserve cardiomyocytes viability during genotoxic stress. In DXR-challenged cells, inhibition of Akt increases apoptosis [START_REF] Sugden | Mechanisms underlying regulation of the expression and activities of the mammalian pyruvate dehydrogenase kinases[END_REF] , while up-regulation of Akt confers significant protection 47 .

Combined inhibition of AMPK and activation of Akt and ERK by DXR will lead to elevated mTOR signaling, as we observed by increased phosphorylation of mTOR (at Ser2448) and its downstream target p70S6K. This will stimulate energetically costly biosynthetic pathways in protein biosynthesis and cell growth, thus generally enhancing DXR-induced energy-deficits, but also generating growth-stimulating signals. Depending on the context, mTOR activation can counteract excessive cardiac atrophy, but also stimulate cardiomyocyte hypertrophy 10,48 . Effects of DXR on mTOR signaling became controversial, with several studies including ours showing DXR-induced increases in mTOR signaling (e.g.

in cultured cardiomyocytes or mouse embryonic fibroblasts 49,50 ), and a recent report on DXRtreated mice showing inhibition of cardiac mTOR and cardioprotective effects of constitutively active mTOR 51 . However, this discrepancy could be explained by a different administration protocol (2 x 10 mg/kg) and probing only four days after the last DXR injection in the latter study. In summary, in DXR-challenged heart, combined energetic, oxidative, and genotoxic stress elicits a specific response where AMPK is paradoxically inhibited at least partially by the known regulatory cross-talk with activated Akt and MAPK pathways, largely triggered by DNA damage signaling via DNA-PK. This occurs in the acute toxicity model, as well as in the more chronic model probed four weeks after the last DXR injection. Though activation of DNA-PK-Akt and MAPK pathways has some protective consequences, impaired energy signaling by AMPK will initiate a negative feedback loop that potentiates DXR-induced energy deficits and seriously compromises heart resistance to additional energy stress.

Activation of growth-stimulating mTOR by combined AMPK inactivation and Akt/ERK activation will also contribute to the pathological cardiac phenotype in chronic DXR toxicity.

Supplement I

Supplementary material and methods

DNA damage quantification

Long PCR was performed in a final volume of 25 µl using a GeneAmp PCRSystem 9700 (PE Applied Biosystems). Specific primers (Table S2-

Immunoblotting and immunoprecipitation

Freeze-clamped hearts were homogenized in 1 M Tris-HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT containing protease and phosphatase inhibitors. Protein concentration was determined with Biorad reagent (Reinach, Switzerland) using BSA as a standard.

Homogenates were either used immediately or stored at -80°C. Fifty µg protein/lane were separated on 7.5%, 10% or 12% polyacrylamide gels and transferred to nitrocellulose membrane (Hybond ECL, GE Healthcare, Buckinghamshire, UK) by wet or semi-dry blotting. After blocking in 5% milk, TBS, and 0.1% Tween 20, blots were immunostained with primary antibody overnight at 4°C, then washed and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody. Immunoreactive bands were detected using a chemiluminescence kit (ECL plus, GE Healthcare) and a CCD camera (ImagerQuant LAS 4000, GE Healthcare). The following primary antibodies were used: from Cell Signaling (Beverly, MA) anti-AMPK rabbit polyclonal 1:1000, anti-phosphoThr-172AMPK 1:400, anti-ACC 1:1000, anti-phosphoSer79-ACC 1:1000, anti-phosphoSer2448-mTOR 1:500; anti-mTOR 1:1000, anti-phosphoSer473-Akt, 1:1000, anti-phosphoThr308-Akt 1:1000, anti-phosphoThr389-p70 S6 kinase 1:1000, anti-phosphoSer9-GSK 3β 1:1000, anti-α-tubulin 1:1000, anti-acetylated-lysine, anti-SAPK/JNK 1:1000, anti-phosphoThr183/Tyr185-SAPK/JNK 1:1000, anti-phosphoThr180/Tyr182-p38 Values are expressed as mean ± SD (n ≥ 4) in µmol/g dry weight.

Supplement II

This supplement includes experiments performed in the context of the study "Inhibition of AMPK signaling by doxorubicin -at the crossroads of the cardiac responses to energetic, genotoxic and oxidative stress" but not included in the submitted manuscript.

As we show in the above-described experiments in DXR-perfused rat hearts and in hearts of chronically DXR-treated rats, DXR provokes an inhibition of AMPK signaling in spite of an increase in the AMP/ATP ratio, which normally should activate AMPK 1 . ERK was among the protein kinases that showed increased activity, especially in the acute DXR toxicity model, suggesting that the known inhibitory cross-talk with AMPK could contribute to AMPK inactivation.

Here we further investigate the DXR-effect on these kinases by addressing the following questions: (i) Does DXR also affect AMPK signaling in cancer cell lines? (ii) Is ERK activation by DXR accompanied by ERK nitration, which was recently shown to activate this kinase 2,3 ? 2.9.1. Effect of doxorubicin on AMPK signaling in cancer cells

The observed inhibition of AMPK signaling in DXR-perfused hearts in situ and in vivo 4 (chapter 2.) raises the question whether this paradox regulation is also present in other tissues, in particular cancer. The AMPK upstream kinase, LKB1 is a known tumor suppressor, acting at least in part via AMPK activation to inhibit mTOR-dependent cell proliferation 5 . Thus, a similar AMPK inhibition in cancer by DXR would be an undesired effect. Here we treated several cancer cell lines with DXR and determined the phosphorylation levels of AMPK and its target ACC.

Materials and methods

Human hepatoma (HepG2 Immunobloting was performed as described earlier (chapter 2.).

Results and conclusions

Incubation of cancer cell lines with 0.1, 1 or 5 µM DXR led to line-dependent effects on AMPK activation (AMPK phosphorylation) and AMPK activity (ACC phosphorylation).

While both phosphorylations were unaffected in HepG2 cells ( Importantly, in all three cell lines and in both incubation periods, no decrease in AMPK or ACC phosphorylation was observed. Thus, the tumor-suppressor signaling axis LKB1-AMPK is intact and no undesired pro-proliferative signaling has to be expected. This also implies a rather specific DXR-effect on cardiac AMPK signaling.

Whether or not a more or less pronounced increase in AMPK signaling can be observed seems to depend rather on the metastatic potential. Hepatoma HepG2 cells show a high degree of morphological and functional differentiation in vitro and are not tumorigenic in nude mice 6 . Colorectal cancer cells of the types HCT8/S11 have limited invasive behavior in vivo 7 , while the breast cancer cell line MDA-MB-231 has the most invasive phenotype and forms mammary fat pad tumors in nude mice 8 . Thus, DXR seems to increase AMPK rather in aggressive types of cancer, which would well correlate with a pronounced anti-cancer effect in these cells by reinforcing the tumor-suppressor signaling axis LKB1-AMPK-mTOR.

Moreover, DXR is known to activate apoptosis via p53, which inhibits Akt signaling in cancer cells under DXR treatment. Thus, the inhibitory cross-talk between AMPK and Akt may be reversed in tumors, leading rather to AMPK activation or at least not its inactivation.

hour treatment 12 hours treatment

Effect of doxorubicin on ERK nitration

In perfused heart, we have observed rapid and strong activation of ERK by DXR (Figure 2-4C,D). One known consequence of ERK activation is to increase generation of reactive oxygen and nitrogen species. In turn, ERK has been recently shown to be activated by nitration 2,3 . Here we have analyzed whether ERK activation by DXR in the perfused heart model is due to DXR-induced nitrosative stress

Materials and methods

Immunoblots were performed as described (Chapter 2.) using a novel NO-ERK (NO-Y 139 for p42 and NO-Y 156 for p44) antibody generated and kindly provided by Dr. Serge Bottari (LBFA, Grenoble).

Results and conclusion

A nitration signal was observed at the level of ERK at 42 kDa, but not of ERK at 44kDa

(Figure 2345678). However, the ERK signal was not affected by DXR treatment. Thus ERK phosphorylation and activation in the DXR perfused heart does not depend on ERK nitration.

Abstract

The successful use of anthracyclines like doxorubicin in chemotherapy is limited by their severe cardiotoxicity. Despite decades of clinical application, a satisfying full description of the molecular mechanisms involved in cardiotoxicity and a consequential successful treatment

have not yet been achieved. Here we address doxorubicin-induced changes in cell signaling as a novel potential mediator of doxorubicin toxicity by applying a non-biased screen of the cardiac phosphoproteome. Two-dimensional gel electrophoresis, phosphospecific staining, quantitative image analysis, and MALDI-TOF/TOF mass spectrometry were combined to identify (de)phosphorylation events occurring in the isolated rat heart Langendorff-perfused with clinically relevant (5 µM) and supraclinical concentrations (25 µM) of doxorubicin. This approach identified 22 proteins with a significantly changed phosphorylation status and thus potentially mediating doxorubicin toxicity, and these results were validated by immunoblotting for selected phosphosites. (reviewed in 3 ). Oxidative stress was supposed to play a major mechanistic role (reviewed in 4- 6 ), but clinical studies with antioxidants were not efficient (reviewed in 3 ). Obviously, DXR cardiotoxicity is a complex, multi-factorial disease, and many mechanisms remain unclear ( 7,8 and reviewed in 3 ).

Comprehensive "omics" approaches are promising tools in the analysis of such complex pathologies, since non-biased screening allows novel mechanistic insight beyond traditionally accepted models. Though some non-biased "omics"-approaches were recently applied to doxorubicin cardiotoxicity [START_REF] Tokarska-Schlattner | Early effects of doxorubicin in perfused heart: transcriptional profiling reveals inhibition of cellular stress response genes[END_REF][10][11][12][13][14] , no attempt has been made so far to identify phosphorylation and dephosphorylation events linked to altered cell signaling by protein kinases and phosphatases. Phosphorylation is by far the most important post-translational modification and key to regulation of most cellular functions (reviewed in 15 ). Cell signaling might amplify the molecular impact of DXR and lead to long-lasting modifications. So far, only few reports on DXR-induced alterations in specific cell signaling pathways are available, such as a paradoxical response of kinases regulating energy supply in the heart that we have reported earlier 16 .

In this study, we applied gel-based phosphoproteomics for non-biased identification of protein (de)phosphorylation events during DXR perfusion of the rat heart, a model already characterized by our previous studies on energy metabolism 16 and the cardiac transcriptome [START_REF] Tokarska-Schlattner | Early effects of doxorubicin in perfused heart: transcriptional profiling reveals inhibition of cellular stress response genes[END_REF] .

It applies two DXR concentrations: the clinically relevant concentration of 5 µM to reproduce maximal plasma drug concentration of patients after standard bolus DXR injection (reviewed in [START_REF] Gewirtz | A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin[END_REF] ), and the supraclinical concentration of 25 µM to reproduce a higher cumulative dose 16 .

Combining powerful two-dimensional gel electrophoresis (2-DE) (reviewed in [START_REF] Rabilloud | Two-dimensional gel electrophoresis in proteomics: Past, present and future[END_REF] ) with a phosphoprotein-specific stain [START_REF] Schulenberg | Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye[END_REF] and mass spectrometry (MS), 22 proteins with altered phosphorylation status were identified, mainly involved in energy metabolism, sarcomeric structure/function and protein synthesis/folding. Some of them have strong functional links to the known cardiotoxic phenotype and represent potential novel targets for protective intervention.

Materials and methods

Materials

Doxorubicin hydrochloride purchased from Fluka (Buchs, Switzerland) was kept as frozen stock solution (10 mM) in water and diluted in Krebs-Henseleit solution just before heart perfusion. Protease inhibitor cocktail tablets were from Roche (Mannheim, Germany), phosphatase inhibitor cocktail from Pierce (Rockford, IL, USA), and trypsin from Promega (Madison, WI, USA). All other chemicals were at least reagent grade.

Rat heart perfusion

Hearts of male Wistar rats (∼300 g) were perfused in a noncirculating Langendorff apparatus without (control) or with 5 µM or 25 µM DXR during 80 min (after 30 min of stabilization with Krebs-Henseleit buffer) and functional parameters were recorded as described earlier [START_REF] Tokarska-Schlattner | Early effects of doxorubicin in perfused heart: transcriptional profiling reveals inhibition of cellular stress response genes[END_REF] . At the end of the perfusion, hearts were rapidly freeze-clamped in liquid nitrogen and stored at -80°C for further analysis. All procedures involving animals were performed according to guidelines of the European Community and approved by the Grenoble Ethics Committee for Animal Experimentation.

Protein sample preparation

Snap-frozen heart tissue was grinded in liquid nitrogen and homogenized with an UltraTurax (Janke and Kunkel, Germany) in 7 M urea, 2 M thiourea, 4% w/v CHAPS, 0.5% v/v IPG Buffer, 40 mM DTT, containing protease and phosphatase inhibitors. Homogenate was incubated during 30 min at 4°C, centrifuged at 15400 g (10 min, 4°C) and the supernatant assayed for protein concentration (2DQuantKit, GE Healthcare, Vélizy, France) and stored at -80°C.

Bi-dimensional polyacrylamide gel electrophoresis and image analysis

Immunoblotting and immunoprecipitation

SDS-PAGE separation of heart homogenates and immunoblotting were performed according to standard protocols [START_REF] Tokarska-Schlattner | Early effects of doxorubicin in perfused heart: transcriptional profiling reveals inhibition of cellular stress response genes[END_REF] . For details including sources and dilutions of antibodies and the immunoprecipitation protocol see online Supporting Information.

Results

DXR induces dose-and time-dependent functional decline in perfused heart

DXR induced a dose-and time-dependent loss of cardiac function, in agreement with earlier reports on such a model of acute DXR cardiotoxicity 16 . Perfusion with 5 µM had no significant effect on heart function after 80 min, while 25 µM DXR decreased developed pressure, +dp/dt, -dp/dt and rate pressure product by more than 50% relative to control and increased end-diastolic pressure 4-fold (Table 3-1S, see online Supporting Information). and the identity of major detected (phospho)proteins [START_REF] De Souza | Preparation of human heart for laser microdissection and proteomics[END_REF][START_REF] Li | Evaluation of several two-dimensional gel electrophoresis techniques in cardiac proteomics[END_REF][START_REF] Mcgregor | Proteomics of the heart: unraveling disease[END_REF] . shown); these were discarded. In two cases, a single protein was identified from two different spots on the same gels, and three proteins were identified in both wide and zoom pattern gels.

This gave a total number of 22 different proteins identified with differential phosphorylation pattern (Table 3-1). Mass spectrometry identification of these proteins satisfied all necessary criteria like sequence coverage of detected peptides and Mascot score, or agreement between theoretical and observed pI and Mr values (Figure 3-2, Table 3-1). Changes in phosphorylation levels were well reproducible, as e.g. seen in cases where proteins were detected twice in wide and zoom pattern gels (Table 3-2).

Almost all 22 proteins with altered phosphorylation levels could be classified according to their function in three main groups: energy metabolism (12), sarcomere structure and function (3), and protein synthesis/chaperones (5) (Figure 3-3, Table 3-2). Phosphorylation increased in 10 and decreased in 12 of these proteins, with increases being most frequent in sarcomeric proteins and decreases in enzymes in energy metabolism (Table 3-2). In contrast to the functional impairment of the heart (Table 3-1S), changes in phosphorylation levels

were not always dose-dependent. Many significant changes were observed already at 5 µM DXR, in particular for diminished phosphorylation, while several enzymes in energy metabolism showed increased phosphorylation in particular at 25 µM DXR. Interestingly, a large proportion of proteins, 10 out of the 22, are located in mitochondria (Table 3-2). Details on the biological functions of the identified proteins are summarized in Table 3-2S. Detailed literature and database search revealed that all identified proteins are known as phosphoproteins with at least one Ser, Thr or Tyr phosphorylated (Table 3-1). Some of them are well characterized in respect to involved site(s) and upstream kinases, as well as the resulting functional changes (Table 3-2S). MS spectra searched for peptides containing the typical mass shift of an additional phosphate moiety suggested some involved phosphorylated amino acids (Ser, Thr, Tyr) or phosphosite(s) (Table 3-1). Since identification of protein phosphatase PP1α could indicate direct effects of DXR on the phosphorylation pattern via activation/deactivation of protein phosphatases, we also analyzed activities of several major phosphatases in control and DXR-perfused hearts.

However, the activities of Tyr phosphatase and Ser/Thr protein phosphatases 2A and 2C were not significantly affected by DXR in our model (Figure 3-1S).

Discussion

This study aimed to identify (de)phosphorylation events involved in the acute cardiac response to DXR to determine early cell signaling phenomena likely contributing to DXR toxicity. The chosen model, isolated rat heart perfused with 5 or 25 µM DXR, mimics either effects of peak plasma DXR concentrations after bolus drug injection or of higher cumulative DXR doses 16 . The applied non-biased and genome-wide phosphoproteomic screen, based on 2-DE and phosphospecific staining, yielded 56 protein spots with DXR-dependent changes in phosphorylation state, out of which 22 different proteins were identified by MS. Specificity of the applied phosphostain is suggested by the staining pattern, not showing most major protein spots, but many others undetectable by protein stain, and was confirmed by MS detection of phosphoresidues and phosphosites within most selected proteins. Certain phosphosites and the validity of our spot quantification in general were confirmed by immunoblot-detection with phosphosite-specific antibodies.

Among the 22 identified differentially phosphorylated proteins, those with functions in energy metabolism and/or mitochondrial location were clearly overrepresented (Figure 345).

The latter coincides with mitochondrial accumulation of DXR, caused by its affinity to cardiolipin 16,[START_REF] Tokarska-Schlattner | New insights into doxorubicin-induced cardiotoxicity: the critical role of cellular energetics[END_REF] , and the functional impairment of mitochondria exposed to DXR (reviewed in [START_REF] Wallace | Doxorubicin-induced cardiac mitochondrionopathy[END_REF] ). Furthermore, this would contribute to the cardiac specificity of DXR toxicity, given the crucial role of mitochondria in cardiac energy generation (reviewed in 8,[START_REF] Ventura-Clapier | Bioenergetics of the failing heart[END_REF] ). Here we suggest that the changes in phosphorylation of mitochondrial proteins, which generally emerge as a key regulator of mitochondrial functions [START_REF] Feng | Phosphoproteome analysis of isoflurane-protected heart mitochondria: phosphorylation of adenine nucleotide translocator-1 on Tyr194 regulates mitochondrial function[END_REF] , mediate toxic DXR effects. In this respect, the identified pyruvate dehydrogenase E1 is a key enzyme, since it represents a metabolic switch shunting glucose metabolites either to oxidative mitochondrial metabolism (via conversion into acetyl-CoA) or to cytosolic lactate production [START_REF] Wang | Metabolic profiling of hearts exposed to sevoflurane and propofol reveals distinct regulation of fatty acid and glucose oxidation: CD36 and pyruvate dehydrogenase as key regulators in anestheticinduced fuel shift[END_REF] ). The E1 protein is the catalytic subunit of the mitochondrial complex, and the identified Ser292 corresponds to the first of three regulatory phosphosites that is targeted by pyruvate dehydrogenase kinases 1, 2 and 4 [START_REF] Sugden | Mechanisms underlying regulation of the expression and activities of the mammalian pyruvate dehydrogenase kinases[END_REF] .

Increased P-Ser292 at higher DXR doses would inactivate the enzyme, thus inhibiting the mitochondrial use of carbohydrate carbons, while increasing their conversion into lactate (reviewed in [START_REF] Holness | Regulation of pyruvate dehydrogenase complex activity by reversible phosphorylation[END_REF] ). Lactate accumulation has indeed been found in hearts of DXR-treated rats [START_REF] Carvalho | Metabolic remodeling associated with subchronic doxorubicin cardiomyopathy[END_REF] .

Pyruvate dehydrogenase inhibition would thus lead to energy deprivation and diminished cardiac contractility, main characteristics of DXR cardiotoxicity [START_REF] Lewandowski | Pyruvate dehydrogenase influences postischemic heart function[END_REF] . Additional up-or mainly down-regulated phosphorylation of enzymes in fatty acid beta-oxidation and glycolysis suggest an entire re-wiring of cellular energy substrate utilization, that is consistent with a switch from mitochondrial to cytosolic glucose metabolization, although the latter is controversial and may depend on the cardiomyopathy stage [START_REF] Tokarska-Schlattner | New insights into doxorubicin-induced cardiotoxicity: the critical role of cellular energetics[END_REF][START_REF] Carvalho | Metabolic remodeling associated with subchronic doxorubicin cardiomyopathy[END_REF][START_REF] Wakasugi | Myocardial substrate utilization and left ventricular function in adriamycin cardiomyopathy[END_REF] .

The second important group of differentially phosphorylated proteins is cytosolic and involved in sarcomere structure and function (Figure 3 -5). Phosphorylation of sarcomeric proteins affects different functional and structural aspects of cardiomyocytes, such as cardiac dynamics downstream of Ca 2+ signals, cellular energy turnover and organization of myofibrils (reviewed in [START_REF] Solaro | Protein phosphorylation and signal transduction in cardiac thin filaments[END_REF][START_REF] Zaugg | Cellular mechanisms in sympatho-modulation of the heart[END_REF] ), all critical for contractile (dys)function and myofibrillar (dys)organization upon DXR treatment. Desmin, an intermediate filament/Z-disc protein, showed a 3-4 fold increase in phosphorylation at probably multiple site, including Thr16, that occurred already at a low DXR dose. Phosphorylation of desmin is known to cause myofibrillar disarray [START_REF] Huang | Protein kinase C-mediated desmin phosphorylation is related to myofibril disarray in cardiomyopathic hamster heart[END_REF][START_REF] Geisler | Phosphorylation of desmin in vitro inhibits formation of intermediate filaments; identification of three kinase A sites in the aminoterminal head domain[END_REF] , a major phenotype in DXR-induced cardiomyopathy [START_REF] Timolati | The role of cell death and myofibrillar damage in contractile dysfunction of long-term cultured adult cardiomyocytes exposed to doxorubicin[END_REF][39][40][41] . Since these changes occurred already [START_REF] Zaugg | Cellular mechanisms in sympatho-modulation of the heart[END_REF]42 ).

A third group of proteins covered diverse functions in protein synthesis and folding.

Phosphorylation was decreased for those enzymes involved in protein synthesis, and increased for those with chaperone-like activity. The latter has likely some compensatory, pro-survival character, in particular in case of α-crystallin B chain. This abundant small cardiac heat shock protein associates in particular with desmin to prevent aberrant desmin aggregation (reviewed in 43 ). The prominent increase in phosphorylation at multiple serines, including Ser59, which occurred already at the low DXR dose, is known to be cardioprotective 44 . It would in particular reduce sarcomere disorganization caused by desmin phosphorylation. In contrast to what has been expected, there was no clear concentration dependence of changes in phosphorylation status. Many changes already seen at 5 µM DXR did not further grow at 25 µM DXR, as e.g. with sarcomeric proteins and chaperones. As already discussed, these changes may reflect not only toxic, but also compensatory adaptations in acute cardiotoxicity, since heart performance is not yet significantly altered. In contrast, some enzymes in energy metabolism, mostly mitochondrial, increased phosphorylation status only at 25 µM DXR. This rather reflects metabolic reprogramming linked to cumulative DXR effects, which are associated with functional alterations of the heart. Finally, the observed differential phosphorylations indicate an altered activity of some signaling protein kinases, including more specific ones like pyruvate dehydrogenase kinases and cardiac myosin light chain kinase, but also more global ones like PKA and PKC (Table 3-2S). Consistently, phosphorylation of e.g. myofibrillar proteins was found to be largely PKC-mediated 41 .

In summary, our unbiased study identified a number of differentially phosphorylated cardiac proteins. These proteins, mostly involved in energy metabolism, sarcomeric function/structure and chaperone activity, potentially mediate the cardiotoxic action of DXR as well as pro-survival responses (Figure 3 -5). Since changes in phosphorylation occurred after only 80 min of DXR perfusion, they represent early events likely to be a cause and no an unspecific consequence of DXR toxicity. Target-oriented functional studies in chronically drug-exposed transgenic animal models and studies with human patients will be needed to further elucidate the contribution of described phosphorylation events to the development of cardiomyopathy.

Supplement

Materials and methods

Bi-dimensional polyacrylamide gel electrophoresis

Isoelectrofocusing (IEF) as first dimension was performed using an Ettan IPGphor 3 IEF system (GE Healthcare). Immobiline DryStrip gels (18 or 24 cm) with immobilized pH 3-10 non linear gradient (pH 3-10NL) were rehydrated for 24 h in 8 M urea, 2% w/v CHAPS, 0.5% v/v IPG buffer, 0.002% v/v bromophenol blue and 18.2 mM DTT using Immobiline DryStrip

Reswelling Tray (GE Healthcare). Then strips were placed in the Manifold multiple strip holder and protein samples (300 µg for 18 cm strips; 500 µg for 24 cm strips) were applied using the sample cup system. Strips were covered by DryStrip cover fluid to minimize evaporation and prevent urea crystallization. IEF was carried out according to the manufacturer's protocols at 20°C until reaching 48.5 kVh or 64.4 kVh for 18 or 24 cm strips, respectively. Strips were then equilibrated in two steps: (i) 15 min incubation with equilibration buffer (6 M Urea, 75 mM TRIS-HCl pH 8.8, 29.3% w/v glycerol, 2% w/v SDS, 0.002% v/v bromophenol blue) containing 1% w/v DTT; (ii) 15 min incubation with equilibration buffer containing 2.5% w/v iodoacetamide. For separation in the second dimension, strips were cut to obtain pH range desired and were placed on the top of SDSpolyacrylamide gels (12% for 18 cm strips, 10% for 24 cm strips) and sealed with an agarose solution (0.5% agarose, 0.002% w/v bromophenol blue). Gels were run in a Protean II xi cell System (Biorad, Reinach, Switzerland) containing running buffer (26.4 mM TRIS base, 192 mM glycine, 0.1% w/v SDS) at 40 V during 1 h and then at 200 V during 5.75 h at room temperature. Phosphoproteins were stained using ProQ Diamond (Invitrogen, Paisley, UK), according to manufacturer's instruction and scanned with a Typhoon9400 (GE Healthcare).

