Emilie Cerf

Rabia Sarroukh

Shiori Tamamizu-Kato

Leonid Breydo

Sylvie Derclaye

Yves Dufrêne

Vasanthy Narayanaswami

Erik Goormaghtigh

Jean-Marie Ruysschaert

Vincent Raussens

Drs Cédric Govaerts

Fabrice Homblé

Drs P Boussard

Y Looze

ANTI-PARALLEL -SHEET -A SIGNATURE STRUCTURE OF THE OLIGOMERIC AMYLOID-BETA PEPTIDE

Keywords: FTIR, Fourier transform infrared spectroscopy, HFIP, hexafluoroisopropanol, TBS, Tris-buffered saline solution, Amyloid-beta, Alzheimer's disease, oligomeric Abeta, anti-parallel beta-sheet, OmpF, secondary structure, infrared spectroscopy. 18

Alzheimer's disease (AD) is linked to amyloid beta peptide (A misfolding. Studies demonstrate that the level of soluble A oligomeric forms correlates better with the progression of the disease than the level of fibrillar forms. Conformation-dependent antibodies have been developed to detect either A oligomers or fibrils, suggesting that structural differences between these forms of A exist. Using conditions which yield well-defined A (1-42) oligomers or fibrils, we studied the secondary structure of these species by attenuated total reflection-FTIR spectroscopy. Whereas fibrillar A was organized in a parallel -sheet conformation, oligomeric A displayed distinct spectral features, which were attributed to an anti-parallel -sheet structure. We also noted striking similarities between A oligomers spectra and those of bacterial outer membrane porins. We discuss our results in terms of a possible organization of the anti-parallel -sheets in A oligomers, which may be related to reported effects of these highly toxic species in the amyloid pathogenesis associated with AD.

Alzheimer's disease (AD) is a widespread form of dementia belonging to the large family of amyloidoses whose common feature is the aggregation of misfolded proteins and/or peptides. AD is a brain-specific degenerative disease neuropathologically characterized by the presence of fibrillar amyloid deposition in extraneuronal spaces/cerebrovascular regions and by neurofibrillary tangles inside the neurons [START_REF] Hardy | The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics[END_REF].

Amyloid plaques are primarily composed of amyloid-(A ) peptide (38 to 43 residue long), which is released after proteolytic cleavage of the amyloid precursor protein (APP) by -andsecretases [START_REF] Selkoe | The cell biology of beta-amyloid precursor protein and presenilin in Alzheimer's disease[END_REF]. The amyloid hypothesis suggests that A accumulation in the brain is the primary event in the pathogenesis. Formation of tau tangles could be one of the consequences of an imbalance between production and clearance of A [START_REF] Hardy | The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics[END_REF]. A and A are the principal components of amyloid plaques [START_REF] Vigo-Pelfrey | Characterization of beta-amyloid peptide from human cerebrospinal fluid[END_REF]. A is the highly amyloidogenic though less abundant form, which appears to be initially deposited [START_REF] Jarrett | The carboxy terminus of the beta amyloid protein is critical for the seeding of amyloid formation: implications for the pathogenesis of Alzheimer's disease[END_REF]. This higher ability of A to aggregate has been related to the two additional hydrophobic amino acids at its C-terminal end [START_REF] Kim | Sequence determinants of enhanced amyloidogenicity of Alzheimer A{beta}42 peptide relative to A{beta}40[END_REF].

Several aggregation states have been identified for amyloidogenic proteins. A can exist as a monomer or larger soluble entities called oligomers and eventually insoluble fibrils. The general term "oligomers" includes different kinds of assemblies such as dimers, trimers, protofibrils, Aderived diffusible ligands (ADDLs), annular or pore-like oligomers [START_REF] Bitan | Neurotoxic protein oligomers--what you see is not always what you get[END_REF] Recently, it has also been reported that oligomers could be classified into prefibrillar or fibrillar oligomers as they have different aggregation pathways [START_REF] Kayed | Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers[END_REF].

A deposition in brain to form fibrillar plaques has been associated for a long time with neurodegeneration, insidious memory loss and cognitive decline [START_REF] Selkoe | The cell biology of beta-amyloid precursor protein and presenilin in Alzheimer's disease[END_REF][START_REF] Selkoe | Amyloid -Protein and the Genetics of Alzheimer's Disease[END_REF]. However, there has been a paradigm shift in this concept and it is now widely accepted that soluble oligomers of A are more neurotoxic than A fibrils and play a direct role in the amyloid pathogenesis [START_REF] Kirkitadze | Paradigm shifts in Alzheimer's disease and other neurodegenerative disorders: the emerging role of oligomeric assemblies[END_REF]. Cognitive decline associated with AD precedes amyloid deposition in human and transgenic mouse models and correlates with soluble A oligomers level rather than APP level or fibrillar amyloid deposits [START_REF] Mucke | High-level neuronal expression of abeta 1-42 in wild-type human amyloid protein precursor transgenic mice: synaptotoxicity without plaque formation[END_REF].

A oligomers are most probably intermediates in the amyloid fibril formation. However, they are not necessarily required to form fibrils [START_REF] Necula | Small molecule inhibitors of aggregation indicate that amyloid beta oligomerization and fibrillization pathways are independent and distinct[END_REF]. Recently, a conformation-dependent antibody has been shown to detect amyloid fibrils with high specificity, regardless of their sequence [START_REF] Kayed | Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers[END_REF]. Previously, using another conformation-dependent antibody raised against A prefibrillar oligomers, it was demonstrated that many amyloidogenic proteins or peptides, which also form oligomers, were recognized by the same antibody. Therefore, different types of amyloid oligomers may adopt a common structural motif postulated to be crucial in their common toxicity [START_REF] Kayed | Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis[END_REF]. Thus, obtaining any new structural information about A oligomers would be an important step in a better understanding of AD etiology and possibly other amyloidogenic diseases. Previous studies carried out on A oligomers reported the dominance of -sheet structures. However, they were unable to distinguish between parallel and anti-parallel structures. In this study, we use attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to compare the structure of A (1-42) oligomers and fibrils. We demonstrate that although both forms adopt a predominantly -sheet structure, A (1-42) oligomers adopt an anti-parallelsheet structure, which is distinctly absent in fibrils.

EXPERIMENTAL Peptide preparation.

A (1-42) was purchased from American Peptide Co. (Sunnyvale, CA). The peptide was dissolved in cold hexafluoroisopropanol (HFIP, Sigma-Aldrich, St. Louis, MO) at a 2 mg/ml concentration, incubated at room temperature for one hour and then aliquoted. HFIP was evaporated under nitrogen flow and residual HFIP removed under vacuum using a Speed Vac. The resulting A (1-42) film was stored at -20°C until further manipulation.

A (1-42) incubations. Prior to any incubation, the peptide was dissolved in dimethylsulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO) at a final concentration of 5 mM and then immediately resuspended using one of the following conditions. To obtain oligomers, the peptide was either dissolved in F12 phenol red-free cell culture medium (Sigma-Aldrich, St. Louis, MO) as developed in Dahlgren et al. [START_REF] Dahlgren | Oligomeric and fibrillar species of amyloid-beta peptides differentially affect neuronal viability[END_REF] and Stine et al. [START_REF] Stine | In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis[END_REF] or in 20 mM Tris-HCl, pH 7.4, 100 mM NaCl (Tris-buffered saline, TBS) as developed by Garzon-Rodriguez et al. [START_REF] Garzon-Rodriguez | Soluble amyloid Abeta-(1-40) exists as a stable dimer at low concentrations[END_REF], at a final concentration of 100 M and incubated at 4°C for 24 h. To obtain fibrils, the peptide was resuspended either in 10 mM HCl at a final concentration of 100 M and incubated at 37°C for 24 h, or in 0.5 mM Hepes pH 7.4 at a final concentration of 500 M and incubated at room temperature under gentle agitation for at least 30 days.

Western blot analysis. Peptide samples were diluted in SDS-PAGE sample buffer and separated on a 12% bis-Tris gel at 4°C for 2 h at 100 V. The separated bands were transferred onto a nitrocellulose membrane which was then blocked for 1 h in 5% nonfat dry milk in TBS/Tween 20 buffer. The membrane was incubated with the mouse monoclonal A antibody 6E10 (1:3000) (Sigma-Aldrich, St. Louis, MO). Detection was carried out using horseradish peroxidase-conjugated anti-mouse antibody (1:2000) and the Supersignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL). Pictures were recorded and analyzed using the ImageQuant 400 gel imager and ImageQuant TL software (GE Healthcare).

Atomic Force Microscopy.

A (1-42) solutions were characterized by atomic force microscopy (AFM) using a Nanoscope IIIa (Veeco Metrology LLC, Santa Barbara, CA) equipped with a 120 x 120 m piezoelectric scanner. Analyses were carried out either in contact or in tapping mode at room temperature in air using AFM cantilevers with spring constant of either 0.01 or 0.03 N/m (Microlevers, Veeco Metrology LLC, Santa Barbara, CA). Before analysis, 100 L of sample (diluted 10x in the case of fibrils and 5x in the case of oligomers) were incubated 10 min at room temperature on freshly cleaved mica and rinsed four times with milliQ water. Excess water was then removed under nitrogen flow.