Total proteins were detected on the same gels using colloidal Coomassie (Roti-Blue, Roth)

and office scanner (360 dpi, Epson perfection V700 photo, Epson, France).

In-gel digestion, mass spectrometry and protein identification

Excised spots were washed twice each with Milli-Q water, with modified digestion buffer (100 mM NH 4 HCO 3 /Acetonitrile (ACN) 1:1 (v/v)), 100% ACN and 100 mM NH 4 HCO 3 .

Subsequently, gel pieces were shrunk with 100% ACN, dried for 20 min at 37°C and swollen with 10 µL of trypsin solution (12.5 ng/µL in digestion buffer) on ice for 15 min. The supernatant was removed and replaced with 50 mM NH 4 HCO 3. The trypsin digestion was performed overnight at 37 °C. The supernatant was then transferred into a fresh tube and the remaining gel pieces were extracted once again with a cycle of 50% ACN (v/v), 0.1% TFA (v/v), and 100% ACN (v/v), each step lasting 20 min. Then, gel pieces were sonicated and the supernatants were pooled. These extracted peptides were dried by vacuum centrifugation, resuspended in 10 µL of 0.1% TFA (v/v), desalted with home-made C18 reverse-phase Zip-Tip, eluted with 1 µL MALDI matrix (10 mg/mL R-cyano-4-hydroxy-cinnamic acid matrix in 70% ACN, 0.1% TFA (v/v)), and spotted on a stainless steel MALDI sample plate (AB Sciex) in triplicate. MS and MS/MS analyses were performed using the 4800 MALDI-TOF/TOF Analyzer (AB Sciex). MS spectra were acquired in positive reflector ion mode using 4000

Series Explorer software (V3.5). MS spectra were acquired between m/z 700 and 4000 using 2000 laser shots. Up to 10 of the most intense ion signals per spot position having an S/N greater than 50 were selected as precursors for MS/MS acquisition to complement the protein identification by PMF. Fragmentation of automatically selected precursors was performed at a collision energy of 1 kV using air as collision gas. For peak detection, an S/N of 10 was applied using cluster area S/N optimization.

Protein identification was performed by PMF and/or specific MS/MS peptide sequencing.

The combined MS and MS/MS spectra from each spot were processed using ProteinPilot software program (v3.0, ABSciex) with Mascot (Matrix Science) as the database search engine (V 2.0). Each MS/MS spectrum was searched against a database of protein sequences

Immunoprecipitation

ND10 was immunoprecipitated from heart homogenate (100 µg protein) using anti-phosphoSer/Thr/Tyr antibody (1:100, Assay Design, Ann Arbor, MI) in PBS, 1% BSA overnight at 4°C (total volume 300 µL). After addition of 120 µg of protein A-sepharose (Sigma Aldrich, Saint Louis, MO), samples were incubated for 1 h at room temperature.

Beads were washed 10 times in PBS, 0.1% Tween 20, resuspended in Laemmli sample buffer, and denatured proteins separated on standard SDS-PAGE gel (12% acrylamide) and immunoblotted as described above, using anti-ND10 antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA) as a primary antibody.

Phosphatase activity

The Tyr and Ser/Thr protein phosphatase activities were determined in heart homogenates (3 µg of proteins) using a kit from Promega (Madison, WI). Briefly, after removing endogenous phosphates with Spin Columns (Sephadex G-25 resin), homogenates were incubated in a 96-well plate with a reaction mix (see manufacturer's instruction for further details) and 100 µM phosphopeptide substrate at 37°C for 45 min. To stop the reaction, 50 µL of molybdate dye/additive mixture was added to the wells, and the absorbance at 600 nm was read using ELx808 Absorbance Microplate Reader (Biotek, Winooski, VT) in at least three independent experiments. Table 3

Figures

Table

Introduction

The anthracycline antibiotic doxorubicin (DXR) is still among the most efficient cancer therapeutics, and has shown additive effects with new, targeted approaches like trastuzumab (Herceptin). Unfortunately, its potent antitumor action is associated with unwanted side effects, especially with serious cardiac toxicity. The mechanisms underlying development of DXR cardiotoxicity are not completely clear [START_REF] De Souza | Preparation of human heart for laser microdissection and proteomics[END_REF]42,[START_REF] Ecroyd | Mimicking phosphorylation of alphaB-crystallin affects its chaperone activity[END_REF] .

Interference of DXR with cardiac gene expression is an important element of the complex response to the drug. However, the impact of DXR on cardiac gene expression is barely explored. DXR was found to inhibit DNA replication in cultured rat cardiomyocytes and to alter cardiac DNA in animals and humans, inducing cardiac-specific lesions [START_REF] Gewirtz | A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin[END_REF][START_REF] Carvalho | Metabolic remodeling associated with subchronic doxorubicin cardiomyopathy[END_REF][START_REF] Lewandowski | Pyruvate dehydrogenase influences postischemic heart function[END_REF]49 .

Development of DXR cardiotoxicity has been associated with important and relatively fast repression of several of cardiac-specific genes [START_REF] Mcgregor | Proteomics of the heart: unraveling disease[END_REF][START_REF] Wallace | Doxorubicin-induced cardiac mitochondrionopathy[END_REF][START_REF] Hoover | alpha B-crystallin gene induction and phosphorylation by MKK6-activated p38. A potential role for alpha B-crystallin as a target of the p38 branch of the cardiac stress response[END_REF] . The few earlier genome-wide studies on the impact of DXR on the cardiac transcriptome focused either on single genes or selected pathways 5,70. The present study aims at a global characterization of the early cardiac transcriptomic response to a low DXR dose to identify the most sensitive genes and groups of genes associated with specific functions or metabolic pathways. Such a systems biology approach is expected to contribute to our understanding of regulatory cross-talk between different involved mechanisms and to identify new elements possibly driving the development of chronic toxicity. The applied experimental model using isolated perfused rat heart together with a low clinically relevant DXR dose (2 µM) has been characterized already in our previous study [START_REF] He | Phosphorylation of mitochondrial elongation factor Tu in ischemic myocardium: basis for chloramphenicol-mediated cardioprotection[END_REF] . Under these experimental conditions, DXR accumulates very rapidly in cardiac nuclei and mitochondria and triggers inhibition of different kinase systems regulating cellular energy homeostasis [START_REF] He | Phosphorylation of mitochondrial elongation factor Tu in ischemic myocardium: basis for chloramphenicol-mediated cardioprotection[END_REF] . In the current study, transcriptomic profiling was performed using both single gene and gene set enrichment analysis (GSEA), the latter being an analytical method to identify coordinated changes in functionally related groups of genes. In addition, energy metabolism was assessed by biochemical and functional assays. Besides confirming some known DXR-responding genes, which validates our approach, the study identified several new genes sensitive to DXR and yielded essentially novel set of DXR-responding pathways. The latter included a detrimental down-regulation of stress-responsive pathways and a modulation of the cardiac remodeling capacity, both potentially also important for long term effects.

Chapter 4.

Doxorubicin effects on the cardiac transcriptome 100

Materials and methods

Chemicals

Perfusion protocol

Hearts from male Wistar rats (ca. 300 g) were perfused in a noncirculating Langendorff apparatus without (control) or with 2 µM DXR (DXR) during 2 h and functional parameters were recorded as described earlier [START_REF] He | Phosphorylation of mitochondrial elongation factor Tu in ischemic myocardium: basis for chloramphenicol-mediated cardioprotection[END_REF] . At the end of perfusion, depending on the experimental series, hearts were either rapidly frozen in liquid nitrogen and stored at -80°C for later analysis (gene and protein expression, metabolite analysis) or immediately used to prepare skinned fibers for respiratory measurements. At least four control and DXR-treated hearts were prepared in each series. All experiments involving animals were performed in compliance with the guidelines of the European Community and the Animal Care and Use Committee of the University of Zurich.

RNA isolation, cDNA synthesis and microarray hybridization

Total RNA was extracted from control and DXR-treated samples using RNAeasy Mini kit (Qiagen, Hilden, Germany). The quality of the isolated RNA was determined with a NanoDrop ND 1000 (NanoDrop Technologies, Delaware, USA) and a Bioanalyzer 2100 (Agilent, Waldbronn, Germany). All the samples had a 260 nm/280 nm ratio between 1.8-2.1 and a 28S/18S ratio within 1.5-2.0 and were further processed. Total RNA samples (2 µg)

were reverse-transcribed into double-stranded cDNA, which was subsequently in vitro transcribed in presence of biotin-labeled nucleotides using an IVT Labeling Kit (Affymetrix Inc., P/N 900449, Santa Clara, CA). The labeled cRNA was purified and quantified using BioRobot Gene Exp -cRNA Target Prep (Qiagen AG, Switzerland); its quality was

Microarray analysis

Microarray analysis was performed following the "minimum information about a microarray experiment" (MIAME) guidelines and data are available at the Gene Expression Omnibus Database (http://www.ncbi.nlm.nih.gov/geo/) under the series number GSE12260.

Normalization and computation of expression values was performed using the Robust

Multichip Average algorithm [START_REF] Li | Evaluation of several two-dimensional gel electrophoresis techniques in cardiac proteomics[END_REF] . In a first step, the statistically most relevant differentially regulated transcripts were identified using Significance Analysis of Microarrays (SAM [START_REF] Kato | Phosphorylation of alphaB-crystallin in mitotic cells and identification of enzymatic activities responsible for phosphorylation[END_REF] ), a method that provides a ranking of the transcripts found to be differentially regulated between any two given protocols. Statistical significance is measured by the q-value, which is the lowest false discovery rate (FDR) at which a gene is described as significantly regulated. The tolerance was set as 5%. FDR is the percentage of genes that are expected to be identified by chance. Here we calculated the local FDR, which is the FDR for genes with scores that fall in a window around the score for a given gene. In SAM, a window of at least 50 genes is taken to estimate the local FDR at each point. In a second step, the association between DXR treatment and functionally related group of genes and pathways was studied using Gene Set Enrichment Analysis (GSEA [START_REF] Kawajiri | Functional significance of the specific sites phosphorylated in desmin at cleavage furrow: Aurora-B may phosphorylate and regulate type III intermediate filaments during cytokinesis coordinatedly with Rho-kinase[END_REF] ). GSEA is a computational method that determines whether an a priori defined set of genes shows statistically significant, concordant differences between two biological states ("phenotypes"). Results are ranked by their normalized enrichment score (NES), a p-value, and the false discovery rate (FDR; q-value). A searchable online collection of gene sets used with GSEA is available at The Molecular Signatures Database (http://www.broad.mit.edu/gsea/msigdb). arrest and DNA-damage-inducible 45α (GADD45α), interleukin 1α, dual specifity phosphatase 1 (DUSP1), and heat shock protein 70kD (HSP70). Primers were purchased from Eurogentec (Seraing, Belgium). cDNA was synthesized from 1 µg total RNA using Superscript III reverse transcriptase and oligo-dT as primer (Invitrogen, Carlsbad, CA). Realtime qRT-PCR was performed in the Roche Light Cycler using Plus Sybr Green 1 Master Mix (Roche, Mannheim, Germany). Amplification reactions were conducted with an initial step at 95°C for 10 min followed by 30-40 cycles. Cyclophilin A and tubulin alpha were used for normalization. The C t difference (ΔΔC t ) was used for quantification of relative gene expression as 2 - ΔΔ Ct37 .

Western blotting.

Forty µg protein was separated by SDS-PAGE and semi-dry blotted onto nitrocellulose membranes. After blocking in 4% milk, TBS, and 0.05% Tween 20, blots were immunostained for 2 h at room temperature with following primary antibodies (dilutions):

anti-CARP (1:100), anti-DUSP1 (1:200), anti-NDUFA10 (1:100), and anti-GAPDH (1:100) from Santa Cruz Biotechnology; anti-HSP70 (1:500), anti-GADD45α/γ (1:500), anti-phosho-p38 MAPK (Thr180/Tyr182, 1:1000), anti-phosphoERK1/2 (Thr202/Tyr204, 1:1000), and anti-phospho JNK (Thr183/ Tyr185, 1:1000) from Cell Signaling; anti-ERK1/2 (1:1000) from Upstate. Anti-PMP24 antibody (1:1000) was generated in rabbits using a synthetic peptide antigen (EYHRPTLQPSLQSSMT) by Eurogentec. Secondary peroxidase-coupled antibodies were diluted (1:2000) in blocking buffer and blots incubated for 1 h at room temperature.

Blots were developed with Amersham ECL Plus™ Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire UK).

Chapter 4.

Early doxorubicin effects on the cardiac transcriptome 103

Determination of ATP and PCr

Adenine nucleotides and phosphocreatine (PCr) were extracted from the freeze-clamped hearts with ice-cold 0.6N perchloric acid, centrifugated at 3000 g for 12 min (4°C), neutralized to pH 6-7 with 5 M K 2 CO 3 and centrifugated again at 3000 g for 12 min (4°C). An enzymatic test was used to determine ATP and PCr [START_REF] Holness | Regulation of pyruvate dehydrogenase complex activity by reversible phosphorylation[END_REF] . Results were expressed in µmol/g dry weight (dw).

Measurement of mitochondrial respiration in skinned fibers

Cardiac fibers were isolated from the hearts immediately after perfusion as described [START_REF] Sugden | Mechanisms underlying regulation of the expression and activities of the mammalian pyruvate dehydrogenase kinases[END_REF]47 , and mitochondrial respiration was measured with 2 mM malate/5 mM glutamate. We determined rates of basal respiration (without addition of external ADP, V 0 ), maximal coupled respiration (in presence of 2 mM ADP, V max ) as well as dependency of respiration on external ADP (0.05-2 mM) and/or creatine (Cr; 1-20 mM) to infer the corresponding apparent Michaelis constants: K m (ADP), apparent K m for ADP; K m Cr (ADP), apparent K m for ADP measured in presence of external Cr (20 mM); K m (Cr), apparent K m for Cr in presence of ATP and an external ADP scavenging system. In the latter case, after initial stimulation of respiration by malate/glutamate and 2 mM ATP, phosphoenol pyruvate (PEP, 5mM) and pyruvate kinase (30 U/ml) were added to trap extramitochondrial ADP. Respiratory stimulation by Cr was then due to ADP regeneration in the mitochondrial intermembrane space by mitochondrial creatine kinase. In some experiments, cardiac fibers were treated with DXR directly in the oxygraph chamber (20 min preincubation and during measurements).

Acceptor control ratio (ACR), calculated as V max /V 0 , served as index of coupling of phosphorylation to oxidation. Normalized respiratory rates are given in nmol O 2 /min g dry weight (dw).

Statistical analysis

If not stated otherwise, data are presented as mean ± SEM. Statistical significance was evaluated by 2-tailed Student's test using Microsoft Office Excel 2003. Differences with p<0.05 were considered significant.

Chapter 4.

Early doxorubicin effects on the cardiac transcriptome 104 4.5. Results

General features of the transcriptomic response

Using a Langendorff model, we have analyzed the early transcriptomic response of rat hearts to 2 h perfusion with 2 µM DXR. This represents a lower limit of DXR concentrations found in plasma of patients after bolus injection of the drug [START_REF] Rabilloud | Two-dimensional gel electrophoresis in proteomics: Past, present and future[END_REF] . This perfusion induced modest but significant hemodynamic dysfunction (Figure 4-1).

Single gene analysis

Selected single genes with a change in expression of at least 50% are shown in Table 4-1.

The corresponding gene products cover a large range of functions, but can be tentatively assigned to some important groups. Many have functions related in a broader sense to cellular stress. Strikingly, expression of most of them was down-regulated in presence of DXR, some even belonged to the group of most strongly down-regulated genes. Consistent downregulation was found for genes whose products are directly involved in stress-responses (like heat shock protein HSP70, the GADD45 family, some immediate early response genes) and antioxidative defense (like glutathione S-transferase M4 and glutamate-cysteine ligase). Many genes related to immune response were also down-regulated, including proinflammatory cytokines and their receptors (like interleukin 1 alpha and tumor necrosis factor α) with multifunctional roles in cell proliferation, induction of apoptosis, and inflammatory response,

or selectin E that recruits neutrophiles at sites of endothelial inflammation. More mixed responses were seen with different chemokine ligands, and up-regulated transcripts included an MHC class Ib antigen and some granzyme transcripts coding for serine proteases of cytotoxic immune cells. Since DXR targets DNA, the response of nucleic acid-related genes is particularly relevant. The group of genes related to nucleic acid synthesis, stability and repair was mostly down-regulated, with genes for histones and Kbtbd5, a potential DNA repair enzyme, belonging to the most strongly inhibited genes. However, two polymerases and the DNA repair enzyme nibrin were up-regulated, possibly as a specific response to DNA damage.

Chapter 4.

Early doxorubicin effects on the cardiac transcriptome 107 DXR-repressed genes in heart remodeling/pathology and myofibrillar structure may reflect early DXR-induced damage occurring in the heart. Their gene products are linked to cardiac signaling and development, including the LIM protein dyxin that modulates cardiomyocyte growth, the actin-binding cardiomyopathy-associated protein Xirp1, and transcriptional coactivator Cited2. Again, some genes in these groups were up-regulated, like osteoglycin and tropomyosin. Importantly, the only group of genes that was consistently upregulated was coding for metabolic enzymes like aldehyde dehydrogenase and NADH dehydrogenase.

Many other DXR-responsive genes code for cell signaling components and were also predominantly down-regulated (Table 4-1). These include protein kinases (e.g. polo-like kinase 2), protein phosphatases (e.g. protein phosphatase 1 regulatory subunit and dual specificity phosphatase 1, DUSP1), G-protein signaling components (e.g. Rho family

GTPases), transcription factors/regulators (e.g. Jun oncogene and activating transcription factor 3, ATF3). The few up-regulated transcripts of these protein families include Jun D proto-oncogene, two phosphatases (PDH phosphatase and dual specificity phosphatase 2) and a developmentally regulated GTP-binding protein. Many of these cell signaling genes also respond to (oxidative) stress (e.g. ATF3 or dual specificity phosphatase) and regulate growth or apoptosis. This is also true for some other DXR-sensitive transcripts like up-regulated quiescin Q6, coding for a sulfhyryl oxidase. The most strongly up-regulated transcripts encode peroxisomal membrane protein 4, 24 kDa (PMP24) and a member of the ATP-binding cassette family. Gene ID, probe identifier as defined by Affymetrix or (*) synonyms according to NCBI-Entrez gene; FC, fold change (DXR value divided by control value); q-value (%), lowest false discovery rate at which a gene is described as significantly regulated; local FDR (%), local false discovery rate. Results for the probe with the highest score are given. Blue, down-regulated compared to control; red, up-regulated compared to control; bold, the three most up-and down-regulated genes.

Gene Set Enrichment Analysis

Gene Set Enrichment Analysis (GSEA) was applied to identify functionally related gene sets which respond to DXR in a coordinated manner. This method can detect relatively weak but significant coordinated changes. It is an especially valuable tool to get insight into biologically relevant signals in the early response to DXR, when changes in single genes are moderate and may escape analysis. Selected gene sets are listed in Table 4- Gene sets and pathways affected by DXR covered most cellular functions identified at the single gene level. They involved stress, defense and repair responses, as well as immune response and some pathways in cardiac remodeling (Table 4-2, Figure 4-4A). Except hypoxia-suppressed genes, all responsive stress-related gene sets were down-regulated, in particular DNA damage signaling and cell signaling involving TNF, interleukin and mitogenavtivated kinases (MAPK). Similarly consistent down-regulation was found for gene sets in apoptotic signaling. Among the few DXR-induced gene sets were cardiac remodeling linked to c-Myc signaling (Table 4-2) and, importantly, two main pathways in energy metabolism, glycolysis and TCA cycle (Table 4-2, Figure 4-4B). Analyzed pathways were from http://www.broad.mit.edu/gsea/msigdb/index.jsp; the original names of pathways were maintained except for those that were not self-explaining (marked with number); for the latter, the original names are as follows: 1 HYPOXIA_REG_UP;

Comparison between mRNAs and protein expression/phosphorylation

For seven DXR-affected transcripts, the corresponding protein abundance after 2h perfusion with 2 µM DXR was analyzed by immunoblotting (Figure 4-5A). Changes were significant only for two proteins, for which they paralleled transcript levels: HSP70 (decrease) and NDUFA10 (increase). Other examined protein levels were not yet altered or showed trends below significance level. We further analyzed the phosphorylation status of three members of the MAPK family (p38, ERK1/2 and JNK), since some transcripts of this pathway were decreased in our model (Table 4-2, Figure 4-4A), while DXR-induced activation of this pathway was reported earlier 39,[START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF][START_REF] Schlattner | Creatine kinase and creatine transporter in normal, wounded, and diseased skin[END_REF][START_REF] Zhu | MAPK superfamily plays an important role in daunomycin-induced apoptosis of cardiac myocytes[END_REF] . Indeed, ERK1/2 phosphorylation was considerably increased, while protein levels remained unchanged (Figure 4-5B).

Phosphorylation of p38 and JNK was neither detectable in control nor in DXR-treated hearts. 4-3). Though reduced cardiac ATP, PCr and PCr/ATP ratios are known to occur after chronic DXR exposure or acutely upon higher DXR doses (for review see [START_REF] Ecroyd | Mimicking phosphorylation of alphaB-crystallin affects its chaperone activity[END_REF] ), the perfusion with only 2 µM DXR did not yet induce significant changes. Maximal ADP-stimulated respiratory rates and the kinetics of stimulation of mitochondrial respiration by ADP or/and creatine (Cr) were determined in permeabilized fibers isolated from the hearts perfused without or with DXR. In an oxidative muscle as heart, the response of respiration to ADP and Cr is a sensitive indicator for the functional state of mitochondria and the efficiency of regulation of mitochondrial respiration and ATP production. Similarly as for high energy phosphates, the tendency to altered respiratory parameters was not statistically significant under the given conditions, i.e. in hearts perfused with 2 µM DXR (Table 4-3, Figure 4-6A).

Discussion

This study aimed at a global characterization of the cardiac transcriptomic response to DXR to identify the most sensitive single genes and functionally related groups of genes.

Such a non-biased systems biology approach is expected to extend our knowledge of molecular events in acute DXR cardiotoxicity and to propose new mechanisms connecting acute DXR exposures with chronic pathology. Indeed, at a clinically relevant DXR concentration found also in patient's plasma after bolus injection [START_REF] Rabilloud | Two-dimensional gel electrophoresis in proteomics: Past, present and future[END_REF] , an extensive transcriptomic reprogramming was observed already after two hours of perfusion, while cardiac dysfunction was statistically detectable but still relatively mild. Transcriptomic profiling by single gene comparison and gene set enrichment analysis identified essentially novel sets of DXR-responding cardiac genes. Roughly, the transcriptomic changes could be classified either as toxic, e.g. by suppressing cellular stress responses, or as compensatory and pro-survival, e.g. by favoring ATP generation. In conclusion, the inability to mount proper stress responses is proposed as a critical mechanism in DXR cardiotoxicity.

Chapter 4.

Early doxorubicin effects on the cardiac transcriptome 117 4.6.1. Cardiac transcriptional reprogramming by doxorubicin reveals similarities to cancer and confirms some known targets

The early transcriptional response of DXR-treated hearts showed prevalent downregulation of gene expression and selective up-regulation of individual genes or gene sets. A similar pattern was reported for human breast carcinoma cells, with rapid gene repression and delayed but persisting gene induction [START_REF] Feng | Phosphoproteome analysis of isoflurane-protected heart mitochondria: phosphorylation of adenine nucleotide translocator-1 on Tyr194 regulates mitochondrial function[END_REF] . Whether a transcriptomic change is specific or rather results from bulk DNA modification may depend on the individual gene. DXR exerts its inhibitory action on rapidly growing cancer cells by DNA intercalation and topoisomerase II inhibition, resulting in repression of DNA transcription and replication, as we have also observed in heart. However, topoisomerase inhibitors act gene-and context-dependent, with some promoter sequences and their target transcription factors being more vulnerable 11,[START_REF] Schulenberg | Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye[END_REF][START_REF] Wang | Metabolic profiling of hearts exposed to sevoflurane and propofol reveals distinct regulation of fatty acid and glucose oxidation: CD36 and pyruvate dehydrogenase as key regulators in anestheticinduced fuel shift[END_REF]41 .

Up-regulation of genes certainly involves more specific mechanisms which are much less understood so far.

Our unbiased study identified a large number of novel and potentially interesting DXR targets at the single gene level. A rather comprehensive coverage of transcriptome changes is suggested by some strongly down-regulated transcripts that were already found in earlier candidate-oriented approaches in heart or cancer. Our study identifies them as very susceptible and early responders. In the heart, these include heme oxygenase 4 , natriuretic peptide precursor type B 9 , TNF-alpha [START_REF] Timolati | The role of cell death and myofibrillar damage in contractile dysfunction of long-term cultured adult cardiomyocytes exposed to doxorubicin[END_REF] and cardiac adriamycin responsive protein (or cardiac ankyrin repeat protein, CARP [START_REF] Tokarska-Schlattner | New insights into doxorubicin-induced cardiotoxicity: the critical role of cellular energetics[END_REF] ; missing in Table 4-1 since SAM fold-change is 0.83). Others were identified in cancer, such as the dual specificity phosphatase 1 (DUSP1 [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF] ), confirming again similarities in the DXR response of heart and cancer.

4.6.2. Stress-response genes are repressed in doxorubicin-treated hearts DXR promotes oxidative stress, DNA intercalation, inflammation and apoptosis.

However, many genes generally induced by these stress conditions and involved in defense or compensatory mechanisms were paradoxically down-regulated, as seen at the single gene and the gene set level.

(i) DXR is considered as a DNA damage-inducing agent, but many pathways and transcripts normally induced by DNA damage were repressed. The entire p53-regulated and DNA damage-inducible Gadd45 family (Gadd45α, Gadd45β, and Gadd45γ) was downregulated. Gadd45 members were recently shown to have critical roles in negative cell growth control and apoptosis, e.g. by affecting the G2/M cell cycle checkpoint 71 .

(ii) Heat shock proteins are molecular chaperones that have cytoprotective effects in a broad variety of adverse conditions. Reduced expression of the stress-inducible family member HSP70 and of the HSP27 pathway will reduce the stress response in the heart, corroborating DXR-induced down-regulation of chaperones observed earlier in cancer [START_REF] Feng | Phosphoproteome analysis of isoflurane-protected heart mitochondria: phosphorylation of adenine nucleotide translocator-1 on Tyr194 regulates mitochondrial function[END_REF] .

(iii) Immediate early response genes are stress-inducible genes associated with the regulation of cell proliferation and apoptosis. They are typically induced by different forms of stress, but were repressed by DXR in the heart.

(iv) DXR with its redox-cycling semiquinone ring is a strong inducer of oxidative stress.

However, DXR reduced several transcripts related to antioxidant response: heme oxygenase, recently shown to protect cells from various insults, including oxidative stress 1,46 , glutamatecysteine ligase, the rate limiting enzyme in glutathione (GSH) biosynthesis, and glutathione S-transferase M4. The latter is a member of the large, multifunctional GST family acting in xenobiotic detoxification (including DXR) by conjugating them with GSH, and also as peroxidase, thus protecting cells from oxidative stress products 54 . Decreased antioxidative capacity of DXR-treated hearts was reported already earlier 15,[START_REF] Solaro | Protein phosphorylation and signal transduction in cardiac thin filaments[END_REF][START_REF] Zaugg | Cellular mechanisms in sympatho-modulation of the heart[END_REF]51 .

(v) The stress-induced MAPK pathway is one of the major signaling pathways activated by DXR both in cardiac and cancer cells 39,[START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF][START_REF] Schlattner | Creatine kinase and creatine transporter in normal, wounded, and diseased skin[END_REF][START_REF] Zhu | MAPK superfamily plays an important role in daunomycin-induced apoptosis of cardiac myocytes[END_REF] . However, we detected a coordinated repression of the MAPK pathway at the level of transcripts, confirming an earlier study 39 .

(vi) Transcription factor ATF3 is a member of the ATF/CREB family of transcription factors. It is rapidly induced by diverse environmental insults including genotoxic stress and affects protein stability and function via regulation of p53, thus maintaining DNA integrity and protecting against cell transformation. In our model, ATF3 was repressed by DXR.

(vii) The hypoxia-inducible stress response plays an essential role in triggering cellular protection under of oxygen deprivation. In DXR perfused heart, the HIF pathway and the related VEGF pathway were repressed.

(viii) Finally, immune and inflammatory systems ensure an efficient response to infections/pathogens. In our study, DXR induced multiple changes in the expression of genes controlling immune and inflammatory response by repressing numerous single genes and pathways and by inducing only few ones (with granzymes B and C probably originating from cytotoxic T lymphocytes and natural killer cells). This mixed response is typical of the immunomodulatory role, mostly immunosuppressive but also immunogenic, often seen with anticancer drugs 2,[START_REF] Wakasugi | Myocardial substrate utilization and left ventricular function in adriamycin cardiomyopathy[END_REF] .

On the basis of these results and earlier reports, we propose that a blunted response to stress or reduced "danger signaling", seen here at the transcript level, is a prime component of toxic DXR action and can drive cardiac cells into pathology. This is consistent with our earlier observation made in a similar model of perfused heart, showing that DXR reduces protein and activation levels of AMP-activated protein kinase (AMPK), a key sensor and signaling hub in cellular and whole body energy homeostasis [START_REF] Huang | A systematic MS-based approach for identifying in vitro substrates of PKA and PKG in rat uteri[END_REF][START_REF] He | Phosphorylation of mitochondrial elongation factor Tu in ischemic myocardium: basis for chloramphenicol-mediated cardioprotection[END_REF] . Importantly, overexpression of some of the targeted stress-related genes (e.g. heat shock proteins, heme oxygenase, or ATF3) has shown promising results for in vitro protection against DXR-induced damage 13,16,[START_REF] Huang | Protein kinase C-mediated desmin phosphorylation is related to myofibril disarray in cardiomyopathic hamster heart[END_REF]43,45,[START_REF] Ande | Insulin induced phosphorylation of prohibitin at tyrosine 114 recruits Shp1[END_REF] .

Doxorubicin affects genes involved in cardiac damage and remodeling

Other transcripts whose down-regulation is directly detrimental to cardiac tissue may represent further potential targets for cardioprotection. These include LIM domain protein dyxin and Cited2, transcriptional regulators essential for normal heart development and function 3,[START_REF] Wu | PP1-mediated dephosphorylation of phosphoproteins at mitotic exit is controlled by inhibitor-1 and PP1 phosphorylation[END_REF] .

Cardiac remodeling is a compensatory mechanism of the heart to maintain cardiac output under stress that compromises cardiac function. It is associated with activation of specific genes that promote cardiac growth. DXR induced transcription of osteoglycin, identified as a major regulator of left ventricular mass 44 . DXR up-regulated also transcripts controlled by c-Myc, a transcription factor whose activity has been associated with hypertrophic response in heart [START_REF] Xiao | Inducible activation of c-Myc in adult myocardium in vivo provokes cardiac myocyte hypertrophy and reactivation of DNA synthesis[END_REF] . Moreover, DXR strongly repressed a group of genes implicated in reverse remodeling in response to mechanical unloading of the failing heart (LVAD, Figure 4-4A). Maintenance of these transcriptional effects may contribute to DXR-induced cardiac hypertrophy/failure.

Up-regulated "energy-related" genes indicate fast compensatory mechanisms

In general, up-regulation of transcripts can have cytotoxic or cytoprotective effects in cardiac and/or cancer cells. Both could be of therapeutic interest. Among the notable upregulated genes with known anti-proliferative action in tumor growth are PMP24 and quiescin Q6. Silencing of PMP24, the most strongly DXR-induced transcript, is associated with cancer progression [START_REF] Wu | PMP24, a gene identified by MSRF, undergoes DNA hypermethylation-associated gene silencing during cancer progression in an LNCaP model[END_REF] . Cytoprotective up-regulation may be found in DNA synthesis/repair with polymerases and nibrin, in cardiac remodeling with tropomyosin 1α and osteoglycin, whose expression correlates with a cardiac hypertrophic response 44 , as well as in immune response and mainly in energy metabolism.

Gene sets related to energy metabolism (glycolysis and Krebs-TCA cycle) belonged to the few up-regulated pathways. This is rather surprising, since acute DXR toxicity in cultured Chapter 4.

Early doxorubicin effects on the cardiac transcriptome 120 cardiomyocytes has been associated with suppression of several energy metabolism-related genes, including phosphofructokinase, ATP/ADP translocase, mitochondrial iron sulfur protein and muscle creatine kinase [START_REF] Mcgregor | Proteomics of the heart: unraveling disease[END_REF][START_REF] Wallace | Doxorubicin-induced cardiac mitochondrionopathy[END_REF] , which was not observed in the model analyzed here.

Our earlier studies in different molecular, cellular and organ model systems also clearly evidenced an impairment of cell energetics by DXR, e.g. inhibition of energy signaling by AMPK or energy buffering and transfer by creatine kinases [START_REF] Inagaki | Intermediate filament reconstitution in vitro. The role of phosphorylation on the assemblydisassembly of desmin[END_REF][START_REF] He | Phosphorylation of mitochondrial elongation factor Tu in ischemic myocardium: basis for chloramphenicol-mediated cardioprotection[END_REF] . In contrast, our transcriptome findings are partially congruent with the transcriptome response to DXR in a rat model, where mitochondrial genes were widely repressed, but glycolytic genes were up-regulated 5 . Upregulation of glycolytic and Krebs cycle genes in the acute situation could be a transient, compensatory mechanism triggered by the very beginning of energetic dysfunction, known to develop during DXR cardiotoxicity [START_REF] Huang | A systematic MS-based approach for identifying in vitro substrates of PKA and PKG in rat uteri[END_REF][START_REF] Ecroyd | Mimicking phosphorylation of alphaB-crystallin affects its chaperone activity[END_REF] . For example in an earlier study in perfused heart we also observed, that a loss in cytosolic muscle-type creatine kinase was compensated by reexpression of brain-type creatine kinase, characteristic for early differentiation states [START_REF] He | Phosphorylation of mitochondrial elongation factor Tu in ischemic myocardium: basis for chloramphenicol-mediated cardioprotection[END_REF] .

In perfused heart, early re-programming in expression of "energy-related" genes was accompanied by fairly normal myocardial metabolic status and mitochondrial function. Even the response of mitochondrial respiration to ADP and creatine, a very sensitive indicator, showed only subtle changes in term of kinetics and related K m s. However, these changes further progressed and reached statistical significance upon higher DXR doses applied directly to the fibers, which is consistent with our previous results obtained with isolated mitochondria [START_REF] Kitamura | Protein kinase C phosphorylation of desmin at four serine residues within the non-alphahelical head domain[END_REF] . Thus, up-regulation of "energy-related" genes and gene sets may precede the known DXR-induced energetic decline and represent a rather transitory compensatory mechanism. Failure of such compensatory mechanisms would then contribute to progressive accumulation of deficiencies in energy metabolism, a critical step in the deterioration of cardiac function.

Mechanisms responsible for specific early up-regulation of energy-related genes would require very sensitive cellular sensors detecting minor changes in oxidative stress or energy state. AMPK is known to respond to minor changes in AMP/ATP ratio that even escape statistical significance, but this pathway is inhibited by DXR [START_REF] He | Phosphorylation of mitochondrial elongation factor Tu in ischemic myocardium: basis for chloramphenicol-mediated cardioprotection[END_REF] . Consistent with this finding, we observed down-regulated expression of a set of AMPK-regulated transcription factors, including PGC1-α (data not shown). Thus, DXR affects energy-related gene expression in an AMPK-independent manner that remains to be elucidated.

Chapter 4.

Early doxorubicin effects on the cardiac transcriptome 121 4.6.5. Complex relationship between mRNA and protein expression/ phosphorylation Some transcriptomic changes already led to altered protein levels under our experimental conditions. Two out of seven proteins examined displayed significant changes that paralleled transcript levels: NDUFA10 and HSP70. The latter was found decreased after DXR treatment in vivo already earlier 50 . Other transcriptomic changes may be translated at a functional protein level only later, since DXR-induced changes in protein abundance are reported for some of them (HO-1 4 , TNFalpha 38 , DUSP1 [START_REF] Schlattner | Creatine kinase and creatine transporter in normal, wounded, and diseased skin[END_REF] ). DXR also affected protein phosphorylation, apparently before emerging transcripomic changes are translated into protein levels. For example, DXR down-regulated transcripts of the MAPK pathway, but increased phosphorylation of extracellular signal-regulated kinase (ERK 1/2), a member of the MAPK family, consistent with earlier reports 39 , and also consistent with down-regulated transcripts of stress-induced DUSP1, a protein phosphatase selective for MAPK isoforms. However, downregulation of the MAPK pathway may become functionally important later. Reduced levels of upstream kinases within the MAPK pathway and increased DUSP2, another MAPK familyspecific protein phosphatase, could lead to ERK inactivation as observed by Lou et al. in DXR-induced heart failure 39 . 4.6.6. Novel anticancer strategies and doxorubicin treatment share similar targets Many identified genes not only respond similarly to DXR in heart and cancer, they also encode proteins that are suggested as targets of new anticancer therapies: heat shock proteins 8 , the signaling pathway of MAP kinases [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF] , certain G-proteins 56 , IGF 10 , Wnt/β-catenin 14,40 , HIF [START_REF] Kwon | Cell cycle-dependent phosphorylation of mammalian protein phosphatase 1 by cdc2 kinase[END_REF] , VEGF [START_REF] Ventura-Clapier | Bioenergetics of the failing heart[END_REF] and TGF-β, the latter playing a dual role of as tumor suppressor and prooncogenic factor [START_REF] Mesnard-Rouiller | Troponin T mRNA and protein isoforms in the human left ventricle: pattern of expression in failing and control hearts[END_REF] . Moreover, DXR has certain mechanistic details in common with a novel class of anticancer drugs, the tyrosine kinase inhibitors like imatinib mesylate (Gleevec) and gefitinib. DXR represses transcripts of pathways targeted by Gleevec and, similarily as gefitinib, induces expression of quiescin Q6 (QSCN6), a gene which may play a role in tissue growth by affecting repartition between cells in the proliferative cycle, in quiescence, and in apoptosis 12,[START_REF] Yano | Identification of genes linked to gefitinib treatment in prostate cancer cell lines with or without resistance to androgen: a clue to application of gefitinib to hormone-resistant prostate cancer[END_REF] .

The induction of c-Myc-activated genes is quite intriguing, given that this protooncogene is already overexpressed in many types of cancer and we identified only a very limited number of up-regulated pathways. Since c-Myc activity has a very complex impact on oncogenic transformation, it is difficult to anticipate the resulting phenotype. As seen in our study, the up-regulated set of myc-related transcripts includes the well established metastasis Early doxorubicin effects on the cardiac transcriptome 122 suppressor NM23A 7 . On the other hand it has been demonstrated that combination of a DXR therapy with an inhibition of c-Myc transcriptional function has an enhanced anti-cancer effect 6. It is to note again, that targeting these genes, which is supposed to be beneficial for cytotoxic action in cancer, may be rather detrimental to the heart.

Perspective and Significance

We have performed a genome-wide screening of DXR-induced acute transcriptome changes in perfused heart, which identified cytotoxic, but also pro-survival responses. The acute repression of stress-responsive transcripts was a hallmark of the toxic response. In Chapter 5.

Summarizing discussion

& Outlook

Context and aim of the thesis project

The chemotherapeutic drug DXR is among the most widely used for treatment of cancer,

although it has serious cardiotoxic side-effects (see Chapter 1.2.). Despite more than forty years of research, DXR cardiotoxicity remains a burden for public health. The underlying molecular mechanisms are not yet entirely clear and efficient preventive or curative treatments are not available. However, it became clear that this is a complex pathology that involves pleiotropic mechanisms. A main feature of DXR at the cellular level is induction of oxidative stress and the development of energy deficits. This is linked, among others, to deleterious effects on the creatine kinase system 1 , and, as our team found more recently, in the signaling of the key energy sensor and regulator AMPK 2 . Inhibition of the latter is a paradox, since the kinase should be activated under energy stress generating higher AMP levels. This finding raised the possibility that DXR has not only general deleterious effects leading to oxidative damage and energy stress, but also interferes specifically with cell signaling events that may prime the acute and chronic phenotype of DXR toxicity.

Our first aim was to establish different models of DXR cardiotoxicity (see Chapter 2.).

The perfused heart model was chosen as an in situ model that allows triggering DXR responses under highly controlled conditions with different, also clinically relevant DXR doses to observe very early responses to DXR. In addition, an in vivo animal model with intravenous drug administration was successfully set up to study toxic DXR effects occurring weeks after completion of the DXR treatment, thus representing chronic DXR toxicity.

Our next aim was then, by using both models, to characterize the inhibition of AMPK in the context of cell signaling, in order to delineate the potentially involved mechanisms (see Chapter 2.). The study focused on AMPK up-and downstream signaling, and in particular on the cross-talk of AMPK energy stress signaling with genotoxic and oxidative stress transmitting DNA-PK/Akt and MAPK signaling pathways. We found that inhibitory crosstalk by the Akt pathway rather than impaired AMPK upstream signaling would lead to AMPK inhibition.

These findings suggested major changes in the cellular signaling network induced by DXR. Our further aims were therefore to study cell signaling with genome-wide, non-biased approaches. For this we used the acute toxicity model, potentially revealing the very early events occurring in response to the drug, before functional alterations become detectable at the macroscopic level. The non-biased approaches are suitable to identify novel targets and mechanisms in complex pathologies such as DXR cardiotoxicity. As a first comprehensive "omics" approach we carried out a screening of the cardiac phosphoproteome to directly identify (de-)phosphorylation events linked to altered cell signaling by kinases and phosphatases due to DXR, an approach that has not yet been used in this field (see Chapter 3.). As a second "omics" approach, aiming at cell signaling in transcriptional control, we applied transcriptomics to analyze changes in gene expression induced by DXR (see Chapter

4.

). Both approaches revealed known as well as novel cellular targets of DXR that are involved in detrimental as well as compensatory mechanisms, most of them belonging to few functional classes: energy metabolism, stress responses, and sarcomere structure and function.

Discussion of models and approaches

Toxicity models

In this thesis, two experimental models of cardiac DXR-toxicity have been used: isolated perfused heart and an in vivo animal model. In the former model, isolated rat hearts perfused with a clinically relevant dose of DXR (2-5 µM) may reveal the molecular effects leading to acute DXR toxicity, in a state before any cardiac dysfunction can be detected 2 . Additional perfusion with a supra-clinical dose (25 µM DXR) showing pronounced functional disorders may give clues about DXR effects at high cumulative dose, although it will not necessarily mimic the long-term drug exposure preceding true chronic DXR toxicity 3 . Perfused heart models will give information about the very early effects of the drug, which are thus likely to be involved in the etiology of DXR toxicity rather than in nonspecific consequences 4 . As further advantages, the experimental approach allows continuous recording of cardiac performance during the treatment, and is not painful for the donor animals.

In the second toxicity model that we established, rats were injected intravenously with the clinically relevant cumulative dose of 12 mg/kg DXR during two weeks and probed four weeks after the last injection. This in vivo model can reveal information relevant to chronic DXR cardiotoxicity, since it will detect modifications that persist several weeks after completion of the DXR treatment 4 . These modifications are not necessarily the cause but can also be consequences of established toxic DXR effects. Although this model is rather painful for the involved animals, it has the important advantage to be close to the clinical situation.

Both experimental models are complementary and taken together can give more comprehensive insight into DXR cardiotoxicity. Interestingly, our targeted approach to cell signaling revealed a large number of similar responses to DXR in both models (see below).

Methodological approaches

Effects of DXR on cardiac cell signaling pathways were analyzed in this thesis with two conceptually different approaches: a focused analysis targeting AMPK-related energy signaling (see Chapter 2.), and a genome-wide, non-biased analysis of the transcriptome and the phosphoproteome (see Chapters 3. and 4.). Targeted approaches allow verifying a precise hypothesis, in our case to answer the question whether AMPK signaling is inhibited in acute and chronic cardiotoxicity, and which upstream or cross-talking signaling elements are involved in this inhibition. Non-biased screening approaches are by definition hypothesisfree, although always a basic assumption is present, e.g. in our case that DXR specifically affects transcriptome and proteome. Since DXR cardiotoxicity is a multi-factorial complex pathology, such non-biased screening in principle allows novel mechanistic insight beyond traditionally accepted models, and the identification of so far unknown targets. However, it will rather generate novel hypothesis than give direct mechanistic insight. Thus, such screening should be followed by classical targeted analysis. It must be also noted that some minor but functionally important changes may escape detection.

For non-biased analysis, two "omics" screening techniques were applied: transcriptomics and phosphoproteomics. The former is the quantitative determination of the complete set of mRNA molecules synthesized in a given cell. However, transcriptomic data cannot be directly interpreted in terms of function, since they do not necessarily reflect protein levels or protein activity 5 . The screening can be automated to reach high-throughput, but should be verified by RT-PCR. Phosphoproteomics is the quantitative study of the complete set of protein phosphorylations occurring in a given cell. This most important post-translational modification directly reflects the functional state of proteins, including enzymatic activation or inhibition, protein-protein interactions, etc. 6 , and therefore carries much more direct information for functional analysis as compared to transcriptomics 7 . However, the 2-DE-MS screening technique that we have applied here is not adaptable to high-throughput.

Discussion of key findings

Energy metabolism

Reduced levels of high-energy compounds such as ATP and PCr and the resulting energy stress are one of the hallmarks of DXR cardiotoxicity. DXR-mediated inhibition of several elements in energy metabolism, mainly by direct oxidative damage, has been shown in the past, including key enzymes like creatine kinases 1 , phosphofructokinase 8 , β-enolase 9 and carnitine palmitoyl transferase I 10 (for review see 11 ), for example. These effects on protein levels and enzymatic activity often concern mitochondrial enzymes, since DXR accumulates at the inner mitochondrial membrane due to its affinity to cardiolipin 12 . In this thesis, we show a novel contribution of cell signaling to the energetic phenotype of DXR cardiotoxicity. We confirm inhibition of AMPK signaling that we have observed earlier 2 , in models of both, acute and chronic, DXR-cardiotoxicity, but not in cancer cells. Our novel data also propose a negative crosstalk with Akt and MAPK pathways, activated by genotoxic and oxidative stresses, rather than inhibition of AMPK upstream signaling, as likely mechanisms for AMPK inhibition. Resulting mTOR activation favors anabolic processes like protein synthesis 13 , which will aggravate energy stress and contribute to the hypertrophic cardiac phenotype 14 , in order to compensate left ventricular failure.

Our novel approach applying phosphoproteomics to analyze acute DXR cardiotoxicity extends the observations made in the AMPK-targeted approach by showing that the predominant changes in phosphorylation status occurs with enzymes in energy metabolism and with proteins that show mitochondrial localization. This lets us propose that phosphorylation of mitochondrial proteins, which emerges as a regulator of many mitochondrial functions 15 , is a mediator of toxic DXR effects. A key target in this respect is PDH, the phosphorylation of which would inhibit pyruvate entry into Krebs cycle, decrease oxidative glucose metabolism and exacerbate energy deficits. However, there are also compensatory mechanisms at work, linked to differential phosphorylation of other energyrelated enzymes and changes in gene or protein expression. For example, higher transcription rate was observed for glycolytic and Krebs cycle enzymes, and increased protein levels for brain-type creatine kinase. This may at least partially explain why in the model of acute DXR toxicity, heart performance parameters were not yet impaired at clinically relevant DXR doses.

Stress response

In addition to energy stress, we observed in both DXR toxicity models more or less pronounced oxidative and genotoxic stress occurring via DNA damage and generation of reactive oxygen and nitrogen species. This would be expected to induce a full-fledged stress response involving multiple signaling cascades. However, observations from the studies presented in this thesis show a rather mixed stress response, with some pathways being effectively activated, and some others being impaired. Among the latter are many pathways responding to energetic, oxidative and genotoxic stress. Combined generation of reactive species and ATP depletion should activate AMPK, which is not the case in either acute or chronic toxicity model. Similarly, the transcriptomics study in the acute toxicity model showed specific strong transcriptional repression of many genes coding for stress response proteins. Genotoxic and oxidative stress should activate expression of genes in DNA damage repair or stabilization and oxidative stress response. However, genes of the entire p53regulated and DNA damage-inducible Gadd45 family, of stress-induced transcription factor ATF3 and of enzymes in anti-oxidative defense such as heme oxygenase or glutathione Stransferase were down-regulated, in line with the known reduced antioxidative capacity of DXR-treated hearts 16,[START_REF] Gewirtz | A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin[END_REF] . Advanced pathway analysis of the transcriptome revealed further down-regulation of typically stress-induced gene groups, including heat shock response, immune response, or the HIF pathway. On the basis of these results we propose a blunted response to stress or reduced "danger signaling" as a prime component of toxic DXR action that can drive cardiac cells into pathology.

Among the stress-responsive pathways that are effectively activated by DXR are the DNA-PK-Akt-GSK3 and ERK pathways as seen in the targeted study, and chaperone-like proteins such as CRYAB in the phosphoproteomic study. DNA-PK apparently transmits the DNA damage signal, and both activated Akt and ERK will limit apoptosis due to genotoxic and oxidative stress. They will also, via mTOR signaling, promote protein biosynthesis and cell growth (see above), thus contributing to cardiac hypertrophy to compensate the decrease of heart contractility caused, among others, by an altered myofibrillar network. The latter may be compensated by the action of chaperones like CRYAB known to be cardioprotective once phosphorylated [START_REF] Rabilloud | Two-dimensional gel electrophoresis in proteomics: Past, present and future[END_REF][START_REF] Schulenberg | Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye[END_REF] .

Sarcomere structure and function

A hallmark of DXR cardiotoxicity is molecular damage and deregulation of the myofibrillar network, leading to myofibrillar disarray [START_REF] De Souza | Preparation of human heart for laser microdissection and proteomics[END_REF][START_REF] Li | Evaluation of several two-dimensional gel electrophoresis techniques in cardiac proteomics[END_REF][START_REF] Mcgregor | Proteomics of the heart: unraveling disease[END_REF][START_REF] Mesnard-Rouiller | Troponin T mRNA and protein isoforms in the human left ventricle: pattern of expression in failing and control hearts[END_REF] This confirms the general, strong homeostatic potential of the heart. However, since these mechanisms already operate under such basic conditions, they may be rapidly overcharged in case of any additional stress like exercise or ischemia. In fact, exercise intolerance and high susceptibility in ischemic situations are known phenotypes of the DXR cardiotoxic phenotype [START_REF] Ventura-Clapier | Bioenergetics of the failing heart[END_REF][START_REF] Feng | Phosphoproteome analysis of isoflurane-protected heart mitochondria: phosphorylation of adenine nucleotide translocator-1 on Tyr194 regulates mitochondrial function[END_REF] .

Preventing mechanisms that contribute to energy deficits and myofibrillar disarray, or reinforcing compensatory functions may represent approaches to decrease DXR cardiotoxicity. Our studies suggest AMPK, PDH and desmin as potential targets that would need to be activated or maintained during DXR treatment.

Outlook

DXR cardiotoxicity is a multi-factorial process involving among others impairment of energy state, stress responses and sarcomere structure and function. We identified different key elements in this context, namely AMPK, PDH, CRYAB and desmin. These require now more detailed mechanistic studies using animal models and material from patients. Moreover, our experiments have also given clues to design future investigations in order to find effective cardioprotective strategies against DXR-induced cardiotoxicity.