Thioflavine T fluorescence. Thioflavine T (ThT, Sigma-Aldrich, St. Louis, MO) fluorescence was used to characterize the different peptide solutions according to LeVine [START_REF] Levine | Quantification of beta-sheet amyloid fibril structures with thioflavin T[END_REF] on a LS55 fluorimeter (Perkin Elmer Instruments). Briefly, 4.5 g of peptide were added to 1 ml of a 5 M ThT solution maintained at 25°C by a circulating water bath and the fluorescence was recorded at 482 nm (excitation wavelength: 450 nm). Dot blot analysis. 1 µg of oligomeric A was spotted onto a nitrocellulose membrane, which was subsequently blocked with 10% nonfat milk in TBS-0.01% Tween 20 for 1 h at 4 C and washed. Then the membrane was incubated overnight at 4°C with rabbit anti-oligomer antibody A11 (1:3000) in 5% nonfat milk TBS-0.01% Tween 20. After washing with TBS-0.01% Tween 20, the membrane was incubated for 1 h at 4°C with horseradish peroxidase-conjugated antirabbit IgG (Cell signaling technology, Danvers, MA) (1:2000). ECL western blot detection system (GE Healthcare, Piscataway, NJ) was used to detect chemiluminescence and pictures were recorded and analyzed using the ImageQuant 400 gel imager and ImageQuant TL software (GE Healthcare).

IR spectroscopy. IR spectra were recorded on an Equinox 55 infrared spectrophotometer (Bruker Optics, Ettlingen, Germany) equipped with a Golden gate reflectance accessory (Specac, Orpington, U.K.). The internal reflection element was a diamond crystal (2 x 2 mm) with an aperture angle of 45° that yielded a single internal reflection. 128 accumulations were performed to improve the signal/noise ratio. The spectrometer was continuously purged with dried air. Spectra were recorded at a resolution of 2 cm -1 . All measurements were made at 24 C. Samples were prepared by spreading 2 L of peptide solution on the diamond crystal surface and by removing the excess water under nitrogen flow. Alternatively, for salt-containing samples, similarly to the AFM sample preparation, 5 L of peptide solution were incubated at the diamond surface for 15-20 min, the sample was then washed three times with excess milliQ water. Finally, excess water was removed under nitrogen flow.

H/D exchange. Hydrogen/deuterium (H/D) exchange experiments were performed on A oligomers and fibrils. The pH of the samples was adjusted in order to perform the exchange on samples with identical pH. The decay of the NH-associated amide II band (1520-1580 cm -1 ) was used to monitor the exchange of the amide group. Results were analyzed as previously described [START_REF] Goormaghtigh | Attenuated total reflection infrared spectroscopy of proteins and lipids in biological membranes[END_REF]. Spectral cluster analysis. Before analysis, a linear baseline was subtracted from all spectra at 1708, 1602, and 1482 cm -1 . Spectra were then rescaled on the amide I area (1708-1602 cm -1 ). Spectra were clustered according to the Euclidian distances for each wavenumber in the amide I and II range (1708-1482 cm -1 ).

Protein spectra used for the clustering analysis were extracted from the RASP50 database [START_REF] Oberg | Rationally selected basis proteins: a new approach to selecting proteins for spectroscopic secondary structure analysis[END_REF]: ADH Alcohol dehydrogenase from horse liver; ALA -Lactalbumin from human milk; APE Apolipoprotein E3 from human; ATX -Hemolysin (alphatoxin) from Staphylococcus aureus; AVI Avidin from hen egg white; BTE Erabutoxin b from Laticauda semifasciata; CAH Carbonic anhydrase from bovine erythrocyte; CNA Concanavalin A from jack bean; COL Colicin A, C-terminal domain from bacterial source; CSA Citrate synthetase from porcine heart; CTG -Chymotrypsinogen A from bovine pancreas; CYC Cytochrome c from horse heart; DPR Dihydropteridine reductase from sheep liver; FTN Ferritin (apo) from horse spleen; GST Glutathione S-transferase from equine liver; HBN Hemoglobin from bovine blood; IGG Immunoglobulin from human; INS Insulin from bovine pancreas; LCL Lectin, lentil from lentil; LOX Lipoxygenase-1 from soybean; LSZ Lysozyme from chicken egg white; MBN Myoglobin from horse heart; MON Monellin from Dioscoreophyllum cumminsii; MTH Metallothionein II from rabbit liver; OVA Ovalbumin (egg albumin) from hen; PAB Parvalbumin from rabbit muscle; PAH Penicillin amidohydrolase from Escherichia coli; PAP Papain from papaya latex; PEP Pepsin from porcine stomach; PER Peroxidase from Arthromyces ramosus; PGK Phosphoglyceric kinase from baker's yeast; PGN Pepsinogen from pig stomach; PLA Phospholipase A2 from bovine pancreas; R61 DD-transpeptidase from Streptomyces r61; REN Rennin (chymosin b) from calf stomach; RIC Ricin from castor bean; RNA Ribonuclease A from bovine pancreas; SBC Subtilisin Carlsberg from Bacillus licheniformus; SBN Subtilisin BPN' (nagarse) from not specified; SDF Superoxide dismutase (Fe) from Escherichia coli; SOD Superoxide dismutase (Cu,Zn) from bovine erythrocyte; TGN Trypsinogen from bovine pancreas; TIB Trypsin inhibitor (soybean, Bowman-Burke) from soybean; TIP Trypsin inhibitor (BPTI) from bovine pancreas; TMT Thaumatin from Thaumatococcus daniellii; TPI Triose phosphate isomerase from baker's yeast; TRO Troponin from chicken muscle; UBQ Ubiquitin from bovine erythrocyte; UOX Glucose oxidase from Aspergillus niger; XYN Xylanase from Trichoderma viride (see [START_REF] Oberg | Rationally selected basis proteins: a new approach to selecting proteins for spectroscopic secondary structure analysis[END_REF] for more details on the RASP50 FTIR database).

In addition to the RASP50 FTIR database spectra, spectrum from OmpF reconstituted in asolectin (kind gift from Dr. F. Homblé) and spectra of both A (1-42) fibrils and oligomers (see Figures 2 and3 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

RESULTS

A (1-42) oligomers and fibrils: formation and assessments. Our goal in this study was to compare the structure of A (1-42) oligomers and fibrils. We used well-defined oligomer-or fibril-forming protocols to obtain the desired A (1-42) aggregates (see experimental section or references therein). Due to the inherent high structural variability of the peptide, we carefully assessed each species using four independent approaches to verify that we obtained the expected entities.

Since A (1-42) is known to produce SDS-resistant oligomers and fibrils, we used bis-Tris SDS-PAGE followed by 6E10 monoclonal antibody recognition to visualize the different species. After incubation at 4°C for 24 h in TBS or F12, large oligomeric species appeared between ~40 and ~170 kDa (Figure 1, Panel A, lane 1). In acidic fibril-forming conditions, after 24 h of incubation, extremely high molecular weight A remained in the stacking gel. This high molecular weight A corresponded to fibrils. A decrease of tri-and tetrameric species was observed (Figure 1A, lane 2). For longer incubation time, in acidic fibril-forming conditions, the large oligomeric bands completely disappeared (Figure 1A, lane 3). Fibrils formed in 0.5 mM Hepes, pH 7.4 after 30 days also revealed an oligomer-free pattern (data not shown). It should be noted that the monomeric A (~4.5 kDa) is present in all conditions. A quantification of the different band intensities using the ImageQuant TL software was carried out. It showed that in the oligomeric conditions (Figure 1A, lane 1), the band corresponding to the monomer represents only 16% of the total intensity while the larger oligomers (~40 to 170 kDa) represent 80% (trimers and tetramers represent 2% each). Such quantification is more difficult to perform for fibril-forming conditions because fibrils remain in the stacking gel and are most likely underestimated. Nevertheless, this result demonstrates that monomers are clearly not the major species in our samples. As a comparison, at t=0 in TBS, pH 7.4, A shows the presence of small oligomers (mainly trimers and tetramers) in addition to monomers which are the major species (~70%) presents on the Western Blot. High molecular weight oligomers are also present on a t=0 gel but do not represent a significant proportion (~3%) of the species formed by A (1-42) (data not shown).

Even though A is known to produce SDS-resistant oligomeric and/or fibrillar species, it has been shown that SDS-PAGE in the study of A oligomers is not devoid of defects and might not always reflect the exact content of the different species present in the sample [START_REF] Bitan | Neurotoxic protein oligomers--what you see is not always what you get[END_REF]. As suggested [START_REF] Bitan | Neurotoxic protein oligomers--what you see is not always what you get[END_REF], we undertook further characterization of both oligomers and fibrils to ensure that our samples were representative of each species.

A second method used to confirm fibril or oligomer formation was AFM. AFM images of oligomers formed after 24 h showed the presence of globular spherical-shaped entities (Figure 1, Panel B, top) with a typical height of 5-6 nm, which is in good agreement with previously characterized oligomers [START_REF] Mastrangelo | High-resolution atomic force microscopy of soluble Abeta42 oligomers[END_REF]. Importantly, it also showed that no fibrils were present in our oligomer-forming conditions. This verification was critical for the subsequent IR analysis, since it excluded potential spectral contributions from fibrils. In fibril-forming conditions, AFM images showed long unbranched fibrils with a typical 7-8 nm width (Figure 1, Panel B, bottom). At higher magnification, these images showed some periodicity along the fibril axes (not shown). All these features are in agreement with previously published data on A fibrils [START_REF] Stromer | Structure and morphology of the Alzheimer's amyloid fibril[END_REF]. To ensure that these fibrils were not formed during AFM sample preparation, especially during the removal of excess water at the mica plate, fibrillar samples were observed in contact mode in buffer. These fibrillar samples were prepared in HCl and then resuspended in TBS pH 7.4 to also ensure that a change in buffer and in pH do not affect the fibril morphology. The fibrils were not as well-resolved as in tapping mode in air. However, the overall morphology and the size of the fibrils resembled those in tapping mode (not shown), demonstrating that the removal of the excess water of the sample at the mica surface and a change in pH do not affect the fibrils once they are formed.

A third assessment was carried out using ThT fluorescence. ThT is known to fluoresce specifically in the presence of amyloid fibrils [START_REF] Levine | Quantification of beta-sheet amyloid fibril structures with thioflavin T[END_REF]. After resuspension (t=0h) of the A (1-42) peptide in all the conditions used here, the samples are ThT negative. Both fibril-forming conditions resulted in a high ThT fluorescence emission intensity. This is already observed after 24h for fibrils formed in 10 mM HCl. On the other hand, all the oligomeric samples showed negligible ThT fluorescence emission intensity even after 48h (Figure 1, Panel C).

Finally, using A11, an oligomeric conformation-specific antibody [START_REF] Kayed | Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis[END_REF], we demonstrated by dot blot analysis that oligomer-forming protocols yielded A11 positive results (Figure 1, Panel D, top for oligomers formed in TBS). Fibrils formed in 10 mM HCl responded poorly or weakly to A11 after one week incubation (Figure 1, Panel D, bottom).

Taken together, these four experiments independently confirm that the oligomeric preparations: (i) do not have any fibrillar species, and, (ii) do indeed contain moieties that are detected by the A11 antibody. Conversely, this analysis also ensures that our A fibril samples are ThT positive and devoid of oligomers. This rigorous pre-analysis is essential in our subsequent FTIR analysis to ensure that our oligomeric A species do not contain any fibrillar species (and vice versa) that may potentially contribute to the spectral signals.

Secondary structure of oligomeric and fibrillar A (1-42) species. We then examined the secondary structure of these well-defined oligomeric and fibrillar A (1-42) species by ATR-FTIR. In FTIR, it has been demonstrated both theoretically and experimentally that parallel and anti-parallel -sheet structures can be distinguished based on the analysis of the amide I (1700-1600 cm -1 ) region [START_REF] Miyazawa | The infrared spectra of polypeptides in various conformations: Amide I and Amide II bands[END_REF]. In anti-parallel -sheet structures, the amide I region displays two typical components. The major component has an average wavenumber located at ~1630 cm -1 while the minor component, about five fold weaker than the major one, is characterized by an average wavenumber at 1695 cm -1 . The 1695/1630 intensity ratio has been suggested to be proportional to the percentage of anti-parallel arrangement of the -strands in a -sheet [START_REF] Chirgadze | Infrared Spectra and resonance Interaction of Amide-I Vibration of the Antiparallel-Chain Pleated Sheet[END_REF]. For parallelsheet structures, the amide I region displays only the major component around 1630 cm -1 [START_REF] Chirgadze | Infrared Spectra and resonance Interaction of Amide-I Vibration of the Parallel-Chain Pleated Sheet[END_REF][START_REF] Goormaghtigh | Determination of soluble and membrane protein structure by Fourier transform infrared spectroscopy. I. Assignments and model compounds[END_REF]. FTIR spectra of A (1-42) in the two fibril-forming conditions employed in this study (Figure 2, spectra a and b) showed typical parallel -sheet features, characterized by a maximum of absorbance at 1630 cm -1 in the amide I region. The relatively narrow width of the major peak at 1630 cm -1 is indicative of stable and/or long -strands and strong hydrogen bonds [START_REF] Goormaghtigh | Determination of soluble and membrane protein structure by Fourier transform infrared spectroscopy. I. Assignments and model compounds[END_REF], as expected for extremely stable structure like amyloid fibrils. FTIR spectra of A (1-42) oligomers were significantly different from those of A (1-42) fibrils, indicating that these entities adopt a different structure. The amide I region is characterized by the presence of the two characteristic components of anti-parallel -sheet structure, at 1630 and 1695 cm -1 (Figure 3, spectrum a-c).

A quantitative analysis of the amide I region by deconvolution followed by curve-fitting showed that for both A (1-42) oligomers and fibrils, -sheet structures were the most abundant (48-57% and ~75% respectively), while random coil and/or helical structures (observed absorbance around 1650-1660 cm -1 ) represented ~20-26 and ~5-10% for the oligomers and fibrils, respectively. The helical content was slightly higher (33% and ~40% if we take into account the random coil contribution) in oligomers incubated less than 1h, indicating a change in structure from -helix to -sheet, with time. -turns content (~1670 cm -1 ) was ~15-20% for all the structures studied here.

Solvent accessibility: hydrogen/deuterium exchange experiments.

To further analyze the structural difference between A (1-42) oligomers and fibrils, we performed hydrogen/deuterium exchange experiments using FTIR spectroscopy. While A (1-42) oligomers showed relatively fast exchange dynamics (about 60% of amide hydrogens exchanged after one hour), A (1-42) fibrils had a slower exchange dynamics (only ~40% of amide hydrogens exchanged during the same period of time) (Figure 4). The initial exchange rate was extremely rapid especially for oligomers where most of the exchange occurred during the first five minutes.

Comparison with other proteins. In order to compare the different structures produced here with known protein structures, we used a protein IR spectra database [START_REF] Oberg | Rationally selected basis proteins: a new approach to selecting proteins for spectroscopic secondary structure analysis[END_REF] designed to cover as well as possible the conformational space as defined by CATH [START_REF] Orengo | CATH--a hierarchic classification of protein domain structures[END_REF], with almost no redundancies. This database was previously shown to contain a number of unique features and allowed an improved structure prediction from infrared spectra [START_REF] Oberg | The optimization of protein secondary structure determination with infrared and circular dichroism spectra[END_REF][START_REF] Goormaghtigh | Evaluation of the information content in infrared spectra for protein secondary structure determination[END_REF].

The overall shape of the A (1-42) spectra in the amide I and II range was compared with the entire database by mean of a cluster analysis based on Euclidian distance measurement (see materials and methods and Figure 5). The results demonstrated that (i) A fibrils spectra do not cluster with other protein spectra and that (ii) A oligomers spectra are clustered with 5 antiparallel -sheet proteins (namely avidin, concanavalin A, lentil lectin, OmpF, and xylanase). From this cluster analysis, it is clear that OmpF spectrum is the closest and most related to the A oligomer ones (Figure 3, spectrum d, and Figure 5). OmpF is folded as a typical anti-parallel -sheet barrel made of 16 anti-parallel strands and is associated with an almost 100% antiparallel arrangement of its -strands [START_REF] Cowan | Crystal structures explain functional properties of two E. coli porins[END_REF]. The spectrum reveals the presence of 1630 and 1695 cm -1 peaks, attributed to anti-parallel -sheet structure. Using the respective 1695/1630 cm -1 intensity ratios [START_REF] Miyazawa | The infrared spectra of polypeptides in various conformations: Amide I and Amide II bands[END_REF] (Figure 6) for the different spectra displayed in Figure 2 and 3, we calculated that in all the oligomer-forming conditions used here, the percentage of anti-parallel arrangement of the -strands was ~100%, while in fibril-forming conditions this percentage was lower than 10%.

DISCUSSION

The objective of our study was to investigate the secondary structure adopted by oligomeric A in comparison with fibrillar A , in an effort to understand the structural basis of the neurotoxicity associated with the former. While models for A (1-40) and A (1-42) in fibrillar conformations, mainly based on solid-state NMR data, are now accessible [START_REF] Antzutkin | Multiple quantum solid-state NMR indicates a parallel, not antiparallel, organization of beta-sheets in Alzheimer's beta-amyloid fibrils[END_REF][START_REF] Petkova | A structural model for Alzheimer's beta -amyloid fibrils based on experimental constraints from solid state NMR[END_REF][START_REF] Petkova | Experimental constraints on quaternary structure in Alzheimer's beta-amyloid fibrils[END_REF][START_REF] Lührs | 3D structure of Alzheimer's amyloid-beta(1-42) fibrils[END_REF], almost no structural information for A oligomers is currently available.