The energy sensor and regulator AMPK is inhibited by DXR in rat heart. It would now be interesting to study the effect of experimentally altered AMPK levels under DXR treatment in genetic or pharmacological animal models. It could be studied whether the use of AMPKα1 -/- and AMPKα2 -/-knockout mice [START_REF] Wang | Metabolic profiling of hearts exposed to sevoflurane and propofol reveals distinct regulation of fatty acid and glucose oxidation: CD36 and pyruvate dehydrogenase as key regulators in anestheticinduced fuel shift[END_REF] would sensitize the heart for a cardiotoxic phenotype in an acute or chronic model. Alternatively, it should be tested whether AMPK activators like AICAR, A-769662 or Metformin show protective effects in animals when given before and/or together with DXR. Metformin has been shown recently to decrease mortality and to improve the outcomes in patients with chronic heart failure (reviewed in [START_REF] Sugden | Mechanisms underlying regulation of the expression and activities of the mammalian pyruvate dehydrogenase kinases[END_REF] ). The drug is already in use for more than 50 years to treat diabetes type II, and anti-diabetic effects in liver are mediated by AMPK activation [START_REF] Holness | Regulation of pyruvate dehydrogenase complex activity by reversible phosphorylation[END_REF] . The mechanism, by which Metformin activates AMPK, involves a direct inhibition of complex 1 [START_REF] Carvalho | Metabolic remodeling associated with subchronic doxorubicin cardiomyopathy[END_REF] , leading to ROS signaling [START_REF] Lewandowski | Pyruvate dehydrogenase influences postischemic heart function[END_REF] and to altered AMP/ATP ratios that activate AMPK allosterically [START_REF] Wakasugi | Myocardial substrate utilization and left ventricular function in adriamycin cardiomyopathy[END_REF] . In addition, Metformin may have AMPK-independent beneficial effects in the heart, like increasing FA oxidation, and thus improving metabolic and energy status [START_REF] Solaro | Protein phosphorylation and signal transduction in cardiac thin filaments[END_REF] . Since at the same time, chronic Metformin treatment inhibits tumorigenesis [START_REF] Zaugg | Cellular mechanisms in sympatho-modulation of the heart[END_REF][START_REF] Huang | Protein kinase C-mediated desmin phosphorylation is related to myofibril disarray in cardiomyopathic hamster heart[END_REF] , this would be a safe treatment to be tested in a clinical trial.

PDH is a metabolic switch shunting glucose metabolites either to oxidative mitochondrial metabolism or cytosolic lactate production. It is inhibited by a DXR-induced increase of its phosphorylation status, which could exacerbate energetic deficits by slowing down the Krebs cycle [START_REF] Geisler | Phosphorylation of desmin in vitro inhibits formation of intermediate filaments; identification of three kinase A sites in the aminoterminal head domain[END_REF] . These changes in PDH and oxidative metabolism will have to be verified in vivo by determination of enzymatic and respiratory activities. Further, inhibitors for the involved PDH kinases (PDKs) have been developed to activate PDH in diabetes, with the beneficial effect to inhibit hepatic gluconeogenesis and to promote glucose disposal in peripheral tissues. The best-known PDK inhibitor is dichloroacetate, which proofed to favor glucose oxidation and thus the energetic status in heart (reviewed in [START_REF] Timolati | The role of cell death and myofibrillar damage in contractile dysfunction of long-term cultured adult cardiomyocytes exposed to doxorubicin[END_REF] ), but other PDK inhibitors have been developed more recently (reviewed in 39 ). Like AMPK activators, they may be tested in DXR toxicity models to study their effect on cellular energy state and the cardiotoxic phenotype in the view of potential clinical studies.

Another preventive treatment could consist in improving the cellular energy state with nutritional supplements. The metabolite Cr has been successfully used as a supplement in this respect in many different pathologies, rehabilitation and sports 40,41 Finally, the role of DXR-induced desmin phosphorylation in myofibrillar disorganization has to be studied. It has been shown earlier in cardiomyocytes that low DXR doses lead to degradation of another sarcomeric protein, titin, by a calpain-dependent mechanism [START_REF] Mcgregor | Proteomics of the heart: unraveling disease[END_REF] . Thus, the relationship between desmin phosphorylation and filament disorganization should be studied by immunocytochemical analysis of cardiomyocytes isolated from in situ or in vivo toxicity models. However, since desmin is phosphorylated at different sites by various kinases [43][44][45] , identification of all sites showing DXR-dependent changes in phosphorylation is required first. This may then also allow testing inhibitors of the involved kinases in DXR toxicity models.

As the etiology of DXR cardiotoxicity is clearly multi-factorial, a combination of different preventive or curative strategies may be a key factor to reduce the side effects of the drug. As an example, co-administration of Metformin and an antioxidant such as dexrazoxane (see Chapter 1.2.) may have cumulative beneficial effects. However, every therapy has to proof that it does not counteract the anticancer effects of DXR.

Concluding remark

Conclusion

Ce travail de thèse apporte de nouveaux éléments de compréhension des mécanismes potentiels de la cardiotoxicité de la DXR. Bien que les principaux phénotypes moléculaires de la pathologie soient connus, notre étude a identifié de nouveaux éléments de signalisation qui pourraient être impliqués dans ces mécanismes. De plus, notre travail fait émerger différentes orientations de stratégies protectrices. Ainsi, il y a encore de l'espoir pour réussir à mieux appréhender la toxicité de la DXR et obtenir ainsi un traitement contre le cancer plus efficace avec moins d'effets secondaires.

Chapter 6.

Compendium of methods

This part is a complete description of experimental procedures that I have used and/or developed in this PhD work, recapitulating, extending and commenting the method chapters of the different parts of the thesis. Methods exclusively developed and applied by collaborators are not included here. Specific materials are quoted (purity, manufacturer)

where appearing in the methods sections.

DXR solutions

Doxorubicin hydrochloride (DXR) was purchased in form of powder from Fluka (Buchs, Switzerland). A stock solution of the drug at 10 mM concentration was prepared in water (Milli-Q), aliquoted and stored at -20°C. Powder was not weighted, but the appropriate water volume was added to the nominal package content. Further dilutions were prepared in Krebs-Henseleit buffer just before heart perfusions or in saline (0.9% NaCl) before injection to animals. All DXR solutions were protected from sunlight.

DXR handling (especially in powder form) was done with care. Powder was dissolved under the hood and by protecting the bench surface by disposable aluminum foil to avoid dispersion.

Heart perfusion protocol

In the present study, hearts of male Wistar rats (3 months old, weighting ca. 300 g) were used for experiments. The animals were housed under controlled light/dark cycle and received a standard chow (A04, Safe, France) and water ad libitum. The rats were anaesthetized with sodium pentobarbital (50 mg/kg ip). After checking adequacy of anesthesia (pedal reflex, muscle relaxation), heparin (1500 IU/kg) was administered in the saphenous vein.

The thorax was opened and the heart was removed quickly and immediately immerged in an ice-cold (4°C) Krebs-Henseleit solution (155 mM Na + , 5.6 mM K + , 138 mM Cl -, 2.1 mM Ca 2+ , 25 mM HCO 3 -, 0.56 mM Mg 2+ , 11 mM glucose, saturated with 95% O 2 and 5% CO 2 , pH 7.4) 1 , if not stated otherwise. The aorta was freed from adjacent tissue. A canula was then placed in aorta and the heart was mounted in a noncirculating Langendorff apparatus.

Hearts were perfused in an isovolumic mode at a constant pressure of 80 mmHg (109 cm In figure "Equilibration" should be changed into "Stabilization" Isolated perfused heart represents a useful in situ/ex vivo preparation because it allows e.g.

the addition of drugs (via the perfusate) and observation of their effects (functional and metabolic) on the heart under strictly controlled experimental conditions. Importantly, its use reduces in vivo experimentation, which is often harmful to the animal. The setup is especially useful to examine the acute direct response of heart to stress or medication. However the choice of a correct, clinically relevant drug concentration requires particular care when applying this model. One has to be aware that this model may not reproduce the full in vivo heart response to the chosen stimuli due to the lack of systemic effects. Despite these limitations, it represents a very valuable tool for the initial analysis of drug effects. For our animal cardiotoxicity model, male Wistar rats were used. The animals were housed under controlled light/dark cycle and received a standard chow (A04, Safe, France)

and water ad libitum. At the beginning of the experiment, 6 weeks old rats (∼175 g) were anaesthetized with an intramuscular injection of ketamine (50 mg/kg)/xylazine (10 mg/kg) and catheterized via jugular vein 2 . Adequacy of anesthesia was carefully monitored by regular checking depth of anesthesia (pedal reflex, muscle relaxation) and physiological parameters (heart and respiratory rate). Anesthetic support such as fluid supplementation and external heating was provided during the whole catheterization procedure: 2.5 mL of saline solution were injected subcutaneously on each side of the spine at the level of kidneys in order to prevent animal dehydration. Body temperature was maintained using a heating pad and overhead lamp. Subsequent monitoring and support during anesthetic recovery was also ensured as described below. The catheters were prepared from polyethylene PE-20 tubing cut to 20 cm length. Before beginning of the surgery, tubing was flushed with saline (0.9% NaCl) using a 1 mL syringe attached to the tubing via a needle connector (size 23G needle with 90° square tip).

Catheter implantation was performed with instruments sterilized by washing with 70% ethanol. The rat was shaved in the ventral neck and interscapular regions. The skin was cleaned with a povidone iodine solution (Bétadine ® , Meda Pharma, Solna, Sweden). The rat was placed on its back with the nose pointing toward the surgeon. A 1.5-to 2-cm incision was made in the ventrolateral aspect of the neck. The right jugular vein was identified and separated from surrounding tissue by removing fascia with forceps and stabilized with a proximally placed knot of 4-0 silk suture (Ethicon, Johnson & Johnson, Belgium). Two silk sutures (distant 7-8 mm from each other) were placed under the isolated part of the jugular vein. The vein was then cut at an angle of 45° with micro-scissors. The catheter was inserted to the vein through the incision in the direction of the heart (not more than 2-3 cm to avoid heart damage). Then the catheter was fixed with the two prealably placed silk sutures.

Catheter functioning was checked by withdrawing blood and flushing it back. To bring the second end of the catheter outside the animal body, it was tunneled subcutaneously with a closed hemostat to exit via an approximately 2.5 cm incision made in the interscapular region.

A few droplets of local anesthetic lidocaine (Xylocaïne, Astrazeneca, Sweden) were applied in the wounds, which were closed with silk sutures. Excess catheter length was cut (to about 5 cm). Catheter was filled with a heparinized saline solution (500 UI/mL) and was heat-sealed.

The rat stayed on the heating pad until the end of anesthesia and then was put in a cage in IMPORTANT: As the cells were not incubated with antibiotic to avoid changes in cell metabolism and signaling not due to DXR, all the procedure until cell lysis had to be performed under strictly sterile conditions.

6.5. Immunoblot

Sample preparation

Western blots were realized using cell lysates prepared as described above or with total heart homogenates. Total heart homogenates were used to minimize problems related to possible differential extractability of proteins from different cellular compartments. Heart tissue was grinded with a mortar in liquid nitrogen and lysed/homogenized in 1 M Tris-HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT containing protease inhibitor cocktail tablets (Roche, Germany) and phosphatase inhibitor cocktail (Pierce, IL, USA) (tissue/buffer (w/v) proportion was 1/7), using an UltraTurax (3 cycles of 10 sec on ice). Protein concentration was determined with Biorad reagent (Reinach, Switzerland) using BSA as a standard.

Homogenates were either used immediately or aliquoted and stored at -80°C. Directly before electrophoresis, heart homogenates (50 µg protein) were lysed directly in Laemmli sample buffer (104 mM Tris-HCL pH 6.8, 3% w/v SDS, 2.5% w/v bromophenol blue, 14.4% glycerol, 5% v/v 2-mercaptoethanol) and heated at 95°C during 5 min.

SDS-PAGE separation

Proteins were separated on 7.5%, 10% or 12% acrylamide gels (depending on the molecular weight of the protein of interest) at 200 V during 45 min using minigels (MiniProtean 3 cell, Bio-rad, CA, USA). Step 1 Gradient 500V 1h

Step 2 Gradient 1000V 1h

Step 3 Gradient 10000V 10h

Step 4

Step & Hold 10000V 2h Six samples were separated each time in parallel: two controls, two 5 µM-DXR and two 25 µM-DXR. Control protein samples were included in each run to verify whether the separation process was correct. Our 2-DE experiments were performed using a high number (n ≥ 4) of biological replicates to obtain a strong statistical gel analysis afterward.

Step 1 Gradient 500V 1h

Step 2 Gradient 1000V 1h

Step 3 Gradient 10000V 10h

Step 4

Step & Hold 10000V 3h per cuvette) was added, the reaction was followed untill PCr exhaustion and the final absorbance was read (A2) The differences A1-A0 and A2-A1 were used to calculate respectively amounts (and corresponding concentrations) of ATP and PCr present in the heart extracts added to reaction mixture. For each sample, measurements were done 3 times and the mean value was calculated for ATP and PCr. It is to note that for ATP, results of quantification by the two applied methods, HPLC and spectrophotometric one, were practically the same.

The data were then normalized as a function of dry weight which was determined in a heart piece dried for 24h at 100°C (weight determined before and after drying).

Phosphatase activity

The Tyr and Ser/Thr protein phosphatase activities were determined in heart homogenates (3 µg of proteins) using a kit from Promega (Madison, WI). Briefly, after removing endogenous phosphates with Spin Columns (Sephadex G-25 resin), homogenates were incubated in a 96-well plate with a reaction mix (see manufacturer's instruction for further details) and 100 µM phosphopeptide substrate at 37°C for 45 min. To stop the reaction, 50 µl of molybdate dye/additive mixture (Promega) was added to the wells, and the absorbance at 600 nm was read using ELx808 Absorbance Microplate Reader (Biotek, Winooski, VT) in at least three independent experiments. (Agilent, Waldbronn, Germany). All the samples had a 260 nm/280 nm ratio between 1.8-2.1 and a 28S/18S ratio within 1.5-2.0 and were further processed. RNA was aliquoted and stored at -80°C.

Reverse transcription

This technique allows synthesizing complementary DNA from mRNA. Each sample was incubated with reverse transcriptase (100 U of M-MLV reverse transcriptase) and oligo-dT as primer (Invitrogen, Carlsbad, CA) in a final volume of 20 µL, following the manufacturer's instruction (Superscript III, Invitrogen). The mix was incubated at 50°C for 45 min to proceed the transcription and at 70°C for 15 min to inactivate the enzyme. The cDNA was kept at -80°C.

Real-time PCR

The RT-PCR allows amplifying of a DNA fragments. Amplification is followed by incorporation of fluorescent primer. The number of cycles necessary for amplification to reach a threshold of 50% of initial primers incorporated is directly proportional to the quantity of originally present mRNA. Primers for RT-PCR were purchased from Eurogentec (Seraing, Belgium). For each gene to be analyzed, two sets of primers were initially designed and the better one was chosen for further experiments (see Table 6-3).

Each primer efficiency was tested using different dilution of sample (1/10, 1/100,1/1000 and 1/10000) to find optimal dilution, using the minimal quantity of sample and optimal for primer efficiency. Moreover, the primer specificity was tested to ensure that the primer pair had specifically amplified only one fragment. To do so, we compared the theoretical melting point (temperature required to separate DNA strand) of the fragment of interest and the experimental one, which must be the same.

Table 6-

  of the anthracycline class of anticancer drugs, which is still among the most used to treat tumors, and still among the most efficient. Anthracyclines were discovered by the end of the 1950s. Di Marco, an Italian scientist, isolated an antibiotic from a species of Streptomyces. This molecule had antibacterial and antifungal activities, but its most remarkable property was its antitumor action against a wide range of murine cancers. The molecule was named daunomycin. Almost at the same time, a French team discovered another new antibiotic, also from Streptomyces, that they named rubidomycin. Both scientific teams recognized that they had isolated the same molecule and the International Nonproprietary Name (INN) convention decided to name this substance daunorubicin (DNR).

Figure 1 - 1 :

 11 Figure 1-1: Structures of DXR and DNR. The residues that are different between DXR and DNR are labeled in red. Figure modified after 4 .

Figure 1 - 2 :

 12 Figure 1-2: Generation of reactive species in a redox cycling involving DXR, and more particularly its quinone and semiquinone part (ring B and C).Figure taken from 25 .

  Figure 1-2: Generation of reactive species in a redox cycling involving DXR, and more particularly its quinone and semiquinone part (ring B and C).Figure taken from 25 .

  ). Merten et al.90 have worked with an acute toxicity model. Mice were treated with an intraperitoneal injection of 20 mg/kg DXR and the proteome was analyzed by 2-DE and MS identification.The level of different groups of proteins was decreased by the drug, particularly those involved in energy metabolism: FA metabolism, glucose metabolism and mitochondrial respiratory chain. The changes in mitochondrial respiratory complexes included an increase in complex III (ubiquinol cytochrome c reductase) and a decrease in cytochrome c and ATP synthase (complex V). These data confirm previous studies including transcriptomic ones on a dysfunction of energy metabolism (see above) and may explain the observed energy depletion. Cellular structure was affected by an increase in the type III intermediary filament vimentin, and cardiac contraction and relaxation by a decrease of troponin T and tropomyosin, both key proteins in sarcomere function. This study has shown that a unique injection of DXR at a supraclinical dose affects protein levels in energy metabolism, calcium homeostasis, and sarcomere structure and function.

  ). In isolated hearts, DXR perfusion led to dose-dependent loss of cardiac function (Figure2-1A-F and Figure S2-1), together with an increase in AMP/ATP ratios (Figure 2-1G) and nuclear and mitochondrial DNA lesions (Figure 2-1J,K), indicating energetic and genotoxic stress. Significant differences were obtained for almost all parameters at 25 µM DXR, except for protein oxidation as revealed by carbonyl formation (Figure 2-1H,I) which was expected to increase due to higher superoxide levels described earlier 16 . Apoptosis was not yet induced, since PARP cleavage was not detectable (Figure 2-1L). In the in vivo animal model, DXR treatment reduced weight gain of the animals, mainly during the treatment period (Figure 2-2A). Four weeks after the end of the treatment, DXRtreated rats also showed impaired heart function (Figure 2-2B-E) and significantly diminished cardiac ATP levels (Figure 2-2F). In contrast to isolated heart, protein carbonyls were increased (Figure 2-2G,H), thus both energetic and oxidative stress were present. DNA damage was less pronounced, below statistically significance, and mainly observed for mitochondrial DNA (Figure 2-2I). However, the onset of apoptosis was clearly revealed by PARP cleavage (Figure 2-2J

Figure 2 - 1 :

 21 Figure 2-1: Characterization of an isolated perfused rat heart model of acute doxorubicin cardiotoxicity. Indicators of cardiac function (A-F), energetic (G), oxidative (H, I), and genotoxic stress (J,K), and apoptosis (L) at the end of an 80 min Langendorff perfusion of isolated rat hearts without (control, label C) or with DXR at 5 µM (D5) or 25 µM (D25). Functional parameters: developed pressure (A), end-diastolic pressure (B), dP/dt (C), -dP/dt (D), heart rate (E), and rate pressure product (F). Indicators of energy stress: AMP/ATP ratio (G), of oxidative stress: protein carbonyls probed by immunoblot (H) and their quantification (I), of genotoxic stress: evaluation of nuclear (J) and mitochondrial DNA damage (K) by Real-Time PCR on nuclear and mitochondrial long and genomic DNA, and of apoptosis: cleaved poly(ADP-ribose)polymerase (PARP) probed by immunoblot and corresponding quantification normalized to tubulin (L); camptothecin-treated Jurkat cells (JC) serve as positive control. *P < 0.05, **P < 0.01, ***P<0.001 vs control (n ≥ 4).

Figure 2 - 2 :

 22 Figure 2-2: Characterization of an in vivo rat model of chronic doxorubicin cardiotoxicity. Indicators of animal growth (A), cardiac function (B-E), energetic, oxidative and genotoxic stress (F-I), and apoptosis (J) in hearts of saline-treated rats (control, label C) or rats treated with a cumulative dose of 12 mg/kg DXR (label D). Body weight (A) during and after DXR treatment (DXR injections marked by arrows). Functional parameters: developed pressure (C), dP/dt (D), -dP/dt (E), heart rate (F). Indicators of energy stress: ATP levels (G), of oxidative stress: protein carbonyls probed by immunoblot (H) and their quantification (I), of genotoxic stress: evaluation of mitochondrial DNA damage by Real-Time PCR on mitochondrial long and mitochondrial genomic DNA (I) and of apoptosis: cleaved poly(ADP-ribose) polymerase (PARP) probed by immunoblot and corresponding quantification (J) normalized to GAPDH. Parameters in (B-J) were evaluated 4 weeks after the end of DXR/saline treatment *P < 0.05, **P < 0.01, ***P<0.001 vs control (n ≥ 4).

Figure 2 - 3 :

 23 Figure 2-3: Doxorubicin reduces phosphorylation of AMPK and its target ACC without affecting AMPK upstream signaling. Phosphorylated and total AMPK and ACC probed by immunoblots in total homogenates from perfused rat hearts (A) and from hearts of in vivo treated rats (B). Effects of DXR on AMPK upstream signaling analyzed in the perfused heart model (C,D). Immunoblots of LKB1, CamKKα, CamKKβ and deacetylase SIRT1, and phosphorylated LKB1 (C). Acetylation of LKB1 (D) probed by immunoprecipitation, controls omitting acetylated lysine antibody (-Ab) or lysate (-Lysate) are shown. Total protein or tubulin signals were used for normalization. *P < 0.05, **P < 0.01 vs control (n ≥ 3).

Figure 2 -

 2 1J,K), the activating phosphorylation of DNA-PK was readily increased after DXR treatment in both of our models (Figure 2-4A,B). Notably, even though measured DNA damage did not reach significance either in isolated heart at 5 µM DXR (Figure 2-1J,K) or in the animal model (Figure 2-2I), an important phosphorylation of DNA-PK was observed (Figure 2-4A,B).

Figure 2 - 4 :

 24 Figure 2-4: Doxorubicin activates DNA-PK-Akt and MAPK pathways. Phosphorylation status (total and phosphorylated proteins) of DNA-PK-Akt (A,B) and MAPK pathways (C,D) probed by immunoblots in total homogenates from isolated perfused rat hearts (A,C) and from hearts of in vivo treated rats (B,D). Total protein, tubulin or GAPDH signals were used for normalization. *P < 0.05, **P < 0.01 vs control (n ≥ 4).

(Figure 2 -

 2 4C), and less so in the chronic model (Figure2-4D). Pro-apoptotic c-Jun N-terminal kinase (JNK) was activated in both models, in particular in the chronic model, where also apoptotic markers were detectable (Figure2-2J). Phosphorylation of the third prominent MAPKs family member, p38, was hardly detectable in the analyzed models (Figure2-4C).2.5.6. Doxorubicin activates mTOR mTOR integrates signals from the Akt, ERK and AMPK pathway and plays an important role in regulating protein biosynthesis, cellular growth and proliferation. Its activation by Akt and ERK is normally restricted by inhibitory AMPK signaling[START_REF] Zaugg | Cellular mechanisms in sympatho-modulation of the heart[END_REF] . As a reliable readout of in vivo mTOR activity, we quantified phosphorylation of the mTOR target p70S6K, which regulates mRNA translation. In both models, increased P-p70S6K revealed an activation of the mTOR pathway by DXR (Figure2-5). Activation of mTOR was also analyzed by its phosphorylation at Ser2448, which depends on active Akt, although its role for mTOR activity is not clearly established. In perfused heart at 25 µM DXR, Ser2448 phosphorylation was increased (Figure2-5A), consistent with other reports on hearts of DXR treated animals[START_REF] Holness | Regulation of pyruvate dehydrogenase complex activity by reversible phosphorylation[END_REF] . Clearly, mTOR activation is expected in a situation where negative control via AMPK is blunted and positive control via Akt and ERK is reinforced.

Figure 2 - 5

 25 Figure 2-5 Doxorubicin activates mTOR. Phosphorylation status (total and phosphorylated proteins) of mTOR and its target p70S6K probed by immunoblots in total homogenates from isolated perfused rat hearts (A) and from hearts of in vivo treated rats (B). Total protein, tubulin or GAPDH signals were used for normalization. *P < 0.05, **P < 0.01 vs control (n ≥ 3).

Figure 2 - 6 :

 26 Figure 2-6: Doxorubicin-induced combined energetic, genotoxic and oxidative stress inhibits AMPK. AMPK inhibition is at least partially due to the regulatory cross-talk with Akt and MAPK pathways, largely triggered by DNA damage signaling via DNA-PK. AMPK inhibition and related activation of mTOR (i) initiate a negative feedback that aggravates DXR-induced energy deficits and makes heart less resistant to additional energy challenge and also (ii) generate growth-stimulating signals. Red arrows indicate up-or down-regulation by DXR; red crosses indicate inhibition of signaling.

  1) amplified a 15.988-kb fragment of the mitochondrial DNA (mtDNA) and a 12.5-k fragment of the nuclear TRPM-2 gene (nDNA). The reaction mixture contained 25-75 ng template total DNA, 2.5 µl buffer 1, 200 µM dNTPs, 0.5 µM of each primer and 0.5 µl of Advantage 2 polymerase (EuroClone, Milan, Italy). Long mt and n PCR products were quantified by Sybr Green Real-Time PCR, using primers localized in the middle of the long PCR fragments: Rattus ND1 F and Rattus ND1R for mtDNA and Rattus 100F and Rattus 100R for nDNA. Quantitative Real-Time PCR was performed in a Bio-Rad iCycler iQ Multi-Color Real-Time PCR Detection System using 2×Quantitect SYBR Green PCR kit (Qiagen). The primer nucleotidic sequences and PCR parameters are given below. The quantitative PCR reaction was performed at 95°C for 10 min to activate HotStart DNA polymerase followed by 50 cycles of the two-step at 95°C for 30 s and at 60°C for 30 s. The specificity of the amplification products obtained was confirmed by examining thermal denaturation plots and by sample separation in a 3% DNA agarose gel.Results were normalized by quantitating each sample for the amount of initial genomic DNA without previous long PCR amplification, in the same real time PCR conditions, using the method described by Pfaffl.[START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF] Appropriate dilutions used were 1x10 -3 for the Long and 0.01 ng of genomic DNA. Each sample was tested in triplicate.