It is recognized that both species are characterized by the presence of -sheet structures. However, fundamental differences in their local structures exist, confering them unique reactivity with conformation-specific antibodies. Using well-defined conditions, we prepared A (1-42) oligomers and fibrils. The formation of A oligomers or fibrils was confirmed with well-established tests that mutually complement each other, such as: (i) the presence of SDSresistant entities of multiple molecular weights by SDS-PAGE, (ii) visualization of oligomeric and fibrillar species by AFM, (iii) confirmation of the absence of fibrils in our oligomer preparations and of the presence of fibrils in our fibril-forming conditions by ThT fluorescence, and (iv) verification of the presence of A11-reactive species in our oligomer preparations and not in our fibril preparations. This suite of assessments was critical for our subsequent FTIR analysis as it was important to demonstrate the absence of fibrillar A in our oligomeric preparations and vice versa. As noted above, in the SDS-PAGE one intriguing point is the presence of monomeric A in each lane. This monomeric band, which is clearly not the most abundant entity (see results), might be in part induced by the presence of SDS. If present in our samples, the monomers would contribute a constant spectral background in all our preparations. After this initial confirmation, we employed ATR-FTIR to compare the structure of A (1-42) oligomers and fibrils. In fibrils, the presence of a major narrow peak located at ~1630 cm -1 was indicative of a highly stable parallel -sheet, in agreement with solid-state NMR models [START_REF] Antzutkin | Multiple quantum solid-state NMR indicates a parallel, not antiparallel, organization of beta-sheets in Alzheimer's beta-amyloid fibrils[END_REF][START_REF] Petkova | A structural model for Alzheimer's beta -amyloid fibrils based on experimental constraints from solid state NMR[END_REF][START_REF] Petkova | Experimental constraints on quaternary structure in Alzheimer's beta-amyloid fibrils[END_REF][START_REF] Lührs | 3D structure of Alzheimer's amyloid-beta(1-42) fibrils[END_REF] and site-directed spin labeling experiments [START_REF] Török | Structural and dynamic features of Alzheimer's Abeta peptide in amyloid fibrils studied by site-directed spin labeling[END_REF], which showed that both A (1-40) and (1-42) adopt a cross-structure with a parallel, in register, -sheet composed of two strands connected by a turn or a loop region. Fifteen to twenty years ago, FTIR was first misleading and demonstrated that A fibrils adopted an anti-parallel conformation. It is not our purpose here to discuss why such a discrepancy occurred in the past. More recently, several groups have demonstrated that for well-characterized parallel fibrils, the corresponding FTIR spectra were typical of parallel -sheet (see for examples for A (1-40), fig. 3d in [START_REF] Habicht | Directed selection of a conformational antibody domain that prevents mature amyloid fibril formation by stabilizing A protofibrils[END_REF], and for A (1-28), fig. 2c in [START_REF] Perálvarez-Marín | Time-Resolved Infrared Spectroscopy of pH-Induced Aggregation of the Alzheimer A 1-28 Peptide[END_REF]). On the contrary, for well-characterized anti-parallel fibrils, FTIR spectra display two peaks (at ~1630 and ~1695 cm -1 ) characteristic of anti-parallel -sheet (see for example [START_REF] Dzwolak | A conformational -helix to -sheet transition accompanies racemic self-assembly of polylysine: an FT-IR spectroscopic study[END_REF][START_REF] Castelletto | Self-assembly in aqueous solution of a modified amyloid beta peptide fragment[END_REF][START_REF] Lee | Steric Zipper of the Amyloid Fibrils Formed by Residues 109-122 of the Syrian Hamster Prion Protein[END_REF]).

A curve-fitting procedure of the infrared spectra revealed that the -sheet content in fibrils was as high as ~75%. Solvent accessibility evaluated by NMR approach, indicated that there are two regions in A (1-42) fibrils protected from the solvent. These two regions encompass 70% of the amino acids and are responsible for the formation of two strands in fibrils [START_REF] Olofsson | The solvent protection of alzheimer amyloid-beta-(1-42) fibrils as determined by solution NMR spectroscopy[END_REF]. Using hydrogen/deuterium exchange, we showed that ~60% of the amide bonds of the peptide were protected against the exchange in the fibril conformation, which is also in very good agreement with data provided by deuterium exchange monitored by mass spectrometry [START_REF] Kheterpal | Abeta protofibrils possess a stable core structure resistant to hydrogen exchange[END_REF].

It is believed that immediately upon its release from the membrane A oligomerizes in dimers and other multimers or soluble oligomers, and possibly forms fibrils. Also referred to as protofibrils or ADDLs, soluble oligomers mediate neuronal toxicity, inhibit long-term potentiation [START_REF] Walsh | Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo[END_REF] and modulate neuronal viability [START_REF] Bitan | Neurotoxic protein oligomers--what you see is not always what you get[END_REF][START_REF] Kirkitadze | Paradigm shifts in Alzheimer's disease and other neurodegenerative disorders: the emerging role of oligomeric assemblies[END_REF], reflecting their inherent toxicity.

Oligomers formed in F12 cell culture medium, using the same protocol as we used here, have been tested for their toxicity on Neuro-2A neuroblastoma cells [START_REF] Dahlgren | Oligomeric and fibrillar species of amyloid-beta peptides differentially affect neuronal viability[END_REF]. The authors showed that A (1-42) oligomers inhibited neuronal viability 10 times more than A (1-42) fibrils. Using the same conditions, the same group has tested the toxicity of A (1-42) oligomers on neurons using primary co-cultures of neurons and glial cells [START_REF] Manelli | A 42 neurotoxicity in primary co-cultures: Effect of apoE isoform and A conformation[END_REF]. They demonstrated that oligomeric A (1-42) but not fibrillar A (1-42) induced a dose-dependent increase in neurotoxicity. Thus, the related toxicity of the A (1-42) oligomers we formed here, has been carefully tested. Unfortunately little structural information is currently available on oligomers. Nevertheless hydrogen-deuterium exchange on A (1-40) protofibrils, which collectively referred to nonfibrillar oligomeric forms of A , has shown that about 40% of the amide backbone are highly resistant to exchange [START_REF] Kheterpal | Abeta protofibrils possess a stable core structure resistant to hydrogen exchange[END_REF]. This is in excellent agreement with the percentage we determined here on A (1-42) oligomers. This also establishes the validity of the approach we employed to assess the oligomeric species. The -sheet content in oligomers was lower than in fibrils but however significant and reached 48-57%. This lower content in secondary structure and faster exchange dynamics probably reflect a higher flexibility of A in soluble oligomers than in fibrils. This higher flexibility (or lower stability) may be a crucial property of such assemblies, especially in their mechanism of toxicity. One of the hypothetical toxicity mechanisms for oligomers is related to their interaction with lipid bilayers in which they might cause perturbation and/or permeabilization [START_REF] Kayed | Permeabilization of lipid bilayers is a common conformation-dependent activity of soluble amyloid oligomers in protein misfolding diseases[END_REF][START_REF] Lashuel | Neurodegenerative disease: amyloid pores from pathogenic mutations[END_REF][START_REF] Kremer | Correlation of betaamyloid aggregate size and hydrophobicity with decreased bilayer fluidity of model membranes[END_REF].

Compared to fibrils, infrared spectra of oligomers revealed the presence of an additional peak located at 1695 cm -1 , indicative of an anti-parallel -sheet conformation in these species. This additional peak has been recently observed in A (1-40) oligomers [START_REF] Habicht | Directed selection of a conformational antibody domain that prevents mature amyloid fibril formation by stabilizing A protofibrils[END_REF] and also in oligomers formed by a prion peptide (PrP82-146) [START_REF] Natalello | Conformational plasticity of the Gerstmann-Sträussler-Scheinker disease peptide as indicated by its multiple aggregation pathways[END_REF]. This structural information suggests that even if fibrils and oligomers are rich in -sheets, they do not adopt the same conformation. This is of significance in the neurotoxicity associated with oligomers. The anti-parallel -sheet organization in oligomers might represent a critical step in perturbation and/or permeabilization of biological membranes [START_REF] Kayed | Permeabilization of lipid bilayers is a common conformation-dependent activity of soluble amyloid oligomers in protein misfolding diseases[END_REF][START_REF] Lashuel | Neurodegenerative disease: amyloid pores from pathogenic mutations[END_REF][START_REF] Kremer | Correlation of betaamyloid aggregate size and hydrophobicity with decreased bilayer fluidity of model membranes[END_REF].

The A11 oligomer-specific antibody does not recognize fibrils or low molecular weight oligomers but is able to recognize higher molecular weight prefibrillar oligomers of a wide variety of amyloidogenic peptides or proteins [START_REF] Kayed | Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis[END_REF]. This suggests that most, if not all, amyloidogenic oligomers adopt the same structure and might share the same mechanism of toxicity. Recently, the same antibody was used with success against the pore-forming bacterial toxin, -hemolysin, and on human perforin [START_REF] Yoshiike | Pore-forming proteins share structural and functional homology with amyloid oligomers[END_REF]. These proteins are known to form oligomeric pores in their toxic conformation, each monomer supplying several -strands which selfassemble to fold into a -barrel. It has been shown that A and other amyloids can form annular or ring-shaped particles [START_REF] Lashuel | Neurodegenerative disease: amyloid pores from pathogenic mutations[END_REF] which are comparable to pore-forming proteins.

Infrared spectra of A oligomers presented striking similarities with bacterial outer membrane porins, which are folded as -barrels. Based on the 1695/1630 cm -1 intensity ratios, we determined that A oligomers were composed of -strands, all of which were in an anti-parallel organization. Interestingly, all pore-forming -barrels adopt an anti-parallel -sheet organization. Based on the secondary structure content (45-50% of extended -sheet structures), we postulate a putative model wherein each A molecule will provide two -strands upon selfassembling during oligomer formation. In our view, the simplest way to accommodate twostrands into the A peptide in oligomeric conformation is to postulate that the two strands present in fibrils and encompassing approximately residues ~11-24 and ~28-42 [START_REF] Lührs | 3D structure of Alzheimer's amyloid-beta(1-42) fibrils[END_REF], will form shorter -strands in oligomers encompassing about 10 to 12 residues each. These strands selfassemble in an anti-parallel -sheet conformation (Figure 3, 5, and 7). Recently, the structure of monomeric A (1-40) in complex with a phage-display selected affibody protein has been solved by NMR spectroscopy [START_REF] Hoyer | Stabilization of a betahairpin in monomeric Alzheimer's amyloid-beta peptide inhibits amyloid formation[END_REF]. This structure clearly showed that monomeric A (1-40) when stabilized by neighbor structural elements can adopt an anti-parallel hairpin involving twostrands encompassing residues 17-23 and 30-36. This is in good agreement with our proposed anti-parallel -sheet model. As suggested by Hoyer and coworkers [START_REF] Hoyer | Stabilization of a betahairpin in monomeric Alzheimer's amyloid-beta peptide inhibits amyloid formation[END_REF], it can be speculated that in our proposed model, a "simple" reorientation of the plane of the strands by 90° will allow a reorganization from an anti-parallel to a parallel -sheet conformation, a conformational change that may be associated with the conversion from oligomeric to fibrillar A and vice versa. Further, it can be suggested that the anti-parallel -sheet conformation of oligomeric A (1-42) might in some case(s) be the early stages in the formation of a -barrel made of amphipathic strands, 10-12 residue long each. This -barrel conformation (see figure 7 for a schematic representation) is ideally suited to insert into a cellular membrane and span a lipid bilayer. Such an organization can potentially lead to permeabilization of cells, thereby contributing to the toxicity associated with these species. We suggest that, in the anti-parallel conformation, thestrands are organized in a manner that allows intra-molecular hydrogen bond formation, and not only inter-molecular ones like in fibrils.

In conclusion, we demonstrated using ATR-FTIR spectroscopy that: (i) A (1-42) oligomers and fibrils can be clearly distinguished from each other and do not adopt the same secondary and tertiary structure, and (ii) A (1-42) oligomers adopt an anti-parallel -sheet conformation. The anti-parallel -sheet structure may thus be a signature structural feature of oligomeric A . (iii) We showed some striking spectral similarities between A oligomers and pore-forming porins and therefore believe that the ability of A oligomers to form a porin-like structure may be associated with their toxicity in AD. fibrils formed in 10 mM HCl during 7 days. Spectral intensities were normalized to the intensity of the 1630 cm -1 peaks. Spectra were shifted for better visualization. Spectra were deconvoluted using a Lorentzian deconvolution factor with a full width at half height of 20 cm -1 , a Gaussian apodization factor with a full width at half height of 13.33 cm -1 to obtain a resolution enhancement factor K = 1.5 [START_REF] Goormaghtigh | Determination of soluble and membrane protein structure by Fourier transform infrared spectroscopy. I. Assignments and model compounds[END_REF]. Spectra are representative of at least 3 independent experiments.

Figure 3. ATR-FTIR spectra in the amide I region of A (1-42

) in oligomer-forming conditions. a and b, A (1-42) oligomers after incubation in TBS for < 1 h and 24 h, respectively. c, A (1-42) oligomers formed in F12 cell culture medium after 24 h. d, bacterial porin OmpF reconstituted in lipid membrane. Spectral intensities were normalized to the intensity of the 1630 cm -1 peaks. Spectra were shifted for better visualization. All spectra were deconvoluted using a Lorentzian deconvolution factor with a full width at half height of 20 cm -1 , a Gaussian apodization factor with a full width at half height of 13.33 cm -1 to obtain a resolution enhancement factor K = 1.5 [START_REF] Goormaghtigh | Determination of soluble and membrane protein structure by Fourier transform infrared spectroscopy. I. Assignments and model compounds[END_REF]. Vertical dashed line was added at 1695 cm -1 to facilitate the identification of differences between spectra. Spectra are representative of at least 3 independent experiments.

) were used. 7 Biochemical

 7 Journal Immediate Publication. Published on 13 May 2009 as manuscript BJ20090379 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090379

Figure 1 .

 1 Figure1. Assessments of A (1-42) in oligomer-and fibril-forming conditions. A. Western blot analysis of A (1-42) oligomers and fibrils separated on a 12% bis-Tris SDS-PAGE and probed with the monoclonal antibody 6E10. Lane 1 shows A (1-42) oligomers formed in TBS after 24 h. Lanes 2 and 3 show A (1-42) fibrils formed in 10 mM HCl after 24 h and 7 days, respectively. A quantification on non-overexposed blots has been carried out using the ImageQuant gel imager and software (see text). B. AFM images of oligomeric and fibrillar A (1-42). AFM images were recorded in 2 x 2 m contact mode with oligomers formed in TBS after 24 h (total z range = 15 nm) (top) and 2 x 2 m tapping mode with fibrils formed in 10 mM HCl after 24 h (total z range = 8 nm) (bottom). C. Relative ThT fluorescence emission intensity after 0, 24, and 48 h for A (1-42) oligomers formed in TBS (black), in F12 medium (light grey), and for A (1-42) fibrils formed in 10 mM HCl (dark grey). D. Dot blot analysis of 1 µg of A (1-42) oligomers formed in TBS after 24h (top) and fibrils formed in 10 mM HCl after 1 week (bottom) with the conformation-dependent A11 antibody. The results shown here are representative from 3-5 independent experiments.

Figure 2 .

 2 Figure 2. ATR-FTIR spectra in the amide I region of A (1-42) in fibril-forming conditions. a, A (1-42) fibrils formed in 0.5 mM Hepes, pH 7.4 under agitation for 36 days. b, A (1-42)fibrils formed in 10 mM HCl during 7 days. Spectral intensities were normalized to the intensity of the 1630 cm -1 peaks. Spectra were shifted for better visualization. Spectra were deconvoluted using a Lorentzian deconvolution factor with a full width at half height of 20 cm -1 , a Gaussian apodization factor with a full width at half height of 13.33 cm -1 to obtain a resolution enhancement factor K = 1.5[START_REF] Goormaghtigh | Determination of soluble and membrane protein structure by Fourier transform infrared spectroscopy. I. Assignments and model compounds[END_REF]. Spectra are representative of at least 3 independent experiments.

Figure 4 .

 4 Figure 4. Hydrogen/deuterium exchange of A (1-42) fibrils (circles) and oligomers (triangles) as a function of time. Fibrils were formed in 10 mM HCl and oligomers in TBS. Before experiments, pH for both samples was adjusted to 6.5. Data are representative of at least 3 independent experiments. Standard deviations between 3 experiments are 5%.

Figure 5 .

 5 Figure 5. Dendrogram of the cluster analysis of RASP50, OmpF, A (1-42) fibril and oligomer spectra in the amide I and II range (1708-1482 cm -1 ). Abscissa is the Euclidean distance between spectra. On the ordinate the protein spectra identification are displayed. The

Figure 6 .

 6 Figure 6. Relative intensity ratios of the 1695 and 1630 cm -1 peaks for A (1-42) fibrils and oligomers. a, for A (1-42) fibrils formed in 0.5 mM Hepes, pH 7.4 under agitation for 30 days. b, for A (1-42) fibrils formed in 10 mM HCl during 1 week, c, A (1-42) oligomers after incubation in TBS for 24 h. d, A (1-42) oligomers formed in F12 cell culture medium after 24 h. e, bacterial porin OmpF reconstituted in lipid membrane. All the 1695/1630 cm -1 ratios have been normalized on the OmpF ratio (taken as 100%). Inset: corresponding FTIR spectra in the 1695 cm -1 region.

Figure 7 .

 7 Figure7. A hypothetical schematic representation of A oligomers in a putative poreforming conformation. The monomer structure of A was taken from the 1Z0Q PDB file[START_REF] Tomaselli | The alpha-to-beta conformational transition of Alzheimer's Abeta-(1-42) peptide in aqueous media is reversible: a step by step conformational analysis suggests the location of beta conformation seeding[END_REF], fibril structure was derived from the 2BEG PDB file[START_REF] Antzutkin | Multiple quantum solid-state NMR indicates a parallel, not antiparallel, organization of beta-sheets in Alzheimer's beta-amyloid fibrils[END_REF]. The size of the pore and the number of strands involved in pore formation are hypothetical. Residues involved in the central strand region are colored in magenta; those in the C-terminal portion of A peptide are in cyan. Nonstructured regions are represented in green. The N-terminal region of the peptide is only represented in the monomer structure. The figure was made using Pymol.