Figure S2- 2 :

 2 Figure S2-2: Doxorubicin does not negatively affect AMPK upstream signaling in vivo. Effects of DXR on AMPK upstream signaling were analyzed in the in vivo animal model. Protein levels of CamKKα, CamKKβ and phosphorylation status of LKB1 probed by immunoblot in total homogenates. The GAPDH signal from immunoblots was used for normalization. *P < 0.05 vs control (n = 7).

Figure S2- 3 :

 3 Figure S2-3: Doxorubicin does not increase the level of protein-HNE adducts. Protein-HNE adducts probed by immunoblot (left) in total homogenates from isolated perfused rat hearts (A; n ≥ 4) and from hearts of in vivo treated rats (B; n=7). Ponceau protein staining is given as loading control (right).

Figure S2- 4 :

 4 Figure S2-4: Doxorubicin does not change activity of protein phosphatase PP2C. Activity of serine/threonine protein phosphatase PP2C was measured by an enzymatic assay in total homogenates from isolated perfused rat hearts (n ≥ 3).

Figure S2- 5 :

 5 Figure S2-5: Doxorubicin induces phosphorylation of multiple Akt substrates. P-Akt substrates were collectively probed by immunoblot with Akt substrate motifspecific antibody (left) in total homogenates from hearts of in vivo treated rats (n=7). Ponceau protein staining is given as loading control (right). *P < 0.05, **P < 0.01 vs control.

Figure S2- 6 :

 6 Figure S2-6: Doxorubicin increases brain-type creatine kinase protein. BCK protein was probed by immunoblot in total homogenates from isolated perfused rat hearts (A; n ≥ 4) and from hearts of in vivo treated rats (B; n=7). Tubulin or GAPDH signals were used for normalization. *P < 0.05, **P < 0.01 vs control.

Figure 2 -

 2 7A,D), they rather increased in HCT8/S11 cells (Figure 2-7B,E) and MDA-MB-231 cells (Figure 2-7C,F)

Figure 2 - 7 :

 27 Figure 2-7: Effect of doxorubicin on AMPK activity in cancer cells. Phosphorylation status (total and phosphorylated proteins) of AMPK and ACC probed by immunoblots in total homogenates of HepG2 (A, D), HCT8/S11 (B, E) and MDA-MB-231 (C, F) cells treated for 1 or 12 h with different DXR concentrations as indicated. Immunobluts were quantified by using the ratio phosphoprotein/total protein. *P < 0.05 for n=3.

Figure 2 - 8 :

 28 Figure 2-8: Effect of doxorubicin on ERK nitration. Nitration status (total ERK and NO-ERK) probed by immunoblots in total homogenates from isolated perfused rat hearts. Right panel: Quantification of the immunoblot using the ratio NO-ERK/total ERK (n ≥ 4).

  Isoelectrofocusing (IEF) was performed using an Ettan IPGphor 3 (GE Healthcare, Vélizy, France) with immobiline DryStrip gels (pH 3-10 non linear) and protein sample loading (300 µg for 18 cm strips; 500 µg for 24 cm strips) with Manifold multiple strip holder and sample cups. Strips were trimmed to obtain the desired pH ranges of pH 4-9 (18 cm strips) or pH5-7,5 (24 cm strips), and applied to large standard 12% or 10% SDS-PAGE, respectively. Gels were stained for phosphoproteins (ProQ Diamond, Invitrogen, Paisley, UK) and total protein (colloidal Coomassie Roti-Blue, Carl Roth, Essen, Germany) and scanned (Typhoon9400, GE Healthcare, or Epson perfection V700 photo, Epson, France). For details see online Supporting Information. Image analysis was performed with ImageMaster 2D Platinum 7 software (GE Healthcare) and included spot detection, matching, normalization, quantification and statistical analysis with one-way ANOVA. All spots, identified by the software as differentially phosphorylated, were visually inspected to avoid artefacts.Differences relative to control with p < 0.05 (n = 4-7 biological replicates) were considered significant.Mass spectrometry and protein identificationSpots were excised from three Coomassie-stained gels, trypsin-digested, subjected to MALDI-TOF/TOF Analyzer (AB Sciex, Foster City, CA) and identified by peptide mass fingerprinting and MS/MS fragmentation, using ProteinPilot software program (v3.0, ABSciex, Foster City, CA) with Mascot (v2.0, Matrix Science, Boston, MA), including screening for Thr, Ser and Tyr phosphorylations. Mascot scores above 51 (p < 0.05) were considered significant. For details see online Supporting Information.

3. 5 . 2 .

 52 The cardiac (phospho)proteome as revealed by 2D gel electrophoresis Cardiac proteins were analyzed by 2-DE combined with sequential staining for phosphoproteins (ProQ Diamond) and total proteins (colloidal Coomassie Blue).Representative protein and phosphoprotein patterns of rat heart are shown in Figure3-1. Two different separation conditions were used in parallel to ensure both maximal coverage and high resolution. First, a wide pattern (Figure3-1A,C) was obtained by separating proteins in large range of pI(4-9) and Mr(10-250). An additional zoom pattern (Figure3-1B,D) was obtained by separation in a narrower range of pI(5-7.5) and Mr(20-100). Within each type of gel, the pattern was highly reproducible. Both protein patterns allowed the detection of about 1000 protein and 350 phosphoprotein spots. The zoom pattern gave markedly better resolution of the protein-rich region at slightly acidic pI and increased the number of detectable spots. About 925 protein and 320 phosphoprotein spots were distinguished in this region in the zoom pattern instead of respectively 415 and 150 spots distinguished in the wide pattern. The major proteins identified by MALDI-TOF/TOF included actin, tropomyosin and muscle creatine kinase (Figure3-1C-D). The cardiac protein and phosphoprotein patterns obtained were consistent with other studies, considering the number and distribution of spots

Figure 3 - 1 :

 31 Figure 3-1: Cardiac (phospho)protein patterns obtained by 2D gel electrophoresis. Representative gels of 2-DE separations of proteins from control rat hearts in a wide pattern (A,C) or zoom pattern system (B,D) followed by ProQDiamond phosphostain (A,B) and colloidal Coomassie Blue protein stain (C,D) (n ≥ 6). The wide pattern 2-DE separates in the range of pI 4-9 and Mr 10-250 (A,C), the zoom pattern 2-DE separates in the range of pI 5-7.5 and Mr 20-200 (B,D; indicated by a square in (A,C)). Major proteins spots in (C,D) were identified by MS and peptide mass fingerprinting. Note that hearts were devoid of blood due to the perfusion and thus blood cell and serum proteins like serum albumin are absent. Abbreviations: Aco, aconitase; ATP 5b, ATP synthase 5b; FABP, fatty-acid binding protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MtCK, mitochondrial creatine kinase; MCK, muscle creatine kinase; Tpm, tropomyosin.

Figure 3 - 2 :

 32 Figure 3-2: Doxorubicin-induced changes in the cardiac phosphoprotein pattern. Representative 2-DE and phosphoprotein stainings of proteins from isolated rat hearts perfused without DXR (A,D, control), with 5 µM DXR (B,E) or with 25 µM DXR (C,F), either using wide range (A-C) or zoom range conditions (D-E). Spots showing significant changes in their phosphorylation status in one of the DXRperfused conditions relative to control (p < 0.05, n ≥ 4) are labeled by number or protein name when identified by combined MS and MS/MS and color-coded: blue, decreased phosphorylation; red, increased phosphorylation. Note that cardiac troponin T is known to have multiple spots in 2-DE with different Mr 23 . For protein abbreviations and further details see Table 3-1.

Figure 3 - 3 :

 33 Figure 3-3: Proportion of identified phosphoproteins in different functional groups. Differentially phosphorylated proteins under control and DXR-perfused conditions were classified according to their biological function (UniProt database, www.uniprot.org).

Figure 3 - 4 :

 34 Figure 3-4: Phosphorylation status of selected identified phosphoproteins as verified by immunoblotting. Phosphorylation of specific sites in desmin (DES), α-crystallin B chain (CRYAB), myosin regulatory light chain 2 (MRLC2), and pyruvate dehydrogenase α subunit (PDH) was probed by immunoblots of total homogenates from isolated rat hearts perfused without (control) or with 5 or 25 µM DXR. Phosphorylation of NADH dehydrogenase 1α subunit 10 (ND10) was probed by immunoprecipitation with anti-phosphoSer/Thr/Tyr antibody and detection with total ND10 antibody; controls omitting phosphoresidue antibody (-Ab) or lysate (-Lysate) are shown. Band intensities were quantified relative to control and normalized to GAPDH (white bars) and compared to 2-DE quantification by ImageMaster Platinum (grey bars). Data are given as means ± SEM for n ≥ 4. Significant differences from control are indicated (* p < 0.05, ** p < 0.01).

at 5

 5 µM DXR, where heart function is not yet impaired, desmin phosphorylation seems to be an early event of the cardiac response to DXR. It is thus rather the cause preceding later functional changes at the macroscopic level, and not an unspecific consequence of established toxicity, thus suggesting a novel mechanism in DXR toxicity. The absence of functional impairment at the organ level may be due to compensatory regulation, such as the over 4-fold increased phosphorylation of myosin regulatory light chain (MRLC) at Ser15. MRLC phosphorylation in heart is critical for normal cardiac dynamics at baseline and in the midst of sympathetic activation, since it potentiates force production and enhances crossbridge cycling kinetics. Animals expressing a non-phosphorylable form of MRLC have depressed contractile function, reduced ejection volume and develop cardiac hypertrophy (reviewed in

Figure 3 - 5 :

 35 Figure 3-5: Localization and function of selected, differentially phosphorylated cardiac proteins affected by doxorubicin. DXR mainly affects phosphorylation status of proteins involved in energy metabolism (yellow), sarcomere structure and function (purple) and preservation of protein structure (green), thus interfering with energy status and muscle contraction. The change in phosphorylation state is indicated (blue ball, decreased phosphorylation; red ball, increased phosphorylation) together with the putative function (when functional data on suggested phosphosites is available). Pyruvate dehydrogenase (PDH) is inhibited by phosphorylation, slowing down pyruvate entry into Krebs cycle. The functional consequence of further phosphorylations of enzymes in energy metabolism may be linked to increased cytosolic glucose metabolization[START_REF] Carvalho | Metabolic remodeling associated with subchronic doxorubicin cardiomyopathy[END_REF] and also altered fatty-acid utilization[START_REF] Tokarska-Schlattner | New insights into doxorubicin-induced cardiotoxicity: the critical role of cellular energetics[END_REF] . Desmin phosphorylation leads to disorganization of desmin filaments and thus myofibrillar disarray. Conformation of desmin filaments is however protected by the activating phosphorylation of chaperone-like α-crystallin-B-chain. The activating phosphorylation of myosin regulatory light chain (MRLC) may also compensate for impaired cardiac contraction. For abbreviations see Table3-1.

  Figure 3-5: Localization and function of selected, differentially phosphorylated cardiac proteins affected by doxorubicin. DXR mainly affects phosphorylation status of proteins involved in energy metabolism (yellow), sarcomere structure and function (purple) and preservation of protein structure (green), thus interfering with energy status and muscle contraction. The change in phosphorylation state is indicated (blue ball, decreased phosphorylation; red ball, increased phosphorylation) together with the putative function (when functional data on suggested phosphosites is available). Pyruvate dehydrogenase (PDH) is inhibited by phosphorylation, slowing down pyruvate entry into Krebs cycle. The functional consequence of further phosphorylations of enzymes in energy metabolism may be linked to increased cytosolic glucose metabolization[START_REF] Carvalho | Metabolic remodeling associated with subchronic doxorubicin cardiomyopathy[END_REF] and also altered fatty-acid utilization[START_REF] Tokarska-Schlattner | New insights into doxorubicin-induced cardiotoxicity: the critical role of cellular energetics[END_REF] . Desmin phosphorylation leads to disorganization of desmin filaments and thus myofibrillar disarray. Conformation of desmin filaments is however protected by the activating phosphorylation of chaperone-like α-crystallin-B-chain. The activating phosphorylation of myosin regulatory light chain (MRLC) may also compensate for impaired cardiac contraction. For abbreviations see Table3-1.

Figure 3 -

 3 Figure 3-1S: Phosphatase activities in isolated perfused rat hearts. Phosphatase activities in rat hearts perfused without (control) or with 5 or 25 µM DXR: (A) tyrosine phosphatase, (B) S/T phosphatase 2A (PP2A) and (C) S/T phosphatase 2C (PP2C); n = 3.

  ND 1000 and Bioanalyzer 2100. Biotin-labeled cRNA samples(15 µg) were fragmented randomly to 35-200 bp at 94°C in fragmentation buffer (Affymetrix Inc., P/N 900371, Santa Clara, CA) and were mixed in 300 µl of hybridization buffer containing a hybridization Control cRNA and Control Oligo B2 (Affymetrix Inc., P/N 900454, Santa Clara, CA), 0.1 mg/ml herring sperm DNA and 0.5 mg/ml acetylated bovine serum albumin in 2-(4-morpholino)-ethane sulfonic acid (MES) buffer, pH 6.7, and hybridized to GeneChip® Rat Genome 230 2.0 arrays for 16 h at 45°C. Arrays were then washed using an Affymetrix Fluidics Station 450 EukGE-WS2v5_450 protocol. An Affymetrix GeneChip Scanner 3000 (Affymetrix Inc., Santa Clara, CA) was used to measure the fluorescent intensity emitted by the labeled target.

  From microarray analysis, 13 genes covering a wide range of fold changes (0.2-4.0) in expression were selected for verification by a two-steps real time RT-PCR: peroxisomal membrane protein 24 kDa (PMP24), tropomyosin 1, neural cell adhesion molecule 1, NADH dehydrogenase 1α subcomplex 10 (NDUFA10), opioid binding protein, muscle phosphofructokinase, carnitine palmitoyltranseferase 2, glyceraldehyde-3-phosphate dehydrogenase, cardiac adriamycin responsive protein (CARP; ankyrin domain 1), growth

Figure 4 - 1 :

 41 Figure 4-1: Effect of DXR on myocardial function in isolated perfused rat hearts. (A) Developed pressure, (B) enddiastolic pressure, (C) coronary flow, (D) heart rate, (E) +dp/dt, (F) -dp/dt. Isolated rat hearts were perfused for 120 min with Krebs-Henseleit solution (control, black dots) or Krebs-Henseleit solution containing DXR at concentrations of 2 µM (DXR, white dots) according to the Langendorff method. Data are given as mean ± SD of four hearts at each time point. Note: A 2 h perfusion with 2 µM DXR already induced some functional alterations in the heart.

Figure 4 - 2 :

 42 Figure 4-2: Comparison of quantitative data obtained by microarray analysis and real-time PCR. For a subset of genes, fold changes (FC) between DXR-treated and control hearts obtained by microarray analysis were related to those determined by real-time PCR by performing regression analysis. A linear relationship was found between the log-transformed variables (

Figure 4 - 3 :

 43 Figure 4-3: Expression of genes in DXR-perfused hearts relative to controls. (A) Distribution of fold changes (FC) in single gene analysis at a 5% false discovery rate (FDR) tolerance. (B) Number of down-and up-regulated gene sets at different p-values.

  2 and individual results of some gene sets are shown in Figure 4-4. Many of the top scoring single genes were also represented within the differentially regulated pathways, showing that the two analytic approaches are consistent and complementary.

Figure 4 - 4 :

 44 Figure 4-4: Representative gene sets showing a coordinated transcriptional response to DXR: cellular stress, cardiac remodeling and energy metabolism. (A) Representative down-regulated gene sets including the MAPK pathway, the "stress" pathway, the tumor growth factor beta (TGFβ) pathway (related to plethora of cell functions including immune response), and the LVAD heart pathway (activated in the human failing heart in response to a left ventricular assist device, LVAD). (B) Representative up-regulated gene sets including the glycolysis pathway and the Krebs (TCA) cycle. Colored squares indicate the expression level of the indicated transcripts in four control and four DXR-treated hearts. Details on enrichment score and statistical significance are given inTable 4-2.

Figure 4 - 5 :

 45 Figure 4-5: Comparison of representative transcript and protein levels. (A) For seven significantly altered transcripts, the mRNA levels determined by microarray analysis (upper panel) were compared with the corresponding protein levels (lower panel: left, Western blot; right, densitometric quantification). (B) DXR-induced changes in phosphorylation and protein levels of ERK1/2 (left, Western blot; right, densitometric qunatification). GAPDH signals were used for normalization. Data from four control and four DXR-perfused hearts are shown. Significant differences from control are indicated: *, p<0,05; **, p<0,01.

  cardiac fibers were exposed to higher DXR doses (200-500 µM) directly in the oxygraph chamber, respiratory parameters were readily affected and supported the tendencies already observed with 2 µM DXR (Figure4-6A,B). DXR at the highest dose significantly reduced the apparent K m (ADP) of respiration, indicating that tight regulation of mitochondrial permeability for ADP is lost. The most striking effects were seen with Crstimulated respiration, where Cr is used by mitochondrial creatine kinase (MtCK) to regenerate ADP within mitochondria. This process involves metabolite channeling inside a microcompartment consisting of VDAC channel (outer membrane), MtCK (intermembrane space) and adenine nucleotide translocator (inner membrane)48 . Due to the intramitochondrial ADP-regeneration in presence of Cr, K m Cr (ADP) is generally much lower than K m (ADP).However, a high DXR dose significantly increased K m Cr (ADP), indicating failure of the MtCK system. This is confirmed by direct analysis of Cr-stimulation of respiration, where a cytosolic ADP-trapping system allows only mitochondrially generated ADP directly channeled from MtCK to ANT to stimulate respiration. The apparent K m (Cr) was already more than 3-times increased at 200 µM DXR, and Cr-stimulation of respiration was even completely abolished at 500 µM DXR (Figure4-6B).

Figure 4 - 6 :

 46 Figure 4-6: Effect of DXR on mitochondrial respiration in permeabilized cardiac fibers. (A) Apparent K m for exogenous ADP in absence (left panel) or presence of Cr (middle panel), and apparent K m for Cr in presence of a cytosolic ADP-scavenging system (right panel). Respiration was analyzed in saponin-permeabilized cardiac fibers isolated from perfused hearts, either controls (white bars) or perfused with 2 µM DXR (black bars), and in cardiac fibers isolated from non-perfused hearts and treated directly in the oxygraph chamber with 200 or 500 µM DXR (hatched bars). Data are given as means ± SEM (n=3-4). (B) Representative oxygraph recordings of Cr-regulated mitochondrial respiration in cardiac fibers isolated from non-perfused hearts and incubated without (control) or with DXR (500 µM DXR) directly in oxygraph chamber. Respiration was stimulated by glutamate/malate and 2 mM ATP, the PEP/PK system was added to trap extramitochondrial ADP, and respiration was stimulated by sequential addition of Cr. Note: In presence of 500 µM DXR, the stimulatory effect of Cr is abolished.

  contrast, induction of glycolytic and Krebs cycle genes and modulation of gene expression for cardiac remodeling is rather a pro-survival response, indicating operation of fast compensatory mechanisms, which support the cardiac energetic network that is essential for heart functioning. The involved sensing and signaling mechanisms triggering the transcriptional reprogramming deserve further elucidation. The response of transcripts involved in cardiac remodeling and down-regulation of stress-response genes could represent a link between acute DXR exposures and development of chronic cardiac injury, and these pathways might serve as targets for the new cardioprotective strategies. Identification of such networks of DXR-responsive targets will facilitate further mechanistic analysis of DXR toxicity in our model system, but is also a prerequisite for future research in chronically drug--exposed animal models and the human hearts.

H 2 O

 2 ) and thermostatized at 37°C throughout the whole perfusion period. A water-filled balloon-tipped catheter was inserted into the left ventricle, and the pressure was adjusted to 4-transducer (MLT844, ADInstrument, Australia) linked to performance analyzer (Powerlab 4SP, ADInstrument, Australia) and a personal computer (Chart v4.2 software, ADInstrument, Australia). Left ventricular systolic pressure (LVSP), end-diastolic pressure (EDP), pressure first derivatives (+dP/dt and -dP/dt) and heart rate were recorded or calculated from recording data with Powerlab 4SP (ADInstruent, Australia). If needed, left ventricular end-diastolic pressure (EDP) was readjusted to 4-7 mmHg at the end of the initial 30 min period of stabilization with Krebs-Henseleit buffer. The volume of the balloon was not changed thereafter. . Coronary flow was measured manually. For each experimental group, at least four hearts were prepared, and functional parameters were recorded.After the initial 30 min period of stabilization, hearts were perfused without (control) or with DXR (DXR at 2, 5, 25 µM) during 80 (proteomics, Figure6-1) to 120 min (transcriptomics). DXR was dispensed directly into perfusion canula by a syringe pump. At the end of perfusion, hearts were rapidly freeze-clamped in liquid nitrogen and stored at -80°C or directly used.

Figure 6 - 1 :

 61 Figure 6-1: Perfusion protocol.

6. 3 .

 3 Animal treatment protocolFull understanding of disease mechanisms requires the use of relevant disease models that possibly closely reflect human pathology, i.e. the use of animal models. Animal models are also indispensable to (re)evaluate mechanisms suggested by in vitro/ex vivo studies. They are especially important when focusing on long-term effects. As stated at an International Workshop on Anthracycline Cardiotoxicity, held 2006 in Como, Italy, also fundamental research in the field of DXR cardiotoxicity needs wider application of long-term animal studies, where anthracyclines would be administered in a similar way as in clinics, i.e. intravenously 2 . Subcutaneous, intramuscular, and intraperitoneal administrations are associated with localized tissue damage that could alter drug distribution and lead to biased conclusions.

  security position with facilitated access to food. Catheterized rats were housed in individual cages.DXR(2 mg/kg; n = 14) or equivalent volume (500 µL) of saline (n = 12) were administered to animals via the implanted catheter three times a week during two weeks for a total dose of 12 mg/kg (Figure6-2). The first injection was realized on the day of catheterization. To maintain the proper functioning of the catheter, it was regularly (in the applied scheme after each injection) flushed with heparinized saline (150 µL). Over the whole experiment, regular observation of animals and weight control were carried out.Four weeks after the last injection, rats were anesthetized again as described previously and left ventricular function was measured with a Millar pressure-volume system 3 in closedchest rats with an ultraminiature catheter pressure transducer inserted into the right carotid artery and advanced downstream to the left ventricle. The catheter PR-249 was attached to control unit model TC100 (Millar Instruments, Inc., Houston, TX) and connected to a recorder (2000 series; Gould Instrument Systems/Data Sciences International, Arden Hills, MN).Subsequently, the hearts were rapidly removed, freeze-clamped in liquid nitrogen and stored at -80°C. All procedures involving animals conform to Directive 2010/63/EU and were approved by the Grenoble Ethics Committee for Animal Experimentation (Com-Eth 15_LBFA-U884-HD-01).

Figure 6 - 2 :

 62 Figure 6-2: Animal treatment protocol (time in days).

6. 4 .

 4 Cell cultureHuman hepatoma (HepG2), breast cancer (MDA-MB-231) and colorectal cancer (HCT8/S11) cell lines, kindly provided by Dr Marie-Lise Lacombe and Dr Mathieu Boissan (INSERM UMRS 938, Faculté de Médecine et Université Pierre et Marie Curie) were first stored in liquid nitrogen. After fast thawing, the vial contents were transferred into 5 mL of culture medium (DMEM, GIBCO complemented with fetal bovine serum 10%) warmed to 37°C. Then the cells were pelleted by centrifugation (1000 g, 5 min). The supernatant was discarded, and the peletted cells were gently resuspended in 10 mL of culture medium and transferred into a 75 cm 2 flask (treated CellBIND ® , Corning, The Netherlands). The flasks were incubated at 37°C, 5% CO 2 , 100% H 2 O. Once the cells had attached the flask, the medium was changed every second day. Passaging was realized when cells reached 70% of confluence. For this, the culture medium was discarded. The cells were washed three times with PBS (sterile), incubated for 2 to 20 min (depending of the cell type) with trypsin until the cells detach; then 10 mL of medium were added to inactivate trypsin. Then cell suspension was gently flushed 30 times to separate the cells, centrifuged (as previously), resuspended into 10mL of culture medium, diluted 10 times in fresh medium and transfer to a new flask.DXR and control treatments were started after the sixth passage. The cells were incubated with medium without (control) or with DXR (0.1-5 µM) during 1 or 12 hours. After the treatment, the cells were washed three times with PBS and lysed in 300 µL of RIPA buffer (NaCl 150 mM, tergicol type NP 40 1 %, deoxycholate 0.5 %, SDS 0.1 %, Tris HCl 50 mM pH 7.5). Protein concentration in lysates was assayed with a BCA protein assay kit (Pierce, USA) using BSA as a standard. Cell lysates were aliquoted and kept in -80°C.

6. 5 . 3 .

 53 Proteins transferProteins were transferred from the gel to a nitrocellulose membrane(GE Healthcare) either by wet or semi-dry blotting. Wet blotting (with cooling) was done overnight at 60 mA per gel in 0.2 M glycin, 25 mM TRIS-Base, 0.01% SDS, 20% methanol (Fisher Scientific, UK). Semi-dry blotting was done during 45 min at 120 mA per gel in 39 mM glycin, 48 mM TRIS-Base, 0.375% SDS, 20% methanol. The choice of the technique depended on the molecular weight of protein of interest: proteins with a high molecular weight were preferentially transferred using wet blotting while those with low and medium molecular weight were transferred with semi-dry blotting. Membranes were subsequently stained for minimize evaporation and prevent urea crystallization. Then strips were placed face up in Manifold track containing 108 mL of DryStrip Cover Fluid and protein samples (300 µg on 18 cm strip; 500 µg on 24 cm strip) were loaded using samples cups, at the acidic side of the strips to limit protein precipitation. Whatman pads were humidified with 150 µL of distilled water and placed at each extremity of the strips (Figure6-3). Then the electrodes were placed on top of all pads.