Figure 1 21

 1 Figure 1

Figure 2

 2 Figure 2

Figure 3 23

 3 Figure 3

Figure 4 24

 4 Figure 4

Figure 5 25

 5 Figure 5

Figure 6

 6 Figure 6

Figure 7

 7 Figure 7

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

for assistance and access to the fluorimeter. E.C. is Research Fellow for the National Fund for Scientific Research (Belgium), R.S. is Research Fellow for the Fund for Research in the Industry and Agriculture (Belgium), Y.D, V.R. are Senior Research Associate, and E.G is Research Director for the National Fund for Scientific Research (Belgium). V.N. was supported by funds from the Grant-In-Aid from the American Heart Association, the Drake Family trust and TRDRP. S.T.K is supported by a fellowship from the American Heart Association.


Bernard Korzeniewski

Véronique Deschodt-Arsac

Guillaume Calmettes

Gilles Gouspillou

Jean-Michel Franconi

Philippe Diolez

Effect of pyruvate, lactate and insulin on ATP supply and demand in unpaced perfused rat heart

Keywords: oxidative phosphorylation, respiratory substrate, hormone, perfusion, metabolic regulation, Proportional Activation Approach 2

Mitochondrial respiration/oxidative phosphorylation is the main source of energy in the form of ATP in heart under physiological conditions. Different respiratory substrates were used in various experiments during heart perfusion: glucose, pyruvate, lactate, glucose + pyruvate, glucose + lactate, glucose + insulin etc. Also under physiological conditions the concentration of respiratory substrates/hormones in blood can vary significantly. In the present study we tested the effect of pyruvate, lactate and insulin (all in the presence of glucose) and glucose (in the presence of pyruvate) on the ATP-producing and consuming blocks in perfused rat heart, in a system where heart rate (HR) was allowed to vary (no pacing). Changes in rate-pressure product (RPP) and phosphocreatine (PCr) concentration were measured. Proportional Activation Approach (PAA) was used to visualize and quantitatively analyze the data. It was demonstrated that addition of glucose (in the presence of pyruvate) exerted essentially no effect on the system. Insulin (in the presence of glucose) activated only the ATP producer. The most interesting finding is that in our system pyruvate and lactate (added in the presence or instead of glucose) activated ATP producer, but significantly inhibited ATP consumer (their effect was quantitatively identical).

INTRODUCTION

Oxidative phosphorylation in mitochondria is the main source of energy in the form of ATP in heart under physiological conditions. Different respiratory substrates can be used by heart depending on their abundance in blood and on physiological conditions: glucose, pyruvate, lactate, fatty acids, keton bodies. The level of insulin affects significantly the glucose uptake. Different combinations of these substrates/hormones were used as components of the perfusion medium in various experiments on perfused heart: glucose (G), pyruvate (P), lactate (L), glucose + pyruvate (GP), glucose + lactate (GL), glucose + insuline (GI) and so on [START_REF] From | 31 P-NMR studies of respiratory regulation in the intact myocardium[END_REF][START_REF] From | Regulation of the oxidative phosphorylation rate in the intact cell[END_REF][START_REF] Zweier | Substrate-induced alterations of high energy phosphate metabolism and contractile function in the perfused heart[END_REF][START_REF] Kobayashi | Control of maximum rates of glycolysis in rat cardiac muscle[END_REF][START_REF] Diolez | Modular regulation analysis of heart contraction: application to in situ demonstration of a direct mitochondrial activation by calcium in beating heart[END_REF][START_REF] Diolez | Mitochondria do not control heart bioenergetics[END_REF][START_REF] Kashiwaya | Control of glucose utilization in working perfused rat heart[END_REF][START_REF] Sharma | Regulation of pyruvate dehydrogenase activity and citric acid cycle intermediates during high cardiac power generation[END_REF]. It has been shown [START_REF] Zweier | Substrate-induced alterations of high energy phosphate metabolism and contractile function in the perfused heart[END_REF] that pyruvate elevated RPP and [PCr] in the paced guinea pig heart system. However, the effects of particular components on the oxidative phosphorylation system have not been systematically studied in unpaced rat heart.

The energetic system of heart can be conceptually divided into two blocks: ATP production and ATP consumption, as it is done within Modular Control Analysis (MoCA) [START_REF] Diolez | Modular regulation analysis of heart contraction: application to in situ demonstration of a direct mitochondrial activation by calcium in beating heart[END_REF][START_REF] Diolez | Mitochondria do not control heart bioenergetics[END_REF] or Proportional Activation Approach [START_REF] Korzeniewski | Proportional activation coefficients during stimulation of oxidative phosphorylation by lactate and pyruvate or by vasopressin[END_REF][START_REF] Korzeniewski | Physiological heart activation by adrenaline involves parallel activation of ATP usage and supply[END_REF]. These blocks "communicate" with each other only through a group of metabolites, the concentrations of which are uniquely related to each other: ATP, ADP, P i , PCr, Cr. In principle, each of these metabolites can be chosen as a representative of the entire group, but in practice it is convenient to choose phosphocreatine because its concentration can be measured noninvasively by NMR and it can change significantly (unlike ATP concentration) under physiological conditions. Generally, changes in [PCr]

involve implicitly changes in [Cr], [ATP], [ADP] and [P i ].

External effectors, such as Ca 2+ ions, are not one of the ways in which the PCr-producing block and PCr-consuming block communicate with each other. Heart activation/inactivation results in variations in work and ATP turnover flux that can be represented approximately by rate-pressure product (RPP). Generally, the recording of changes in RPP and [PCr] caused by some external factor enables to find out if ATP producer and consumer are activated or inhibited by this factor, and to which extend [START_REF] Diolez | Modular regulation analysis of heart contraction: application to in situ demonstration of a direct mitochondrial activation by calcium in beating heart[END_REF][START_REF] Korzeniewski | Physiological heart activation by adrenaline involves parallel activation of ATP usage and supply[END_REF].

It has been demonstrated that both elevated [Ca 2+ ] in the perfusion medium [START_REF] Diolez | Modular regulation analysis of heart contraction: application to in situ demonstration of a direct mitochondrial activation by calcium in beating heart[END_REF] and adrenaline [START_REF] Korzeniewski | Physiological heart activation by adrenaline involves parallel activation of ATP usage and supply[END_REF] activate the ATP producer and consumer to a similar level. However, it was also observed that, in unpaced heart, [PCr] is much higher in heart perfused with pyruvate, than with glucose [START_REF] Diolez | Modular regulation analysis of heart contraction: application to in situ demonstration of a direct mitochondrial activation by calcium in beating heart[END_REF]. Nevertheless, few studies [see e.g. [START_REF] Zweier | Substrate-induced alterations of high energy phosphate metabolism and contractile function in the perfused heart[END_REF][START_REF] Kobayashi | Control of maximum rates of glycolysis in rat cardiac muscle[END_REF][START_REF] Santos | Metabolic control of contractile performance in isolated perfused rat heart. Analysis of experimental data by reaction:diffusiom mathematical model[END_REF] have been conducted using the same beating hearts on the effect of replacing glucose with pyruvate (or inversely) (or other changes of substrate/hormone composition of the perfusion medium). Respiratory substrate can be considered as an external effector equally well as calcium ions or adrenaline. For instance, it was demonstrated that lactate/pyruvate added to suspension of isolated hepatocytes activate both ∆Ψ (electrical potential across the inner mitochondrial membrane)-producing and ∆Ψ-consuming system (the former to a slightly greater extent) [START_REF] Korzeniewski | Proportional activation coefficients during stimulation of oxidative phosphorylation by lactate and pyruvate or by vasopressin[END_REF].

It has been demonstrated that Proportional Activation Approach (PAA) [START_REF] Korzeniewski | Proportional activation coefficients during stimulation of oxidative phosphorylation by lactate and pyruvate or by vasopressin[END_REF] is a very useful theoretical method for quantitative analysis of the proportional activation of the producer and consumer of some metabolite (group of metabolites) M by some external effector [START_REF] Korzeniewski | Proportional activation coefficients during stimulation of oxidative phosphorylation by lactate and pyruvate or by vasopressin[END_REF][START_REF] Korzeniewski | Physiological heart activation by adrenaline involves parallel activation of ATP usage and supply[END_REF]. In the present study PAA was used to determine quantitatively the effect (activation or inhibition) on the ATPproducing and ATP-consuming system of the following changes in the substrate/hormone composition of the perfusion medium: 1. replacement of glucose with pyruvate (G → P transition); 2. addition of pyruvate in the presence of glucose (G → GP transition); 3. addition of lactate in the presence of glucose (G → GL transition); 4. addition of glucose in the presence of pyruvate (P → GP transition); 5. addition of insulin in the presence of glucose (G → GI transition). We hypothesize that the substrate/hormone composition of the perfusion medium has a significant effect on the kinetic properties of the bioenergetic system of the cardiac myocyte.

METHODS

Experimental Procedures

Langendorf perfusion and NMR measurements procedures for perfused rat (Male Sprague-Dawley rats : 350-400g) heart were described previously [START_REF] Diolez | Mitochondria do not control heart bioenergetics[END_REF]. Hearts were perfused by an aortic canula at constant pressure (100 mm Hg) with Krebs-Henseleit solution (95% O 2 and 5% CO 2 , pH 7.35, temperature 37°C) containing 10 mmol/L Na-pyruvate as substrate and actual free calcium concentration set to 2.0 mmol/L. Left ventricle systolic pressure (LVP) of unpaced heart was measured under isovolumic conditions of contraction thanks to a latex balloon inserted into the left ventricle and connected to a pressure transducer. Mechanical performance was evaluated as the product of heart frequency and developed pressure (RPP, in mmHg.beat.min -1 ). Generally, we used the unpaced heart system. This differs our system from some other perfusion systems where pacing was applied [START_REF] Zweier | Substrate-induced alterations of high energy phosphate metabolism and contractile function in the perfused heart[END_REF][START_REF] Kobayashi | Control of maximum rates of glycolysis in rat cardiac muscle[END_REF].

To study the effects of external parameters under physiological conditions, pacing was not applied.

Perfused heart was placed into a 20 mm NMR tube inserted into a 9.4-T super-conducting magnet equipped with a 20-mm bore (Bruker DPX400 Avance) for 31P NMR spectroscopy. In order to eliminate endogenous substrates and remaining anesthetics, at least 20 minutes were allowed for stabilization of heart activity before any measurement was made.

For the study of steady state modifications four NMR spectra (5 min duration time for each spectrum) were acquired before modulation was applied, then after a transition time equivalent to one spectrum (5 min) four new spectra (20 min) were recorded. Each steady state (before and after modulation) was characterized by the mean values of the four spectra for phosphorylated compounds and the corresponding four RPP values.

The effects of the following changes in the substrate/hormone composition of the perfusion medium were studied: 1. G → P transition, 16.7 mM of glucose was replaced with 10 mM of pyruvate; 2. G → GP transition, 10 mM of pyruvate was added to 16.7 mM of glucose; 3. G → GL transition, 20 mM of lactate was added to 16.7 mM of glucose; 4. P → GP transition, 16.7 mM of glucose was added to 10 mM of pyruvate; 5. G → GI transition, 20 IU of insulin was added to 16.7 mM of glucose.

The elasticity of the ATP/PCr producer to PCr can be determined by a direct change of the activity of the consumer. This causes a change in the PCr concentration which affectes the flux through the producer. The elasticity of the ATP/PCr consumer to PCr can be determined in the opposite way. To perform this kind of analysis one must use agents that directly increase/decrease (modulate) the activity of the consumer and at the same time have no effect on the activity of the producer, or inversely.

NaCN, an inhibitor of cytochrome oxidase, was used to directly decrease the activity of the ATP/PCr producer. The linearity of the response of the consumer to PCr for glucose as the respiratory substrate was tested by applying three different NaCN concentrations: 0.1, 0.2 and 0.3 mM. The obtained results confirmed the assumption concerning the linearity -see Fig. 2, 3 and 4 (compare also the discussion below). The response of the ATP producer to PCr, measured by an increase in the balloon volume in the left ventricle (direct activation of the ATP/PCr consumer), was calculated from averaged relative changes in RPP and [PCr] given in Table 2 in [START_REF] Diolez | Modular regulation analysis of heart contraction: application to in situ demonstration of a direct mitochondrial activation by calcium in beating heart[END_REF] -that experiment was carried out for the same substrate (glucose), rat breed and experimental conditions as the present experiment. The linearity of the response of the ATP/PCr producer to PCr was confirmed in [START_REF] Korzeniewski | Physiological heart activation by adrenaline involves parallel activation of ATP usage and supply[END_REF] by applying both an increase in the balloon volume in the left ventricle (activation of the consumer) and BDM administration (inhibition of the consumer) (see discussion below). The elasticities of the producer and consumer were calculated as the ratios of the relative changes in RPP to the relative changes in [PCr]: (∆RPP/RPP) / (∆PCr/PCr) after an increase in the balloon volume and after NaCN administration, respectively.

Technically, the measurements of the producer and consumer elasticities to PCr, and of the responses to an external effector must be performed on different hearts.

Theoretical Methods

The Proportional Activation Approach (PAA) [START_REF] Korzeniewski | Proportional activation coefficients during stimulation of oxidative phosphorylation by lactate and pyruvate or by vasopressin[END_REF] was used in the present work to analyze, interpret and visualize the experimental data. PAA defines the proportional activation coefficient as the ratio of the relative direct activation by some factor F (it may be e.g. hormone, electrical/neural stimulation or respiratory substrate) of the consumer (C) of some metabolite M (for instance PCr or ATP) to the relative direct activation of the producer (P): (∆C/C)/( ∆P/P) [START_REF] Korzeniewski | Proportional activation coefficients during stimulation of oxidative phosphorylation by lactate and pyruvate or by vasopressin[END_REF]. It was shown [START_REF] Korzeniewski | Proportional activation coefficients during stimulation of oxidative phosphorylation by lactate and pyruvate or by vasopressin[END_REF] that this coefficient equals: The values of the elasticities (defined within the so-called Metabolic Control Analysis: see e.g. [START_REF] Fell | Metabolic Control Analysis: a survey of its theoretical and experimental development[END_REF] for review) of the M producer and M consumer blocks to [M] ( and , respectively) quantify the relative change in the flux through these blocks in response to a given relative change in M concentration. The higher the absolute value of the elasticity, the more sensitive is the flux to changes in [M]. In principle elasticities are defined as infinitesimal coefficients, but when the responses of fluxes to M are (nearly) linear, as it takes place in our system, they can be identified with large-scale responses of the producer and consumer blocks to [M]. Under typical conditions is negative (M inhibits its own production), while is positive (M activates its own consumption).

τ ε τ ε ⋅ - ⋅ - = P M C M PC F P 1 1 ( 1 ) 4
P M ε C M ε P M ε C M ε
All possible cases of the effect of some external factor on the system of production and consumption of some metabolite (group of metabolites) M are presented in Fig. 1. When 1/τ equals (the relevant curves overlap) only P is activated or inhibited, depending on the fact whether flux increases or decreases. When 1/τ equals (the relevant curves overlap) only C is activated or inhibited. In the special case when τ = 0 (no changes in M; horizontal line in Fig. 1), both P and C are directly activated or inhibited to the same extent ( =1). In all other cases both C and P are activated and/or inhibited to the relative extent characterized by , as indicated in Fig. 1. In particular, when is positive both P and C are either activated or inhibited, while if is negative P is activated and C is inhibited, or inversely.

C M ε P M ε PC F P PC F P PC F P PC F P
In the present work PAA was used to determine quantitatively the respective direct activation or inhibition of the ATP producer and ATP consumer blocks in the result of changes in the substrate/hormone composition of the perfusion medium, as indicated above.

Statistics and calculations

Results are presented as means ± SD. The effects of changes in the substrate/hormone composition of the perfusion medium were compared using one-way ANOVA. Significant differences were considered at P < 0.05. coefficients were calculated in the following steps. For each particular heart the values of RPP and [PCr] for control (glucose) and modified substrate/hormone composition were used for calculation of the relative (in %) change in RPP and [PCr] during a given transition. The responses τ were calculated as indicated above. Proportional activation coefficients were calculated from the responses and elasticities according to Equ. 1 (using the values of the elasticities of the producer and consumer to [PCr] equal to -5.34 and 0.96, respectively, determined as described above). Using this method, only RPP and [PCr] and relative changes (in %) in these variables can be presented as means ± SD.

RESULTS AND DISCUSSION

Effect of pyruvate, lactate and insulin

In the present study we measured heart rate (HR), left ventricular pressure (LVP), and thus rate-pressure product (RPP), as well as phosphocreatine (PCr) concentration in the same hearts in control conditions (with glucose as the respiratory substrate) and after a given transition (modification of the substrate/hormone composition in the perfusion medium). The results of these measurements for different transitions are presented in Tables 1 and2. One can easily see (Table 1) that pyruvate, lactate and insulin caused statistically significant changes in both RPP and [PCr]. Pyruvate and lactate decreased RPP, while insulin increased this variable. In all cases a significant increase in [PCr] was observed. As to the components of RPP, both HR and LVP were affected, although the effect on LVP seems to be generally greater than on HR (Table 2). Averaged relative changes (in %) in RPP and [PCr] were used for calculation of the responses τ of the system to modifications of substrate/hormone composition, and subsequently for calculation of proportional activation coefficients (Table 3).

The values of RPP and [PCr] for control conditions (with glucose) were used as reference values and scaled for 100 % for each transition. This is what 100% (the 'reference point') means in Figs. 234 3.

The data concerning the G → GP and G → P transitions clearly demonstrated that, in our system, while pyruvate significantly activates the ATP producer, it also surprisingly significantly inhibits the ATP consumer. This can be seen in Fig. 2, where both transitions are directed up and left from the initial point (compare Fig. 1). The calculated coefficients equal -0.307 and -0.296 for G → GP transition and G → P transition, respectively. This means that in both cases the activation of the producer was about three times higher than the inhibition of the consumer. It may seem paradoxical that, although the activation of the producer is about three times greater than the inhibition of the consumer, the flux through the system decreases and not increases. However, one should bear in mind that the consumer has a much greater (3.7 times with glucose as the substrate) control over the flux than the producer [START_REF] Diolez | Modular regulation analysis of heart contraction: application to in situ demonstration of a direct mitochondrial activation by calcium in beating heart[END_REF]. The effect on the flux is proportional to the product of the relative activation/inhibition and the control over flux. For this reason in our system the smaller relative inhibition of the consumer prevails over the much higher relative activation of the producer.