Figure 6 - 3 :

 63 Figure 6-3: Organization of manifold tray

6. 10 .

 10 Analysis of gene transcription 6.10.1.RNA extraction Total RNA was extracted from control and DXR-treated samples using RNAeasy Mini kit (Qiagen, Hilden, Germany). The quality of the isolated RNA was determined with a NanoDrop ND 1000 (NanoDrop Technologies, Delaware, USA) and a Bioanalyzer 2100

3 :

 3 Sequences (5'-3') of primers used for RT-PCR -GTG-TCA-CTG-CAA-AA GCA-TCT-GTG-GCC-TTT-TTC-TC Neural Cell adhesion Molecule 1 1379389_at TGG-AAC-CAT-ACT-CCA-GCA-CA CTT-CTT-CAC-CCA-GCG-ACT-TC NADH Dehydrogenase 1 aplha subcomplex 10 1389334_at ACA-ATC-CCA-AAA-GCA-ACG-AC TGT-CCT-GTG-CTC-AAG--CGG-CTC-TTG-ATG-AC AGC-TAT-GCG-CGA-GTT-TTT-GT Growth Arrest and DNA-damage-inducible 45 alpha 1368947_at GCG-AGA-ACG-ACA-TCA-ACA-TC ATG-AAT-GTG-GGT-TCG-TCA-CC Interleukin 1 alpha 1368592_at GGC-CAT-AGC-CCA-TGA-TTT-AG TGA-TGA-ACT-CCT-GCT-TGA-CG Dual Specifity Phosphatase 1 1368147_at ACA-AGA-GCA-TCC-CTG-TGG-AG GAG-GTA-AGC-AAG-GCA-GAT-GG Heat Shock 70kD protein 1 1370912_at TGC-TTC-ACC-CGG-AAA-TAC-AC CTC-CGC-TGA-TTG-TGT-GAC-TG

Table 1 - 1 :

 11 Summary of DXR-induced changes in mRNA levels identified in transcriptomics studies PAGE in a second dimension. This is still a major technique used in proteomic research due to its ability to separate the entire proteome in a two-step procedure and to allow different detection methods, e.g. for high sensitivity total protein detection (silver stain, some thousand proteins per gel) or visualization of post-translational modifications. Only some types of proteins remain difficult to separate in 2-DE. In particular in the first dimension gel, basic proteins tend to precipitate and membrane proteins often do not quantitatively enter into the gel (reviewed in85,86 ).

		Mice	Hypertensive rats	Rats
	DXR toxicity model:	Acute,	Chronic,	Acute,	Chronic,	Chronic,
		15 mg/kg 81	36 mg/kg 81	3 mg/kg 82	9 mg/kg 82	12 mg/kg 83
	Functional category of
	the encoded protein:
	Energy metabolism
	FA oxidation					
	Glycolysis					
	Oxidative phosphorylation					
	Electron transport chain
	Krebs cycle					
	Defense against stress
	Oxidative stress					
	DNA damage			
	Immun response			
	Inflammatory process				
	Other stress mechanisms.				
	Others
	Mitochondrial function					
	Apoptosis				
	Calcium handling				
	Muscle contraction/				
	cardiac remodelling

: Expression of most genes in this category is up-regulated : Expression of most genes in this category is up-regulated, while a minority is down-regulated : Expression of most genes in this category is down-regulated : Expression of most genes in this category is down-regulated, while a minority is up-regulated

1.3.2. Proteomics

Proteomics is the study of all protein variants synthesized in a given cell. This is complementary to transcriptomics, since mRNA and protein abundance are not necessarily correlated. In addition, as compared to transcripts, proteins have a much-increased diversity due to maturation and degradation processes, and are the cellular actors that mediate cellular structure and function. Powerful analytical tools have been developed for proteomics, which involve separation, identification and quantification of a large number of proteins from a single sample. 2D electrophoresis (2-DE) is still one of the main techniques in proteomics. It allows an immediate readout of the overall protein pattern and can be accommodated in standard biochemical laboratories. Proteins are separated according to their surface charge (isoelectric point, pI) in a first dimension by isoelectrofocusing, and according to their size by SDS-reproducibility, good resolution power and high sensitivity. To overcome persisting limitations due to inter-gel variations, the difference gel electrophoresis (DIGE) has been developed. Two different samples are labeled with different fluorescent dyes and separated on the same gel. The gel is then scanned by exciting both fluorophores, yielding two perfectly superimposable images for exact quantitative comparison between both samples (reviewed in 87

Table 1 - 2 :

 12 Summary of protein functions affected by DXR as shown by proteomics studies

		Mice		Rats
	DXR toxicity model: Proteins involved in:	Acute, 15 mg/kg 92	Acute, 20 mg/kg 90	0,5 µM, 48h 93	Chronic, 20 mg/kg 93
	Energy metabolism
	FA metabolism

Table 1 -3: Glossary

 1 of selected proteins in cell signaling mentioned in this thesis.

.5. Aim of the work

	1The aim of this thesis work is to provide new molecular insight into mechanisms involved
	in DXR-cardiotoxicity, in particular into potential mechanisms by which the drug disturbs
	cardiac cell signaling. Understanding these mechanisms is a first step towards novel
	preventive or curative treatments of cardiotoxicity that preserve the efficient anti-cancer
	activity of the drug.
	•		•
	• 4E-BP1 We use two experimental models of DXR cardiotoxicity (Chapter 2.). Acute toxicity is • eukaryotic translation inhibition factor 4E-binding protein 1: 121 • anti-apoptotic factor while unphosphorylated studied by using Langendorff-perfusion of rat hearts with clinically relevant DXR doses and
	• ACC with higher DXR doses mimicking cumulative effects. For the study of chronic drug toxicity, • acetyl CoA carboxylase: • biogenesis of fatty acids 122
	an in vivo model is established in form of DXR-treated rats. Both models are thus • Akt / PKB • protein kinase B: 108 • critical signaling node, pro-survival factor, participating in growth and proliferation complementary, describing either very early effects or effects that persist weeks after
	• AMPK completion of a chronic treatment. In the experimental sections, two different experimental • AMP-activated protein kinase: • critical signaling node, key regulator of energy homeostasis by controlling ATP 55 supply and consumption, growth and proliferation approaches are used: a targeted approach, focusing on activation of specific signaling
	• BAD pathways, and two non-biased "omics" approaches, focusing on the cardiac phosphoproteome • Blc2 antagonist of cell death: • promotes cell death 123
	• CamKK and transcriptome to potentially identify so far unrecognized DXR targets. • calmodulin kinase kinase:
		• involved in many cellular processes like in the calcium/calmodulin-dependent (CaM) kinase cascade, or in AMPK regulation 124 The first experimental section (Chapter 2.) will focus on some signaling pathways in
	stress-sensing, energy metabolism and proliferation. It is based on an earlier finding of our • DNA-PK • DNA dependent protein kinase: 125 • senses and signals DNA double-strand breaks group, the inhibition of AMPK signaling by DXR 50 . This study aims to confirm AMPK
	• ERK 1/2 inhibition in the two different experimental models and to find some clues on the involved • extracellular signal-regulated kinases 1 and 2: • members of the MAPK family, regulators of cell cycle progression, proliferation, 105 transcription, differentiation, senescence, cell death, migration and cell adhesion molecular mechanisms.
	• GSK	• glycogen synthase kinase : • regulator of glycogen synthase, pro-apototic factor The second experimental section (Chapter 3.) is a non-biased study of the cardiac 126
	• JNK / SAPK • c-Jun NH2-terminal kinase: phosphoproteome, a novel approach in the field. The appropriate experimental approach
	• member of the MAPK family, regulator of cell proliferation, differentiation, apoptosis, cell survival and cell mobility 105 including 2-DE and image analysis has to be established with the aim to identify specific,
	• LKB1 early changes in protein phosphorylation that are involved in DXR cardiotoxicity. • Ser/Thr protein kinase 11: 127 • tumor suppressor, AMPK kinase, involved e.g. in metabolic regulation The last experimental section (Chapter 4.) is a non-biased, genome-wide transcriptomic
	• MAPK study in order to identify genes that are specifically and strongly regulated by DXR, since • mitogen activated protein kinases: • includes ERK1/2, JNK/SAPK and p38
	• mTOR DXR is known to affect cardiac gene expression. • mammalian target of rapamycin: • critical signaling node, regulates cell growth and proliferation	128
	• p70S6K	• ribosomal protein S6 kinase, 70kD: • regulator of protein synthesis, cell proliferation, cell growth and cell survival	129
	• PARP	• poly-ADP-ribose polymerase: • trigger of DNA strand break repair	130
	• SIRT1	• sirtuin 1:
			• deacetylase, tumour suppressor, with protective functions in metabolic syndrome, cardiovascular diseases and neurodegeneration 131
	•		•

Chapter 2. Inhibition of AMPK signaling by doxorubicin - at the crossroads of the cardiac responses to energetic, oxidative and genotoxic stress

	2.1. Abstract
	Cardiotoxic side-effects of anthracyclines, the most widely used anticancer drugs, are
	well documented, while involved mechanisms are not fully elucidated. The cellular energy
	sensor and regulator AMP-activated protein kinase (AMPK) was suggested as a putative
	mediator of cardiotoxicity of doxorubicin, the leading anthracycline drug, by our earlier work.
	Here we study the interference of doxorubicin with AMPK signaling and potentially involved
	mechanisms. Effects of doxorubicin on cell signaling are studied in isolated Langendorff-
	perfused Wistar rat hearts and in hearts from doxorubicin-treated Wistar rats. In both models,
	doxorubicin induces energetic, oxidative and genotoxic stress. Despite energy depletion and
	unaffected AMPK upstream signaling, doxorubicin does not activate the AMPK pathway and
	even reduces basal phosphorylation of AMPK and its downstream target acetyl-CoA
	carboxylase. By contrast, oxidative and genotoxic stress do well activate pro-survival MAPK
	and Akt pathways, the latter via DNA-PK activation triggered by DNA damage. Combined
	inhibition of AMPK and activation of Akt and MAPKs leads to activation of growth-
	stimulating mTOR signaling. Our results suggest that in doxorubicin-challenged heart, a
	combined energetic, oxidative and genotoxic stress elicits a specific, hierarchical response
	where AMPK is inhibited at least partially by the known negative cross-talk with Akt and
	MAPK pathways, largely triggered by DNA damage signaling. Though such signaling can be
	protective, e.g. by limiting apoptosis, it primarily induces a negative feedback that increases
	cellular energy deficits, and via activation of mTOR signaling, it also contributes to the
	Séverine Gratia 1,2 , Laurence Kay 1,2 , Lucia Potenza 3 , Amal Seffouh 1,2 , Valérie Novel-pathological cardiac phenotype in chronic doxorubicin toxicity.
	Chaté 1,2 , Coralie Schnebelen 1,2 , Piero Sestili 3 , Uwe Schlattner 1,2 , and Malgorzata
	Tokarska-Schlattner 1,2
	1 University Joseph Fourier, Laboratory of Fundamental and Applied Bioenergetics,
	Grenoble, France; 2 Inserm, U1055, Grenoble, France. 3 Dipartimento di Scienze
	Biomolecolari, Università degli Studi di Urbino "Carlo Bo", Urbino, Italy
	Running head: Inhibition of AMPK signaling by doxorubicin
	Keywords: Adenosine 5'-monophosphate-activated protein kinase; anthracyclines; cardiac
	energetics; cardiac signaling; cardiotoxicity.
	Status: Under revision for Cardiovascular Research

  ), breast cancer (MDA-MB-231) and colorectal cancer (HCT8/S11) cell lines, kindly provided by Dr Marie-Lise Lacombe (INSERM UMRS 938,

	Faculté de Médecine et Université Pierre et Marie Curie) were cultivated with DMEM
	(GIBCO) complemented with fetal bovine serum 10% until 70% confluence and then treated
	without or with DXR (0.1-5 µM) during 1 or 12 hours. Then cells were lysed in RIPA buffer
	(NaCl 150 mM, tergicol type NP 40 1 %, deoxycholate 0.5 %, SDS 0.1 %, Tris HCl 50 mM
	pH 7.5) and proteins were assayed using a BCA protein assay kit (Pierce, USA).

  Doxorubicin (DXR) is one of the most efficient drugs against several types of solid cancer, leukemia and lymphomas (reviewed in 1 ). However, its clinical use is limited by severe cardiotoxicity and the lack of adequate preventive treatment. An acute toxicity, which occurs immediately after treatment, causes transient arrhythmias, reversible hypotension, and pericarditis. The chronic toxicity, which occurs months to years after the treatment, causes chronic cardiomyopathy and congestive heart failure (reviewed in 2 ). Many hypotheses have been proposed to explain the cardiotoxic side-effects, but none of them is fully satisfying

	3.2. Résumé
	3.3. Introduction
	Le succès de l'utilisation de la DXR en chimiothérapie est limité par ses effets
	cardiotoxiques très prononcés. Malgré des décennies d'utilisation clinique, aucun modèle
	complet et satisfaisant des mécanismes moléculaires impliqués dans cette cardiotoxicité n'a
	été décrit, et ainsi aucun traitement cardioprotecteur efficace n'a été établi. Dans cette étude, à
	l'aide d'une analyse systémique du phosphoprotéome cardiaque, nous étudions les
	changements de la signalisation cellulaire induits par la DXR dans le modèle de coeur isolé et
	perfusé, avec des doses de doxorubicine cliniques (5 µM) ou supérieures (25 µM). Nous
	combinons les techniques de gels d'électrophorèse en deux dimensions, coloration
	phosphospécifique, analyse d'images quantitative et spectrométrie de masse par MALDI-

Overrepresentation of mitochondrial proteins (>40%) identified this compartment as a prime target of doxorubicin. Identified proteins were mainly involved in energy metabolism (e.g. pyruvate dehydrogenase, α-enolase, acyl-CoA dehydrogenase), sarcomere structure and function (e.g. desmin) or chaperone-like activities (e.g. α-crystallin B chain, prohibitin). Increased phosphorylation of pyruvate dehydrogenase, regulating pyruvate entry into the Krebs cycle, and desmin, maintaining myofibrillar array, are relevant for main symptoms of cardiac dysfunction that develop during doxorubicin treatment, namely energy imbalance and myofibrillar disorganization. TOF/TOF pour detecter les évènements de (dé)phosphorylation de protéines. Cette approche nous a permis d'identifier 22 protéines dont le niveau de phosphorylation varie significativement sous l'action du médicament, ce qui pourrait partiellement expliquer la toxicité de la DXR. Pour certains sites de phosphorylation sélectionnés, ces résultats ont été validés par immunoblot. Le grand nombre de protéines mitochondriales (> 40%) affectées montre que cette organelle est une cible majeure du médicament. Les protéines identifiées sont, pour la plupart, impliquées dans le métabolisme énergétique (pyruvate déshydrogénase, α-enolase, acétyl-CoA déshydrogénase), dans la fonction et la structure du sarcomère (desmine, chaine B de l'α-crystaline) ou sont des protéines chaperonnes (chaine B de l'αcrystaline, prohibitine). La phosphorylation de la pyruvate déshydrogénase, contrôlant l'entrée du pyruvate dans le cycle de Krebs, et celle de la desmine, maintenant le réseau de myofibrille, sont augmentées. Ces deux modifications sont en accord avec les principaux symptômes de la cardiotoxicité induite par la DXR : le déséquilibre énergétique et la désorganisation des myofibrilles.

Table 3 - 1 :

 31 Alphabetical list of identified, differentially phosphorylated cardiac proteins ) UniProt (www.uniprot.org) accession number; b) abbreviation used in Figure3-2; c) pI and Mr calculated (www.uniprot.org) and observed (2-DE); d) number of peptides and sequence coverage in MALDI-TOF/TOF MS; e) MASCOT score > 51 is significant (p < 0.05); f) phosphosites suggested by MS analysis or verified by immunoblot (see Figure3-4); g) in italic type: sites found only in databases (www.phosphosite.org); h) synonym: alcohol dehydrogenase class-3; i) synonym: myosin light chain 7, regulatory. su, subunit; j) presence of different splice variants.[START_REF] Mesnard-Rouiller | Troponin T mRNA and protein isoforms in the human left ventricle: pattern of expression in failing and control hearts[END_REF]

	Accession	Annotation	Abbr. b) pI	Mr	pI	Mr	No. of	Co-	MS	Phosphosites	Immuno	Known phosphosites
	no. a)			calc.	calc.	obs.	obs.	pep-	verage	score	suggested by	-blot f)	from literature and databases g)
				c)	c)	c)	c)	tides d)	(%) d)	e)	MS f)
	P15650	Acyl-CoA dehydrogenase (long-chain specific) LCAD	6.3	45	7.1	38	41	50	269 S		S54,S55,S191,S193,S200,S362
	P08503	Acyl-CoA dehydrogenase (medium-chain spec.) MCAD	7.7	44	7.9	45	26	37	297 Y		S70,T351
	P15651	Acyl-CoA dehydrogenase (short-chain specific) SCAD	6.4	42	7.7	40	35	58	543 S,T		T27,T305
	P12711	Alcohol dehydrogenase 2 h)	ADH	7.6	39	6.9	36	8	25	79 -		T127,S128,S247,T303
	P23928	α-crystallin B chain	CRYAB 6.8	20	7.6	20	19	61	189 S	S59	S19,S45,S59
	P48675	Desmin	DES	5.2	55	5.2	60	40	68	716 S,T	T17	S12,S13,T17,S45,S48,S60,S68,T76,T77,S81
	Q07803 Elongation factor G (mitochondrial)	EF-Gmt	6.4	83	6.3	75	18	26	215 S,T,Y		S92,S276
	P85834	Elongation factor Tu (mitochondrial)	EF-Tu	6.2	45	6.8	40	43	60 1180 -		T273,S312
	P04764	α-enolase	NNE	6.2	47	7.0	45	32	51	544 -		Y44
	P15429	β-enolase	MSE	7.1	47	6.9	46	25	45	271 S,T		S37,S40,T41,Y44,S83,Y131,S263,S362,T390
	P14604	Enoyl-CoA hydratase 1	ECH	6.5	28	7.3	26	15	43	300 T		S45,S118,T194
	P42123	L-lactate dehydrogenase B chain	LDH-B	5.7	36	6.0	35	19	34	355 S,T,Y		S44,Y240,Y248,T249
	B1WC41 Myosin regulatory light chain 2, atrial i)	MRLCa	4.4	17	4.3	21	15	49	353 S	S15	S15,S41
	Q561S0 NADH dehydrogenase 1 α subcomplex su 10	ND10	6.0	37	6.8	34	35	74	590 -	S/T/Y	T,S59,S231
	Q641Y2 NADH dehydrogenase iron-sulfur protein 2	ND2	6.0	49	6.5	42	18	33	83 -		T258
	P38652	Phosphoglucomutase 1	PGM1	6.3	61	6.9	60	35	56	329 S,T,Y		T19,T115,S117,Y353,S369,S505,T507
	P67779	Prohibitin	PHB	5.6	30	5.6	27	26	62	809 -		S101,Y114,S121,Y249,T258,Y259
	P26284	Pyruvate dehydrogenase E1 su α	PDH	6.8	40	7.7	38	44	61	816 S	S293	Y227,T231,S232,Y289,S293,S295,S300,Y301,
	Q8VIF7 Selenium-binding protein 1	SBP	6.1	53	6.7	50	17	34	60 S,T,Y		Y12,S111,Y335,S422,S467
	P62138	Serine-threonine protein phosphatase PP1 α	PP-1A	5.9	38	6.1	35	16	50	103 S,T		S2,S4,S11,S22,T320,S325
	Q6P502 T-complex protein 1 su γ	TCP-1	6.2	61	7.1	65	29	36	256 S,T		S11,T14,S24,T60,S170,T225,S244,S252
	P50753	Troponin T, cardiac	TnTc	5.0-	30-	5.2	27-	22	36	609 S,T		S2,S191,T205,S209,T214,S286,T295
				5.2	36 j)		38

a

Table 3

 3

-1.

Table 3 - 2 :

 32 Doxorubicin-induced changes in phosphorylation levels of identified cardiac proteins

	Annotation	Abbr.	Accession Spot	5 µM DXR	25 µM DXR
		a)	no. b)	no. c)	FC d)	p e)	FC d)	p e)
	Energy metabolism
	Acyl-CoA dehydrogenase (medium-chain spec.) MCAD	P08503	24_196 0.20	0.036	0.38	0.209
	Acyl-CoA dehydrogenase (short-chain specific ) SCAD	P15651	24_194 0.32	0.049	0.27	0.065
			P15651	18_80	0.24	0.011	0.57	0.339
	Alcohol dehydrogenase 2	ADH	P12711	24_191 0.28	0.015	0.49	0.164
	α-enolase	NNE	P04764	18_104 0.59	0.045	0.81	0.232
	L-lactate dehydrogenase B chain	LDH-B	P42123	18_72	0.48	0.098	0.39	0.039
	NADH dehydrogenase 1 α subcomplex su 10	ND10	Q561S0 24_43	0.30	0.030	0.54	0.133
	NADH dehydrogenase iron-sulfur protein 2	ND2	Q641Y2 18_95	0.38	0.003	0.75	0.368
	Phosphoglucomutase 1	PGM1	P38652	18_117 0.51	0.013	0.52	0.056
	Acyl-CoA dehydrogenase (long-chain specific)	LCAD	P15650	24_63	0.59	0.180	1.71	0.009
	β-enolase	MSE	P15429	24_82	1.12	0.681	1.86	0.034
	Enoyl-CoA hydratase 1	ECH	P14604	18_208 0.47	0.185	3.06	0.042
	Pyruvate dehydrogenase E1 su α	PDH	P26284	24_51	0.94	0.918	3.73	0.004
	Sarcomeric structure and function
	Troponin T, cardiac	TnTc	P50753	18_340 < 0.1	0.005	0.26	0.098
			P50753	18_8	1.14	0.822	0.18	0.021
			P50753	24_327 0.93	0.802	0.42	0.046
	Myosin regulatory light chain 2, atrial	MRLCa	B1WC41 18_341 4.53	0.049	1.32	0.519
	Desmin	DES	P48675	24_330 4.05	0.034	1.31	0.674
	Desmin	DES	P48675	24-331 3.05	0.043	2.70	0.100
	Protein biosynthesis, chaperones
	Elongation factor G (mitochondrial)	EF-Gmt	Q07803 18_125 0.45	0.024	0.50	0.023
			Q07803 24_136 0.45	0.020	0.71	0.053
	Elongation factor Tu (mitochondrial)	EF-Tu	P85834	24_74	0.32	0.025	1.06	0.908
	T-complex protein 1su γ	TCP-1	Q6P502 18_178 0.11	0.044	0.36	0.262
	α-crystallin B chain	CRYAB P23928	18_2	1.70	0.001	1.66	0.055
	Prohibitin	PHB	P67779	24_12	3.78	0.049	1.19	0.753
	Others
	Selenium-binding protein 1	SBP	Q8VIF7 24_100 0.64	0.123	1.61	0.027
	S/T protein phosphatase PP1 α	PP-1A	P62138	24_37	1.30	0.402	1.72	0.003
	Selected spots meet significance criterion (p < 0.05, DXR vs. control) for at least one condition: 5 or 25 µM

DXR; blue numbers, decreased phosphorylation; red numbers, increased phosphorylation; bold numbers, p < 0.05; normal numbers, p < 0.1. a) Short abbreviation of protein name; b) UniProt accession number; c) number encoding IEF strip type (18 or 20 cm) and ImageMaster spot number; d) fold change relative to control (DXR value divided by control value); e) probability obtained by ANOVA test with n ≥ 4 for each group. su, subunit.

  MS/MS fragment tolerance set to 0.1 Da, and charge set to +1. Identification was assigned to a protein spot feature if (i) at least two MS/MS spectra with ion scores >20 and MASCOT identify were assigned; (ii) sequence coverage was at least 25% in cases where only one MS/MS spectrum was available. Moreover, the protein score calculated must be greater than 50, correlating to a confidence interval greater than 95%.

	Immunoblotting
	50 µg proteins per lane were separated on standard 12% polyacrylamide gels and
	transferred to Hybond ECL nitrocellulose membranes (GE Healthcare, Buckinghamshire,
	UK) by wet blotting (90 min at 75 mA per gel, in 0.2 M glycin, 25 mM TRIS-Base, 0.01%
	SDS, 20% methanol) or semi-dry blotting (60 min at 120 mA per gel in 39 mM glycin, 48
	mM TRIS-Base, 0.375% SDS, 20% methanol). After transfer, membranes were stained with
	Ponceau-Red to check gel loading and the quality of transfer. Subsequently, membranes were
	blocked (2 h at room temperature in 0.15 M NaCl, 20 mM TRIS-Base pH 7.5, 0.1% v/v
	phosphoαB crystalline (Ser59; 1:1000); from Santa Cruz Biotechnology (Santa Cruz, CA)
	anti-MYL7 (1:250) and anti-GAPDH (1:250). Antibody anti-phosphoMLC2 (Ser15) was
	made and kindly provided by Dr. ND Epstein (Laboratory of Molecular Cardiology, NHLBI,
	Bethesda).
	(UniProtKB, release 2011-06 (May 31, 2011) 529056 sequences entries), resulting in a set of

trypsic peptides matched with confidence values. The Mascot searches were run using the following parameters: methionine oxidation, phosphorylations (Thr, Ser, Tyr) as variable and cysteine carbamidomethylation as fixed; one missed cleavages allowed; precursor tolerance at 50 ppm, Tween20, 5% w/v skimmed milk), incubated with primary antibodies (overnight at 4°C) and after washing with horseradish peroxidase-conjugated secondary antibodies (1 h at room temperature). Immunoreactive protein bands were detected using a chemiluminescence kit (ECL plus, GE Healthcare, Buckinghamshire, UK) and an ImagerQuant LAS 4000 camera (GE Healthcare, Buckinghamshire, UK). Band intensities were quantified with ImageQuantTL (GE Healthcare) using the GAPDH signal for normalization. Statistical significance was evaluated using Student T-test. Data are given as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001. Following primary antibodies were used: from Abgent (San Diego, CA) anti-phosphodesmin (Thr16; 1:500); from Assay Designs (Ann Arbor, MI) anti-

  -1S: Changes in cardiac functional parameters in response to 80 min perfusion with doxorubicin LVDP, left ventricular developed pressure; LVEDP, left ventricular end-diastolic pressure; CF, coronary flow; HR, heart rate; RPP, rate pressure product. Data are given as means ± SD, ***p < 0.001 vs. control, n= 4 to 7.

		LVDP	LVEDP	+dP/dt	-dP/dt	CF	HR	RPP
		(mmHg)	(mmHg)	(mmHg/s)	(mmHg/s)	(ml/min)	(beats/min)	(mmHg/min)
	Control	141 ± 19 5.8 ± 4.5	2732 ± 366	2012 ± 278	14.3 ± 2.8	254 ± 23	36191 ± 6788
	DXR 5 µM	153 ± 18 8.3 ±3.2	2981 ± 296	2129 ± 145	13.1 ± 0.9	248 ± 17	38063 ± 4164
	DXR 25 µM	53 ± 6 *** 24.6 ± 8.9 ***	988 ± 112 ***	584 ± 87 *** 12.8 ± 1.8	240 ± 30	12857 ± 2692 ***

Table 3 -

 3 2S: Localization, biological function and phosphorylation sites of identified proteins

	Protein	a	Local. b Protein function	Function of phosphorylation(s)	Upstream kinase(s)
	Energy metabolism
	Acyl-CoA dehydrogenase (medium-chain sp.)  Mt-Mx Lipid (C4 to 16) metabolism, β-oxidation	Increase of protein solubility 45	-
	Acyl-CoA dehydrogenase (short-chain spec.)  Mt-Mx Lipid (C2 to C3) metabolism, β-oxidation	-	-
	Alcohol dehydrogenase 2	 C	Long-chain primary alcohol oxidation	-	-
	α-enolase	 C, CM Glycolysis, gluconeogenesis, hypoxia and ischemia tolerance	Inhibition 46	pPKL12 47 , cABL 48
	L-lactate dehydrogenase B chain	 C	Fermentation of pyruvate to lactate and reverse	-	-
	NADH dehydrogenase 1 α subcomplex su 10  Mt-Mx Respiratory chain (complex 1, accessory subunit)	-	-
	NADH dehydrogenase iron-sulfur protein 2  Mt-IM Respiratory chain (complex 1, essential subunit)	-	-
	Phosphoglucomutase 1	 C	Breakdown and synthesis of glucose	-	CamKII 49
	Acyl-CoA dehydrogenase (long-chain spec.)  Mt-Mx Lipid (C12 to C16) metabolism, β-oxidation	-	-
	β-enolase	 C	Glycolysis, development and regeneration of striated muscle	-	-
	Enoyl-coA hydratase 1	 Mt-Mx Lipid (C4 to C16) metabolism, β-oxidation	-	-
	Pyruvate dehydrogenase E1 su α	 Mt-Mx Regulates the use of pyruvate to fuel the mitochondrial TCA	Inactivation (S293) 29	PDK 50
				cycle
	Sarcomeric structure and function
	Troponin T, cardiac	 CS	Muscle contraction, tropomyosin-binding subunit of troponin	Decrease contractile activity 51	TnTc kinase, PKC,
						ASK1 52,51
	Myosin regulatory light chain 2, atrial	 CS	Muscle contraction, regulatory subunit of myosin	Increase calcium sensibility and myocardium	MLCK 53
					force (S15) 42,53
	Desmin	 C, CS Connection of myofribrils to each other and to membrane,	Desorganization of desmin filament and of
				maintaining cell integrity and myocyte contraction	myofibrils (S12,S13,T17,S45,S48,S60,S68,
					T76,T77,S81)

Table 4 - 1 :

 41 DXR-induced changes in the expression of single genes identified by Significance Analysis of Microarrays (SAM). Well annotated genes whose expression changes by at least 50% are listed according to their general function.

	Gene Name	Gene Abbr.	Gene ID	FC	q-value	local
					(%)	FDR
	Stress / early response					(%)
	Growth arrest and DNA-damage-inducible 45 γ	GADD45G	1388792_at	0,09	0,00	0,00
	Heat shock 70kDa protein 1B *	HSPA1B	1368247_at	0,20	0,00	0,00
	Immediate early response 5	IER5	1389355_at	0,33	0,00	0,00
	Immediate early response 3	IER3, IEX1	1388587_at	0,38	0,00	0,00
	Growth arrest and DNA-damage-inducible 45 α	GADD45A	1368947_at	0,46	0,00	0,00
	Growth arrest and DNA-damage-inducible 45 β	GADD45B	1372016_at	0,49	0,00	0,00
	Antioxidant defense
	Heme oxygenase (decycling) 1	HMOX1	1370080_at	0,45	0,00	0,00
	Glutathione S-transferase M4 *	GSTM4	1375909_at	0,48	0,00	0,00
	Glutamate-cysteine ligase, catalytic subunit	GCLC	1372523_at	0,49	0,00	0,00
	DNA / RNA synthesis / stability / repair
	Kelch repeat and BTB (POZ) domain containing 5	KBTBD5	1395851_at	0,10	0,00	0,00
	Histone cluster 1, H2bh *	HIST1H2BH	1374832_at	0,12	0,00	0,10
	Histone cluster 2, H2aa *	HIST2H2AAA 1371959_at	0,29	0,00	0,03
	TCDD-inducible poly(ADP-ribose) polymerase	TIPARP	1374446_at	0,39	0,00	0,00
	Histone cluster 2, H4a *	HIST2H4A	1392476_at	0,41	0,00	0,00
	polymerase (RNA) II (DNA directed) polypeptide A	POLR2A	1382976_at	2,02	0,73	9,74
	polymerase (DNA directed), α 2	POLA2	1387795_at	1,56	0,73	14,07
	Nibrin	NBN, NBS1	1387977_at	1,51	1,85	21,14
	Immune response
	Selectin E * (endothelial cell)	SELE	1388018_at	0,24	0,00	0,00
	Chemokine (C-C motif) ligand 3	CCL3	1369815_at	0,29	0,00	0,00
	Tumor necrosis factor receptor superfamily, member 12a TNFRSF12A	1371785_at	0,31	0,00	0,00
	Interferon-related developmental regulator 1	IFRD1	1367795_at	0,32	0,00	0,00
	Interleukin-1 receptor-associated kinase 2	IRAK2	1383474_at	0,34	0,00	0,00
	Chemokine (C-C motif) ligand 4 *	CCL4	1370832_at	0,36	0,00	0,00
	Tumor necrosis factor superfamily, member 2 (α)	TNF	1387691_at	0,37	0,00	0,00
	Chemokine (C-C motif) ligand 7	CCL7	1379935_at	0,42	0,00	1,90
	Interleukin 1, α	IL1A	1368592_at	0,42	0,00	0,11
	RT1 class I, CE5 (MHC class Ib antigen)	RT1-CE5	1371209_at	2,23	0,41	6,83
	Granzyme C	GZMC	1368377_at	2,03	0,24	4,27
	Chemokine (C-X-C motif) ligand 9	CCL9	1382454_at	1,92	4,43	31,06
	Granzyme B	GZMB	1370628_at	1,72	0,24	5,76
	Chemokine (C-X-C motif) ligand 12	CCL12	1388583_at	1,62	1,54	19,63
	Neural cell adhesion molecule 1	NCAM1	1379389_at	1,61	1,54	17,31
	CD69 antigen	CD69	1384837_at	1,51	2,34	22,84
	Cardiac pathology / remodeling
	LIM and cysteine-rich domains 1 (dyxin)	LMCD1	1376632_at	0,15	0,00	0,10
	Xin actin-binding repeat containing 1 *	XIRP1	1389119_at	0,24	0,00	0,00
	Cbp/p300-interacting transactivator 2	CITED2	1367602_at	0,40	0,00	0,00
	Natriuretic peptide precursor type B	NPPB	1367616_at	0,49	0,44	15,72
	Osteoglycin	OGN	1383263_at	2,02	1,85	21,41
	Myofibrillar / cytoskeletal proteins
	Filamin C, gamma (actin binding protein 280)	FLNC	1388496_at	0,27	0,00	0,00
	Titin immunoglobulin domain protein (myotilin)	MYOT	1392950_at	0,43	0,00	0,00
	Tropomyosin 1 (α)	TPM1	1395794_at	2,07	1,54	17,76
	Dynein, axonemal, heavy polypeptide 7	DNAH7	1383943_at	1,86	0,00	3,21
	Metabolism
	Aldehyde dehydrogenase family 1, member A1	ALDH1A1	1387022_at	1,77	0,73	11,06
	Electron-transfer-flavoprotein, beta polypeptide	ETFB	1381840_at	1,67	3,57	28,39

Table 4 - 2 :

 42 DXR-induced changes in the expression of selected gene sets as identified by Gene Set Enrichment Analysis (GSEA). For details on representative gene sets seeFigure 4-4.

	GSEA pathway

Table 4 -

 4 2.

Table 4 - 3 :

 43 Energy status and mitochondrial function after DXR-perfusion.

	control	DXR	p

  . It increases the intracellular creatine pool and thus, via the CK reaction, generates energy reserves in form of PCr (see Chapter 1.2.2.). The PCr can then be used by the cell in situations of energy deficits, again via the reversible creatine kinase reaction, to regenerate ATP, which could be very beneficial in DXR carditoxicity. Indeed, creatine-supplementation in rats treated with high DXR doses decreased various parameters of DXR cytotoxicity 42 . Thus, nutritional creatine supplementation may trigger protective mechanisms against DXR toxicity. It would be interesting to further investigate this effect more mechanistically and with clinically relevant doses of DXR, e.g. in perfused rat heart from Cr-fed animals.

. Contexte et but du projet de thèse

  This thesis work provided new insight into potential mechanisms of DXR-induced cardiotoxicity. While the main molecular phenotypes of DXR cardiotoxicity are known, our study identified interesting novel signaling elements as part of potentially important toxicity mechanisms. Moreover, the work suggests targets that may lead to novel protective strategies.La DXR est l'un des médicaments les plus efficaces dans le traitement de différents types de cancers, mais son application clinique est limitée par ses effets cardiotoxiques. Malgré des décennies de recherche, la cardiotoxicité de la DXR reste un enjeu pour la santé publique, les mécanismes sous-jacents ne sont pas entièrement compris et aucun traitement satisfaisant, curatif ou préventif, n'existe à ce jour. Cependant, il est désormais établi qu'il s'agit d'une pathologie complexe, qui implique des mécanismes pléiotropiques. Au niveau cellulaire, l'induction du stress oxydant et du déficit énergétique sont les principales caractéristiques des effets de la DXR. Ceci est lié, entre autres, aux effets délétères sur le système créatine kinase (CK) 1 et, comme notre équipe l'a montré récemment, sur le senseur et le régulateur clé de la signalisation énergétique, l'AMPK 2 . L'inhibition de cette dernière est paradoxale : la kinase devrait en effet être activée en cas de stress énergétique. Ainsi, outre les dommages oxydatifs et le stress énergétique qu'elle induit, la DXR pourrait interférer spécifiquement avec la signalisation cellulaire, amorçant ainsi les phénotypes aigu et chronique, typiques de la toxicité du médicament.Ces résultats laissent supposer des changements majeurs, induits par la DXR, dans le réseau de la signalisation cellulaire. Ainsi, notre dernier objectif a été d'étudier la signalisation cellulaire à l'aide d'approches non biaisées. Nous avons alors utilisé le modèle de toxicité aiguë afin de mettre en évidence les événements précoces induits en réponse à la DXR, avant que les altérations fonctionnelles physiologiques soient détectables. Les études systémiques sont appropriées pour identifier de nouvelles cibles et les mécanismes à l'oeuvre dans des pathologies complexes telles que la cardiotoxicité induite par la DXR. La première étude a consisté à cribler le phosphoprotéome cardiaque afin d'identifier directement les événements de (dé)phosphorylation liés à l'altération de la signalisation cellulaire (l'activité des kinases et des phosphatases) par le médicament. Cette approche phosphoprotéomique n'avait encore jamais été utilisée dans ce contexte (Chapitre 3.). La seconde étude systémique a eu pour objectif d'étudier les modifications du transcriptome cardiaque induites par la DXR (Chapitre 4.) afin d'analyser les changements d'expression génique. Ces deux approches ont mis en évidence de nouvelles cibles cellulaires de la DXR en plus de celles déjà connues, La toxicité chronique est étudiée chez le rat recevant des injections de DXR par intraveineuse, à des doses cumulatives correspondant aux doses cliniques (12 mg/kg) pendant deux semaines, les animaux étant ensuite sacrifiés quatre semaines après la dernière injection.Ce modèle permet de visualiser les modifications induites pas la DXR qui persistent plusieurs semaines après le traitement4 . Ces changements ne représentent pas nécessairement la cause de la cardiotoxicité, mais peuvent en être des conséquences, une adaptation des cellules cardiaques aux différents stress engendrés par le traitement. Ce modèle est malheureusement plus douloureux pour les animaux mais il reste très proche de la situation clinique. Cependant, ces résultats ne peuvent pas être directement interprétés en terme de fonction, car ils ne reflètent pas systématiquement le niveau de protéines ou leur activité enzymatique5 . Ce criblage peut être automatisé pour atteindre un très bon rendement, mais les données doivent néanmoins être vérifiées par RT-PCR. La seconde approche non biaisée est l'analyse du phosphoprotéome qui consiste en une étude du panel complet de l'état Dans cette thèse, nous montrons comment les voies de signalisation sont impliquées dans le phénotype énergétique de la cardiotoxicité induite par la DXR. Nous confirmons l'inhibition de la voie de l'AMPK dans les deux modèles de cardiotoxicité utilisés (in situ et in vivo), fait que nous n'avons pas retrouvé dans les cellules cancéreuses. Ces Notre approche phosphoprotéomique, nouvelle dans le contexte de la cardiotoxicité de la DXR, élargit les observations faites dans l'approche ciblée sur l'AMPK en montrant que les principales modifications de l'état de phosphorylation concernent les enzymes impliquées dans le métabolisme énergétique et les protéines localisées dans les mitochondries. Ceci nous conduit à considérer que la phosphorylation des protéines mitochondriales, qui émerge comme un élément clé dans la régulation de nombreuses fonctions de la mitochondrie15 , relaye la toxicité de la DXR. Ainsi, une cible clé serait la PDH, dont la phosphorylation augmente sous l'effet du médicament, inhibant ainsi l'entrée du pyruvate dans le cycle de Krebs, diminuant donc le métabolisme oxydatif du glucose, et aggravant davantage le déficit énergétique. Cependant, des mécanismes compensatoires sont également mis en place en modulant les niveaux de phosphorylations, de transcrits ou de protéine totale de certaines enzymes du métabolisme énergétique. Par exemple, le taux de transcription augmente pour certaines enzymes de la glycolyse ou du cycle de Krebs, et la quantité totale de BCK est plus grande. Ceci pourrait, au moins partiellement, expliquer pourquoi dans le modèle de toxicité aiguë, les performances cardiaques ne sont pas affectées lors de perfusions avec des stress ATF3 et pour les enzymes de défense contre le stress oxydant telles que l'hème oxygénase, ou la glutation-S-transférase. Ces éléments viennent confirmer les conclusions de précédentes études qui montrent une diminution des capacités des cellules cardiaques à réagir au stress oxydant16,[START_REF] Gewirtz | A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin[END_REF] . De plus, l'analyse avancée du transcriptome montre une diminution typique de l'expression de groupes de gènes induits par le stress, parmi lesquels les gènes de réponse aux chocs thermiques, de réponse immunitaire ou la voie de HIF. A partir de ces résultats, nous suggérons que la réponse émoussée au stress ou la réduction de la signalisation au « danger » est un élément majeur de la toxicité de la DXR et peut mener les cellules cardiaques vers un état pathologique.Parmi les voies de réponse au stress qui sont activées par la DXR, nous retrouvons les voies DNA-PK-Akt-GSK3 et ERK mentionnées dans l'étude ciblée (Chapitre 2.) ou certaines protéines chaperonnes mises en évidence dans l'étude phosphoprotéomique (Chapitre 3.) telles que CRYAB. La voie de DNA-PK transmettrait le signal que l'ADN est endommagé et l'activation des voies d'Akt et de ERK limiterait l'apoptose due aux stress oxydant et génotoxique. De plus, par l'activation de la voie de mTOR, ces kinases vont induire la synthèse de protéine et la croissance cellulaire, ce qui contribuerait au développement de l'hypertrophie cardiaque pour compenser la baisse de contractilité causée, Bien qu'une partie des voies de réponse au stress soit défaillante dans le coeur traité à la DXR, il existe certains mécanismes préventifs et compensatoires qui maintiennent une fonction cardiaque normale sous travail modéré. Ceci confirme le fort potentiel homéostatique du coeur. Cependant, dans la mesure où ces mécanismes opèrent déjà lors de l'activité basale du coeur, lorsque surviennent de nouveaux stress tels que l'exercice ou l'ischémie, ces mécanismes sont dépassés et le coeur ne peut plus assurer correctement sa fonction. Ainsi, l'intolérance à l'exercice et la sensibilité à l'ischémie sont des caractéristiques bien connues du phénotype observé dans la cardiotoxicité de la DXR[START_REF] Ventura-Clapier | Bioenergetics of the failing heart[END_REF][START_REF] Feng | Phosphoproteome analysis of isoflurane-protected heart mitochondria: phosphorylation of adenine nucleotide translocator-1 on Tyr194 regulates mitochondrial function[END_REF] . Des souris déficientes en certaines isoformes d'AMPK (AMPKα1 -/-, AMPKα2 -/-) 28 pourraient être traitées à la DXR de façon aiguë ou chronique afin d'étudier si cette déficience sensibiliserait le coeur au médicament. Alternativement, nous pourrions traiter des animaux avec la DXR et un activateur de l'AMPK comme AICAR, A-769662 ou la Metformin, et voir si un effet protecteur apparaît lorsque les animaux sont traités avant et/ou pendant le traitement à la DXR. De récentes études ont mis en lumière des effets intéressants de la Metformine dans le traitement des insuffisances cardiaques, notamment la diminution de la mortalité et l'amélioration de l'état des patients souffrant d'insuffisance cardiaque chronique[START_REF] Sugden | Mechanisms underlying regulation of the expression and activities of the mammalian pyruvate dehydrogenase kinases[END_REF] . De plus, depuis une cinquantaine d'années, ce médicament est utilisé dans le traitement du diabète de type II, et ses effets antidiabétiques dans le foie impliquent l'activation de l'AMPK[START_REF] Holness | Regulation of pyruvate dehydrogenase complex activity by reversible phosphorylation[END_REF] . Le mécanisme par lequel la Metformine active la kinase implique une inhibition directe du complexe 1[START_REF] Carvalho | Metabolic remodeling associated with subchronic doxorubicin cardiomyopathy[END_REF] , ce qui conduit à la signalisation via les espèces réactives de l'oxygène 32 et à une altération du rapport AMP/ATP qui active l'AMPK de façon allostérique[START_REF] Wakasugi | Myocardial substrate utilization and left ventricular function in adriamycin cardiomyopathy[END_REF] . De plus, la Metformine pourrait présenter des effets bénéfiques pour le coeur, indépendamment de l'AMPK, en augmentant l'oxydation des acides gras et donc en améliorant le statut métabolique et énergétique[START_REF] Solaro | Protein phosphorylation and signal transduction in cardiac thin filaments[END_REF] . En outre, la Metformine étant connue pour inhiber la croissance tumorale[START_REF] Zaugg | Cellular mechanisms in sympatho-modulation of the heart[END_REF][START_REF] Huang | Protein kinase C-mediated desmin phosphorylation is related to myofibril disarray in cardiomyopathic hamster heart[END_REF] , cela pourrait être un traitement sûr à tester en clinique. Cette supplémentation augmente la réserve intracellulaire de créatine et, via l'action de la CK, les réserves d'énergie sous forme de PCr. La forme phosphorylée est disponible pour des situations de déficit énergétique, pour régénérer l'ATP, ce qui pourrait être très favorable dans le contexte de la toxicité de la DXR. En effet, une étude sur des rats complémentés en Cr et traités avec de fortes doses de DXR 42 montre une diminution des effets cytotoxiques du médicament. Il serait intéressant d'étudier d'avantage cet effet bénéfique sur le plan mécanistique, avec des doses cliniques de DXR, par exemple avec des perfusions de coeurs de rats nourris avec des suppléments de Cr.Enfin, nous pourrions étudier le rôle de la phosphorylation de la desmine induite par la DXR dans la désorganisation myofibrillaire. Il avait déjà été démontré dans des cardiomyocytes que de faibles doses de DXR pouvaient conduire à la dégradation d'une autre protéine sarcomérique, la titine, par mécanisme dépendant de la calpaine[START_REF] Mcgregor | Proteomics of the heart: unraveling disease[END_REF] . La relation entre la phosphorylation de la desmine et la désorganisation des filaments pourrait être étudiée par une analyse immunocytochimique de cardiomyocytes isolés de coeurs de rats traités avec la DXR in situ ou in vivo. Cependant, la desmine étant phosphorylée sur différents sites par différentes kinases, il faudrait auparavant identifier tous les sites affectés par la DXR[43][44][45] .

	Discussion et perspectives Comme il a été exposé plus haut, les dommages de la fonction myofibrillaire pourraient
	être prévenus ou compensés par différents mécanismes : (i) l'activation des chaperonnes pour
	Ces deux modèles expérimentaux sont complémentaires et permettent une vision plus complète des événements cardiaques déclenchés par la DXR. Il est intéressant de noter que notre étude ciblée sur la signalisation cellulaire donne de nombreux résultats similaires dans les deux modèles. 5.2.2. Approches méthodologiques de phosphorylation des protéines dans un tissu donné. La phosphorylation est la plus importante modification post-traductionnelle, qui reflète l'état fonctionnel des protéines : l'activation ou l'inhibition enzymatique, les interactions protéine-protéine, etc. 6 . Son étude apporte par là même plus d'informations fonctionnelles que l'analyse transcriptomique 7 . Cependant, la 2D-PAGE-MS telle qu'utilisée dans ce travail n'est pas adaptée à des analyses à haut débit. 5.3. Discussion des principaux résultats 5.3.1. Métabolisme énergétique empêcher l'agrégation de la desmine (phosphorylation de CRYAB), (ii) l'amélioration de la contractilité cardiaque par la phosphorylation de MRLC et (iii) le remodelage du coeur par l'induction des gènes codant pour l'osteoglycine and c-Myc. 5.3.4. Potentiel homéostatique du coeur 5.1Notre premier objectif était d'établir différents modèles de cardiotoxicité (Chapitre 2.). Un modèle de coeur perfusé a été choisi comme modèle in situ, permettant de déclencher des réponses à la DXR sous des conditions hautement contrôlées avec différentes doses de DXR (clinique ou supra clinique), afin d'observer les effets toxiques les plus précoces. De plus, un modèle in vivo chez le rat traité en intraveineuse par des doses cliniques de DXR a été développé, afin d'étudier les effets du médicament qui apparaissent quelques semaines après la fin du traitement, et représentent la cardiotoxicité chronique. Ensuite, le deuxième objectif était de caractériser l'inhibition de l'AMPK au sein de la signalisation cellulaire dans ces deux modèles, afin de décrire les éventuels mécanismes impliqués (Chapitre 2.). Ce travail a conduit à étudier les enzymes impliquées en amont et en aval de l'AMPK, et plus particulièrement les interactions entre la voie de signalisation de l'AMPK, induite par le stress énergétique, et celles impliquées dans la réponse aux stress oxydant et génotoxique (DNA-PK-Akt et MAPK). Cette inhibition d'AMPK semble être Le premier est un modèle de coeur isolé et perfusé de rat. Tout d'abord, la perfusion à des doses cliniques de DXR (2-5 µM) permet l'identification des mécanismes moléculaires conduisant à la toxicité aiguë, avant l'apparition du dysfonctionnement cardiaque 2 . Ensuite, la perfusion à une dose supra clinique (25 µM de DXR) ayant des effets fonctionnels prononcés, aide à comprendre les effets cumulatifs du médicament, mais ne reflète pas nécessairement la toxicité chronique 3 . Ainsi, le modèle du coeur perfusé va apporter des informations concernant les effets précoces de la DXR, lesquels sont probablement impliqués dans l'étiologie de la toxicité plutôt que dans des effets non spécifiques 4 . Ce modèle permet également du médicament est une pathologie complexe multifactorielle, ce type d'approche systémique permet une vision mécanistique nouvelle, au-delà des modèles traditionnellement acceptés, et l'identification de cibles jusqu'alors inconnues. Cependant, les renseignements obtenus vont plutôt permettre de générer de nouvelles hypothèses que de donner des détails sur les mécanismes impliqués. Ainsi, de tels cribles doivent être complétés par des approches ciblées plus traditionnelles. De plus, il faut noter que des changements mineurs, mais néanmoins importants sur le plan fonctionnel, peuvent échapper à la détection de ces analyses globales. nouvelles données laissent supposer que cette inactivation est induite par les voies de signalisation d'Akt et des MAPK, elles mêmes activées par les stress énergétique et génotoxique, plutôt que par l'inhibition des kinases en amont de l'AMPK. Il en résulte l'activation de mTOR qui va alors déclencher des processus anaboliques (synthèse de Additionnellement au stress énergétique, nous avons observé, dans les deux modèles de toxicité, des stress génotoxique et oxydant plus ou moins prononcés, induits par des dommages à l'ADN d'une part et la génération d'espèces réactives de l'oxygène ou de l'azote d'autre part. Ainsi, nous pourrions supposer que la totalité des voies de réponses au stress soit activée. Or, les résultats obtenus dans nos études montrent plutôt une réponse au stress mixte, certaines voies de signalisation étant effectivement activées, alors que d'autres sont diminuées. Parmi ces dernières se trouvent plusieurs voies de réponse aux stress énergétique, génotoxique et oxydant. La présence de radicaux libres associée à l'augmentation du niveau d'AMP devrait activer l'AMPK, ce qui n'est le cas ni dans le modèle aigu ni dans le modèle chronique. De même, l'étude transcriptomique dans le modèle de coeur perfusé a mis en évidence une répression spécifique de l'expression des nombreux gènes codant pour des protéines de réponses au stress. Les stress oxydant et génotoxique devraient activer l'expression des gènes impliqués dans la réparation ou la stabilisation des dommages à l'ADN compensé par l'action de chaperonnes telles que CRYAB, connue pour être cardioprotectrice une fois phosphorylée 18,19 . 5.3.3. Structure et fonction des sarcomères La cardiotoxicité induite par la DXR est caractérisée pas des dommages moléculaires et une dérégulation du réseau de myofibrilles, entraînant ainsi une désorganisation myofibrillaire 20-23 . Nos études systémiques ont mis en évidence un certain nombre de mécanismes qui pourraient être responsables de ce phénotype. Tout d'abord, l'analyse transcriptomique a montré une diminution du niveau d'expression des gènes codant pour les facteurs de transcription impliqués dans le développement normal du coeur. L'étude phosphoprotéomique a révélé une altération du niveau de phosphorylation d'une protéine myofibrillaire clé, la desmine, ce qui serait un facteur important dans la déstructuration du réseau myofibrillaire induite par la DXR. La phosphorylation de la desmine provoque la désorganisation des filaments de desmine, ce qui affecte le réseau entier de myofibrilles, ainsi 5.4. Perspectives La cardiotoxicité de la DXR est un processus multifactoriel impliquant entre autres la dégradation de l'état énergétique, l'altération des différentes réponses cellulaires au stress et la détérioration de la structure et de la fonction du sarcomère. Nous avons identifié plusieurs éléments clés dans ce processus tels que l'AMPK, la PDH, CRYAB et la desmine. Une complète compréhension du phénomène nécessite maintenant des études mécanistiques détaillées utilisant des modèles animaux ou des biopsies de patients. De plus, notre étude apporte de nouveaux indices permettant de concevoir des investigations futures afin de trouver certaines stratégies protectrices efficaces contre la cardiotoxicité de la DXR. phosphorylation oxydative mitochondriale ou vers la production cytosolique de lactate. Elle est inhibée par la DXR, via l'augmentation de son niveau de phosphorylation, ce qui pourrait intensifier le déficit énergétique en ralentissant le cycle de Krebs 37 . Ces changements d'activité de la PDH et du métabolisme oxydatif devraient sans doute être vérifiés in vivo par une détermination de l'activité enzymatique et respiratoire. De plus, de nombreux inhibiteurs des PDH kinases (PDKs) ont été développés avec succès pour activer la PDH dans le diabète afin d'inhiber la gluconéogenèse hépatique et augmenter la disponibilité du glucose pour les organes périphériques. Le plus connu de ces inhibiteurs est le dichloroacétate, qui favorise l'oxydation du glucose et ainsi le statut énergétique du coeur 38 . D'autres inhibiteurs ont cependant été récemment développés 39 . Comme les activateurs de l'AMPK, ils pourraient être testés dans différents modèles de toxicité à la DXR pour étudier leurs effets sur le statut énergétique cellulaire et sur le phénotype cardiotoxique dans le but d'étendre leurs applications à des études cliniques. Cela permettrait également de tester des inhibiteurs des différentes kinases impliquées dans protéines) concentrations cliniques de DXR. 5.3.2. Réponse au stress entre autres, par l'endommagement du réseau de myofibrilles. Ce dernier serait également La PDH est une enzyme permettant d'orienter le produit de la glycolyse vers la des modèles de toxicité à la DXR.
	qu'il a été observé dans des études précédentes 24,25 , et par conséquent la contractilité
	cardiaque.

provoquée par une régulation inhibitrice via la signalisation d'Akt plutôt que par une modification de kinases en amont de l'AMPK. impliquées dans les mécanismes délétères d'une part et compensatoires d'autre part. Ces cibles appartiennent plus particulièrement à certaines classes fonctionnelles telles que le métabolisme énergétique, les réponses au stress et la structure/fonction des sarcomères. 5.2. Discussion des différents modèles et approches expérimentaux 5.2.1. Modèles de toxicité Dans cette thèse, deux modèles expérimentaux distincts in situ et in vivo ont été utilisés. l'enregistrement continu des performances cardiaques lors du traitement et n'est pas douloureux pour l'animal donneur. Dans cette thèse, les effets de la DXR sur la signalisation cellulaire cardiaque ont été analysés à l'aide de deux concepts différents : une étude ciblée sur les voies de signalisation énergétique liées à l'AMPK (Chapitre 2.) d'une part, et des analyses systémiques du phosphoprotéome et du transcriptome (Chapitre 3. et 4.) d'autre part. Les approches ciblées permettent la vérification d'une hypothèse précise : ici la question était de savoir si la voie de l'AMPK était inhibée dans le contexte de toxicité aiguë et chronique, et quels étaient les éléments de signalisation en amont ou de contrôle impliqués dans cette inhibition. Les approches de criblage non biaisées ne répondent pas par définition à une hypothèse prédéfinie, bien qu'il y ait toujours un postulat de départ : dans notre cas, nous supposions que la DXR affectait spécifiquement le transcriptome et le protéome. Comme la cardiotoxicité Ce type d'approche a été mené ici à l'aide de deux techniques de criblage différentes. La première est l'étude du transcriptome, c'est à dire la quantification de l'ensemble des ARNm dans un tissu donné. La diminution des métabolites « hautement énergétiques », tels que l'ATP ou la phosphocréatine (PCr), et le stress énergétique qui en résulte sont des caractéristiques de la cardiotoxicité de la DXR. Il est bien connu que le médicament inactive partiellement des éléments du métabolisme énergétique, principalement par des dommages oxydatifs directs, tels que des enzymes clés comme la CK 1 , la phosphofructokinase 8 , la β-enolase 9 et la carnitine palmitoyl transférase I 10,11 ). Ces effets sur le niveau de protéines et l'activité enzymatique concernent le plus souvent des enzymes mitochondriales, puisque la DXR s'accumule dans la membrane interne mitochondriale en raison de son affinité pour les cardiolipines 12 . 13 , consommateurs d'énergie, aggravant ainsi le déficit énergétique. De plus, l'activation de mTOR va contribuer à la mise en place du phénotype d'hypertrophie cardiaque 14 afin de compenser l'insuffisance ventriculaire gauche provoquée par la DXR. 144 et dans la réponse au stress oxydant. Cependant, la DXR induit une diminution d'expression des gènes codant pour la famille entière des protéines régulées par p53 et la famille de protéines induites par les dommages à l'ADN Gadd45, pour le facteur de transcription induit par le Prévenir les mécanismes qui contribuent au déficit énergétique et à la désorganisation des myofibrilles, ou renforcer les mécanismes compensatoires pourrait constituer d'intéressantes approches thérapeutiques afin de diminuer la cardiotoxicité de la DXR. Les résultats de cette thèse laissent penser que l'AMPK, la PDH et la desmine seraient des cibles thérapeutiques potentielles, qui pourraient être activées ou maintenues actives durant le traitement à la DXR. L'AMPK, senseur et régulateur de l'énergie cellulaire, est inhibée dans les coeurs de rats traités à la DXR. Il serait intéressant d'étudier l'incidence de niveaux d'AMPK expérimentalement modifiés sur des modèles animaux génétiques ou pharmacologiques sous traitement à la DXR. D'autres traitements préventifs pourraient consister à améliorer le statut énergétique cellulaire avec des compléments nutritionnels. La créatine (Cr) a été utilisée avec succès comme complément dans de nombreuses pathologies, la réhabilitation et le sport 40,41 . L'étiologie de la cardiotoxicité de la DXR étant clairement multifactorielle, une combinaison de différentes stratégies préventives ou curatrices pourrait être un facteur clé pour diminuer les effets secondaires du médicament. Par exemple, la co-administration de Metformine et d'un antioxydant tel que le dexrazoxane (voir Chapitre 1.2.) pourrait avoir des effets cumulatifs bénéfiques. Cependant, chaque thérapie devra prouver au préalable qu'elle n'interagit pas avec les effets anticancéreux de la DXR.

Table 6 -

 6 2: Guidelines for running 18 and 24 cm strips

	18 cm strip	24 cm strip

  La doxorubicine (DXR) est l'un des médicaments les plus efficaces en chimiothérapie, mais son application clinique est limitée par ses effets cardiotoxiques. Malgré des décennies de recherche, ses mécanismes pathogéniques ne sont toujours pas entièrement compris. Il s'ensuit qu'aucun traitement satisfaisant, curatif ou préventif, n'existe. Dans cette étude, nous recherchons les mécanismes de signalisation cellulaire impliqués. Deux modèles expérimentaux de toxicités, aigue d'une part (coeur isolé et perfusé de rat avec la DXR), et chronique d'autre part (rat traité à la DXR), ont permis de réaliser une étude ciblée (sur les voies de signalisation énergétiques) et deux études systémiques (phosphoprotéomique et transcriptomique). Les résultats combinés de ces travaux ont montré que la DXR modifiait le niveau de phosphorylation (activation) ou l'expression génique de protéines impliquées dans trois domaines fonctionnels distincts : métabolisme énergétique, réponses au stress, et structure/fonction du sarcomère. (i) Métabolisme énergétique : nous avons confirmé la surprenante inhibition de l'AMPK, probablement provoquée par un contrôle négatif exercé par des partenaires de signalisation (Akt et ERK), plutôt que par une modification des kinases activatrices en amont. Nous avons également montré l'augmentation du niveau de phosphorylation de la PDH, ce qui, en inhibant l'enzyme, ralentit le cycle de Krebs. Cependant, nous avons également observé un phénomène compensatoire de surexpression de gènes codant pour des enzymes de la glycolyse et du cycle de Krebs ; (ii) Réponses au stress : dans nos modèles, la DXR génère des stress énergétique, génotoxique et oxydatif. Cependant, seuls quelques mécanismes compensatoires sont activés (les voies de signalisation de DNA-PK-Akt-GSK3, diverses chaperonnes). Les autres semblent être inhibées suggérant que l'amoindricement des réponses au stress serait un des mécanismes de la cardiotoxicité de la DXR; (iii) Structure/fonction du sarcomère: L'augmentation de la phosphorylation de la desmine ainsi que la réduction du nombre de transcrits codant pour des protéines essentielles au développement cardiaque normal pourraient être la cause de la désorganisation du réseau myofibrillaire. En conclusion, ces résultats révèlent potentiellement de nouveaux mécanismes de la cardiotoxicité induite par la DXR et permettent d'envisager de nouvelles cibles moléculaires pour le développement de stratégies protectrices.

	Annexe 1: Abbreviations Left ventricular developed pressure La cardiotoxicité de la doxorubicine LVDP
	LVEDP	Left ventricular end-diastolic pressure
	2-DE MAPK ACC MCK ACR MES AICAR MS Akt (PKB) MS-TOF/TOF AMPK MRLC ANT MtCK APN mTOR ATF3 mTORC1 ATP, ADP, AMP ND2 BCK ND10 CamK NES CamKK NMR CARP PARP CF PCr Cr PCR CRYAB RT-PCR DIGE PDH DNA-PK PDK DNR PEP +dP/dt PFK -dP/dt PGM1 DUSP1 pI DXR PKA DXZ PKC ECH PMF ERK PMP24 FA PP2C FDR PTP GADD45α RNS GAPDH ROS GSEA RPP GSK3 RT-PCR HIF SAM HR SDS-PAGE HSP27 SIRT1 HSP70 SR IEF TnTc INN VDAC	Mitogen-activated protein kinase Two-dimensional polyacrylamid gel electrophoresis Muscle-type creatine kinase Acetyl-CoA carboxylase 2-(4-morpholino)-ethane sulfonic acid Acceptor control ratio Mass spectrometry Aminoimidazole carboxamide ribonucleotide Mass spectrometry using two time-of-flight analysis consecutively Ser/Thr protein kinase B Myosin regulatory light chain AMP-activated protein kinase Mitochondrial creatine kinase Adenosine nucleotide translocator Mammalian target of rapamycin Adiponectin Mammalian target of rapamycin complex 1 Activating transcription factor 3 NADH dehydrogenase iron-sulfur protein 2 Adenosine tri-(di-, mono-)phosphate NADH dehydrogenase 1 α subcomplex subunit 10 Brain-type creatine kinase Normalized enrichment score Calmodulin-dependent protein kinase Nuclear magnetic resonance Calmodulin-dependent protein kinase kinase Poly(ADP-ribose) polymerase Cardiac adriamycin-responsive protein Phosphocreatine Coronary flow Polymerase chain reaction Creatine Real-time polymerase chain reaction α crystallin B chain Pyruvate dehydrogenase Difference gel electrophoresis Pyruvate dehydrogenase kinase DNA dependent protein kinase Phosphoenolpyruvate Daunorubicin Phosphofructokinase Inotropy Phosphoglucomutase 1 Lusitropy Isoelectric point Dual specifity phosphatase 1 Protein kinase A Doxorubicin Protein kinase C Dexrazoxane Peptide mass fingerprint Enoyl-CoA hydratase Peroxisomal membrane protein 4, 24 kDa Extracellular signal-regulated protein kinase Protein phosphatase 2C Fatty acids Permeability transition pore False discovery rate Reactive nitrogen species Growth arrest and DNA-damage-inducible 45α Reactive oxygen species Glyceraldehyde-3-phosphate dehydrogenase Rate pressure product Gene set enrichment analysis Real-time polymerase chain reaction Glycogen synthase kinase 3 Significance analysis of microarrays Hypoxia-inducible factor Sodium dodecyl sulfate polyacrylamide gel electrophoresis Heart rate Sirtuin 1 Heat shock protein 27 kDa Sarcoplasmic reticulum Heat shock protein 70 kDa Troponin T, cardiac Isoelectrofocusing International Nonproprietary Name Voltage-dependent anion channel
	JNK	c-Jun-N-terminal kinase
	LC	Liquid chromatography
	LCAD	Acyl-CoA dehydrogenase (long-chain specific)
	LDH	Lactate dehydrogenase
	LKB1	Serine/threonine protein kinase 11

2.2. RésuméBien que les effets secondaires cardiotoxiques des anthracyclines soient largement documentés, les mécanismes impliqués ne sont pas entièrement compris. Nos précédents travaux laissent supposer que la protéine kinase activée par l'AMP (AMPK), senseur et régulateur de l'énergie cellulaire, pourrait être impliquée dans la médiation de la toxicité cardiaque de la doxorubicine (DXR). Dans cet article, nous étudions les interférences du médicament avec la voie de l'AMPK et les mécanismes potentiellement impliqués. Les effets de la DXR sur les voies de signalisation cellulaire sont étudiés dans un modèle de coeur isolé et perfusé de rat avec ou sans DXR (5et 25µM) et dans un modèle chronique de rat traité à la DXR (dose cumulative 12mg/kg). Dans les deux modèles, le médicament induit des stress énergétique, oxydatif et génotoxique. Bien que a DXR induise un déficit énergétique et qu'elle n'affecte pas les kinases activatrices de l'AMPK, elle n'active pas pour autant l'AMPK et diminue même sa phosphorylation basale ainsi que celle de sa cible, l'acétyl-coA carboxylase. En revanche, les stress oxydatif et génotoxique activent les voies de la survie cellulaire des MAPK et d'Akt, cette dernière par la DNA-PK elle-même activée par les dommages à l'ADN. L'inhibition de l'AMPK et l'activation des voies des MAPK et d'Akt conduisent à l'activation de mTOR, stimulateur de la croissance et donc de l'hypertrophie cardiaque. Nos résultats laissent supposer que la combinaison des stress énergétique, oxydatif et génotoxique dans les coeurs soumis à la DXR, provoquent une réaction spécifique et hiérarchique dans laquelle l'AMPK est inhibée. Cette inhibition est au moins partiellement provoquée par un contrôle négatif exercé par les voies d'Akt et des MAPK, elles-mêmes largement induites par la voie de signalisation des dommages à l'ADN. Bien que ces voies de signalisation puissent être protectrices en limitant l'apoptose, elles induisent également l'inhibition de l'AMPK, augmentant ainsi le déficit énergétique. De plus, via l'activation de mTOR, elles peuvent aussi contribuer à la mise en place du phénotype pathologique du coeur lors du traitement par la DXR.

Supplementary figures

proteins with Ponceau red (Sigma) to check the quality of transfer and equal protein loading.

Membranes, destained in T-TBS (0.15 M NaCl, 20 mM TRIS-Base pH 7.5, 0.1% Tween20), were blocked in blocking buffer (T-TBS and 5% skimmed milk) for 2 h at room temperature.

After blocking, membranes were incubated overnight at 4°C with primary antibodies (dilutions listed in Table 6-1). After washing with T-TBS (3 times for 15 min), membranes were incubated for 1 h at room temperature with horseradish peroxidise-conjugated secondary antibodies diluted in blocking solution. Immunoreactive bands were detected by enhanced chemiluminescence kit (ECL plus, GE Healthcare, Buckinghamshire, UK) using a camerabased system ImagerQuant LAS 4000 (GE Healthcare, Buckinghamshire, UK). Quantification of band intensities was made with ImageQuantTL (GE Healthcare).

Normalization was done using GAPDH or tubulin signals or in case of some phosphorylated proteins using the total protein signals. Values are given relative to control.

Immunoprecipitation

Immunoprecipitation is a technique to separate (precipitate) a particular protein from a sample containing potentially many thousands of different proteins by using an antibody that specifically binds to that particular protein. In the present work, the technique was applied to analyze the acetylation status of LKB1 and phosphorylation of ND10.

To analyze the acetylation status of LKB1, we first immunoprecipitated lysine-acetylated proteins from heart homogenate using anti-acetylated lysine antibody. For this, heart homogenate (70 µg protein) was incubated with anti-acetylated lysine antibody (1:100, Cell Signaling) in PBS, 1% BSA for 2 h at room temperature (total volume 300 µl). After addition of 200 µg of magnetic beads with immobilized sheep anti-rabbit IgG (Dynabeads M-280

Sheep anti-Rabbit IgG, Invitrogen) samples were incubated for another 1 hour at room temperature. The beads were washed 6 times using PBS + 0,1% Tween 20, resuspended in Laemmli sample buffer, and denatured proteins were separated on standard SDS-PAGE.

LKB1 was detected by immunoblotting as described above.

To analyze phosphorylation status of ND10, we first immunoprecipitated phosphorylated (on Ser/Thr/Tyr residues) proteins from heart homogenate using anti-phosphoSer/Thr/Tyr antibody. For this purpose, heart homogenate (100 µg protein) was incubated with anti-phosphoSer/Thr/Tyr antibody (1:100, Assay Design, Ann Arbor, MI) in PBS, 1% BSA overnight at 4°C (total volume 300 µL). After addition of 120 µg of protein A-sepharose (Sigma Aldrich, Saint Louis, MO), samples were incubated for 1 h at room temperature.

Beads were washed 10 times in PBS, 0.1% Tween 20, resuspended in Laemmli sample buffer, and denatured proteins were separated on standard SDS-PAGE gel (12% acrylamide) and immunoblotted as described above, using anti-ND10 antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA) as a primary antibody.

In order to ensure the correct interpretation of the results, two negative controls were run in parallel with experimental samples. The first control was realized by omitting the precipitating antibody (anti-acetylated lysine antibody or anti-phosphoSer/Thr/Tyr antibody) during immunoprecipitation. It allowed verifying that antigen protein was not precipitated by a non-specific reaction. The second control was realized by omitting the lysate during immunoprecipitation, in order to verify that neither the antibody (used to precipitate) nor the beads reacted with primary antibody during immunobloting.

Bidimensional electrophoresis

The bidimensional electrophoresis is a technique of protein separation in two dimensions:

1) in the first dimension in function of their isoelectric point and subsequently 2) in second dimension in function of the molecular weight.

6.7.1.Heart proteins extraction

Heart tissue was grinded with mortar in liquid nitrogen and proteins were extracted using a buffer composed of 7 M urea, 2 M thiourea, 4% CHAPS, 0.5% IPG Buffer (GE Healthcare), 40 mM DTT, containing protease and phosphatase inhibitors (tissue/buffer proportion 1:10 w/v). This extraction buffer contained chaotropic agents (urea, thiourea, CHAPS) in order to extract the maximum of proteins, including membrane proteins. The mixture was homogenized using an UltraTurax (3 cycles of 10 sec on ice). Homogenate was incubated 30 min at 4°C and centrifuged at 15400 g for 10 min at 4°C. The supernatant was assayed for protein concentration using 2DQuantKit (GE Healthcare) and stored at -80°C. The extraction step is crucial for first dimension separation: proteins must be completely denatured, not charged artificially (SDS) and salt concentration must be very low (because of high voltage used) 4 .

First dimension: IsoElectroFocusing (IEF)

Proteins were first separated according to their isoelectric point (pI) using Ettan IPGphor II Isoelectrofocusing System with Ettan IPGphor Manifold (GE Healthcare). First, the protective cover was removed from the Immobiline DryStrip gels (18 or 24 cm) with immobilized pH 3-10 non-linear gradient (pH 3-10NL). Then, strips were rehydrated for 10 to The software did spot detection automatically. To perform matching, a reference gel was defined for each series; this gel was chosen because its spots number was in the average of the series. Though the software is designed to work with minimal user intervention, it was usually necessary to define its landmarks, i.e. points that relate corresponding spots in each of the gels to be matched, to correct for gel differences and distortions. The number of landmarks was limited to a strict minimum necessary to obtain proper matching of all gels. In general, 5-10 landmarks were defined for one experimental set of gels. Landmarks are placed on protein spots that are clearly visible, small and sharp on all gels analyzed and are covering the entire image surface (both X and Y directions). Once this step made, the matching proceeded automatically, as well as normalization and quantification steps. were excised in triplicate and subjected to mass spectrometry analysis, using the 4800 MALDI-TOF/TOF Analyzer (AB Sciex).

Quantification of nucleotide and phosphocreatine

Freeze-clamped hearts (≈ 200 mg) were grinded in a mortar in presence of 0.6 N perchloric acid in liquid nitrogen (7 fold the heart volume). The resulting white powder was transferred in 15mL plastic tube and thawed on ice with frequent vortexing. The heart powder containing tubes from one series stayed in liquid nitrogen until the last heart sample was powdered. After thawing, the tubes were incubated on ice for 15 min more and then were centrifuged (12 min, 3000 g, 4°C). Pellets were discarded. The volumes of protein-free supernatants (containing the nucleotides) were determined and were neutralized by addition of 5 M potassium carbonate (to pH 6-7), incubated 30 min in ice and centrifuged again (12 min, 3000 g, 4°C). The final supernatants were aliquoted and kept at -80°C for maximally 2 weeks.

Nucleotide separation was performed using High Performance Liquid Chromatography (HPLC, Varian Inc). 30 µl of protein-free extract was diluted in the same volume of pyrophosphate buffer (28 mM Na 4 P 2 O 7 , pH 5.75) and separated on a C18 HPLC column (Polaris 5C18-A, S250*4.6 Repl, Varian, France) in pyrophosphate buffer at 1 ml/min flow rate and 30°C. ATP, ADP and AMP eluted at 3, 5 and 9 ml, respectively. A standard curve was made with different concentrations of the nucleotides from 1 to 100 µM. ATP, ADP and AMP were purchased from Sigma (Saint Louis, MO). Elution peaks were integrated with STAR software (Varian, France). For each sample, HPLC separation was done 3 times and the mean value was calculated.

Phosphocreatine (and also ATP independently to HPLC method) was quantified with a spectrophotometric test following the production of NADPH at 340 nm and using a three enzyme coupled reaction scheme involving creatine kinase (CK), hexokinase (HK), and glucose-6-phosphate dehydrogenase (G6PDH).

The quantities of NADPH produced (calculated from the measured changes in absorbance at 340 nm and the known NADPH extinction coefficient ε NADPH, NADH, 340 nm =6,22 mmol -1 •l•cm -1 )

in the two steps (first after HK and then after CK addition) reflect the quantities respectively of ATP and PCr in added heart extracts.

In the enzymatic test performed with a Specord 210 spectrophotometer (Analytik Jena, Germany), neutralized and protein-free heart extract (30-50 µl) was added to the cuvette containing reaction mixture consisting of 40 mM D-glucose, 1 mM NADP and 5 mM MgCl 2 in 0.1 M triethanolamine buffer pH 7 (to total volume of 1 ml). G6PDH (3 U) was added and the baseline absorbance (A0) was read at 340 nm. Then HK (5 U) was added, the reaction was followed till ATP exhaustion and the absorbance was read again (A1). Subsequently CK (5 U Real-time qRT-PCR was performed in the Roche Light Cycler using Plus Sybr Green 1

Master Mix (Roche, Mannheim, Germany). The PCR cycle is resumed in the Table 6-4. If not stated otherwise, the t-student test was used to determine the significance of each experiment. We considered a difference as being significant when the p-value was less than 0.05. Data were given mean ± SD (if not stated otherwise) and significant differences were given: * P < 0.05, ** P < 0.01, *** P < 0.001.

Annexes