PC F P

We tested both transitions, because, although we used in our previous studies [START_REF] Diolez | Modular regulation analysis of heart contraction: application to in situ demonstration of a direct mitochondrial activation by calcium in beating heart[END_REF] either G or P system, the G → P transition comprises the combined effect of pyruvate addition and glucose removal. On the other hand, the G → GP transition is equivalent to a pure effect of pyruvate addition. Anyway, both transitions are similar, indicating that the effect of glucose removal (when pyruvate is added) is minor.

This conclusion is supported by the studies on the P → GP transition, where no statistically significant effect on either RPP or [PCr] was observed (data not shown). This result strongly suggests that in the presence of saturating concentrations of pyruvate, glucose has negligible influence on the kinetic properties of the system.

The effect of lactate (G → GL transition) was very similar to that of pyruvate, although both metabolites exert opposite effect on the cytosolic NADH/NAD + ratio. This is demonstrated in Fig. 3.

The calculated proportional activation coefficient

for lactate equals -0.310. Again, as in the case of pyruvate, this means that the inhibition of the consumer is about three times smaller than the activation of the producer.

PC F P

In the presence of glucose, insulin activates directly ATP producer and has no direct effect on ATP consumer. This is clearly seen in Fig. 4 where the G → GI transition is presented. It is evident that the effect of insulin addition overlaps (within experimental error) with the consumer elasticity to PCr. Thus, the coefficient equals (approximately) zero (see Table 3). As it is explained in Fig. 1, this fact means a pure activation by insulin of ATP producer without the inhibiting effect on the consumer observed with pyruvate and lactate. In fact, when the metabolic control is shared by both the producer and consumer, a direct activation of the producer only will cause an increase in [PCr], which in turn indirectly activates the consumer and increases RPP. In such a case the slope of the response to some external effector (hormone, substrate) overlaps with the elasticity of the consumer to [PCr], as it can be seen in Fig. 4. In other words, this result simply means that the consumer is not activated directly, but only via the increase in [PCr] caused by the direct activation of the producer.

Generally, the present results demonstrate that the substrate/hormone composition of the perfusion medium has pronounced impact on the kinetic properties of oxidative phosphorylation in perfused heart.

Mechanism of pyruvate/lactate effect

The fact that pyruvate/lactate activates the ATP producer is something that could be expected, because as a substrate of the tricarboxic acid cycle it accelerates NADH production in the mitochondrial matrix, elevates the mitochondrial NADH/NAD + ratio and thus activates oxidative phosphorylation through a faster electron supply to the respiratory chain. However, perhaps the most surprising finding of the present study is the significant inhibition of the ATP consumer by pyruvate and lactate. This inhibition has nothing to do with the cytosolic redox potential, because pyruvate, being the 'oxidized form of lactate', lowers, through the lactate dehydrogenase, the cytosolic NADH/NAD + ratio, while lactate has the opposite effect. It is also not likely that the inhibition of the consumer results from a decrease in calcium transient, because this would also inhibit the producer. Finally, pyruvate/lactate did not change the cytosolic pH (roughly estimated from the hardly seen P i peak in the NMR spectrum). Therefore, it is difficult to determine the mechanism responsible for the inhibition of the PCr producer by pyruvate/lactate. We can only speculate that heart pacing, absent in our system, somehow prevents the inhibition of heart contractile activity by pyruvate/lactate (see below).

Our previous paper [START_REF] Diolez | Modular regulation analysis of heart contraction: application to in situ demonstration of a direct mitochondrial activation by calcium in beating heart[END_REF] and the present study demonstrate that, in our system, the PCr concentration in a heart perfused with pyruvate is much higher than in a heart perfused with glucose. Recently [START_REF] Korzeniewski | Physiological heart activation by adrenaline involves parallel activation of ATP usage and supply[END_REF] we speculated that with pyruvate the PCr/Cr ratio can be even as high as 10. (These intuitive expectations are probably somewhat exaggerated -computer simulations using the computer model of oxidative phosphorylation in heart [START_REF] Korzeniewski | Regulation of oxidative phosphorylation in intact mammalian heart in vivo[END_REF] suggest that the PCr/Cr ratio for pyruvate of about 2.5 would be able to account for our results [data not shown]). The present study offers a reliable mechanism that could be responsible for the discussed phenomenon. Namely, both activation of ATP producer and inhibition of ATP consumer increase the PCr/Cr ratio. The concomitant action of these two effects is able to elevate the PCr/Cr ratio to very high levels.

Reasons of lack of glucose effect

The lack of significant effects on the system of the addition of glucose in the presence of high levels of pyruvate, evidenced in the present study, is not surprising. Glucose is the substrate of glycolysis that produces just pyruvate. Continuous supply of pyruvate constitutes a metabolic 'shortcut' -glycolysis is not needed any more and it can be inhibited (for instance by a high ATP/ADP ratio, linked to a high PCr/Cr ratio, or low P i and AMP concentration). As it was formulated in [START_REF] Zweier | Substrate-induced alterations of high energy phosphate metabolism and contractile function in the perfused heart[END_REF], in the presence of pyruvate 'the numerous rate-limiting steps in glycolysis are avoided'. It was suggested in [START_REF] Kobayashi | Control of maximum rates of glycolysis in rat cardiac muscle[END_REF] that glycolysis is self-limiting at high fluxes by increasing the cytosolic NADH/NAD + ratio and thus blocking glyceraldehyde-3-phosphate dehydrogenase. Generally, in the presence of pyruvate glucose loses its significance as a respiratory substrate.

Of course it regains its importance in the absence of pyruvate and other respiratory substrates. In such a case an increase of glucose supply will of course mean a significant activation of the ATP producer. This is most probably the reason why insulin accelerates ATP synthesis, without having any direct influence on ATP usage (Fig. 4). Namely, it is a well known fact that one of the main functions of this hormone is to increase the glucose uptake by cells from blood. Indeed, it was observed that in isolated perfused heart insulin elevates glucose utilization [START_REF] Kashiwaya | Control of glucose utilization in working perfused rat heart[END_REF]. Even in our system, where glucose concentration in the perfusion medium is high, the effect of insulin is quite significant. Therefore, one could expect a stronger effect at lower, more physiological glucose concentrations.

It is worth to mention that the contribution of different substrates to ATP synthesis and oxygen consumption may change with workload. For instance, in the perfusion medium used in [START_REF] Santos | Metabolic control of contractile performance in isolated perfused rat heart. Analysis of experimental data by reaction:diffusiom mathematical model[END_REF] (glucose + insulin, lactate, oleate) lactate and oleate predominated at low workload (with negligible contribution of glucose, in agreement with our results), while after transition to high workload induced by adrenaline lactate and glucose became to have the highest contribution. This suggests that with adrenaline in the perfusate glucose addition in the presence of pyruvate or lactate would have a significant effect in our experimental system.

Effect of pyruvate/lactate -other studies

It has been shown, using PAA, that in isolated hepatocyte system a mixture of lactate and pyruvate activates both the ∆Ψ-producing and ∆Ψ-consuming block [START_REF] Korzeniewski | Proportional activation coefficients during stimulation of oxidative phosphorylation by lactate and pyruvate or by vasopressin[END_REF]. This finding differs from what we observe in the present study. However, one should bear in mind that the ATP-consuming system is completely different in beating heart and in liver. In particular, one of the most important ATP-using processes in liver is gluconeogenesis that is activated by its substrates, pyruvate and lactate. Additionally, gluconeogenesis is very sensitive to the ATP/ADP ratio [START_REF] Buttgereit | A hierarchy of ATP-consuming processes in mammalian cells[END_REF], unlike ATP usage in our system. Finally, hepatocytes are cells isolated from non-excitable tissue. This example demonstrates clearly that it makes little sense to compare the effect of the same factor (lactate/pyruvate) in completely different systems/conditions, and that different tissues may answer in a completely different way to such a factor.

It has been demonstrated by Zweier and Jacobus [START_REF] Zweier | Substrate-induced alterations of high energy phosphate metabolism and contractile function in the perfused heart[END_REF] that pyruvate, when compared with glucose, causes an increase in both [PCr] and RPP. A rough estimation by means of PAA using this effect and our elasticities of the producer and consumer to PCr leads to the conclusion that in this case pyruvate activated the producer, but did not have any effect on the consumer. Therefore, the authors did not obtain the inhibition of the consumer encountered in our present study. However, the perfusion systems used in both experiments were very different. In particular, in [START_REF] Zweier | Substrate-induced alterations of high energy phosphate metabolism and contractile function in the perfused heart[END_REF] electrically paced guinea pig hearts were used. In our opinion, this difference between [START_REF] Zweier | Substrate-induced alterations of high energy phosphate metabolism and contractile function in the perfused heart[END_REF] and our experiment would be mainly explained by this kind of stimulation. Moreover, we observed [unpublished data] that the kinetic properties could change in regard to the animal species (rat or guinea pigs).

Kobayashi and Neely [START_REF] Kobayashi | Control of maximum rates of glycolysis in rat cardiac muscle[END_REF] found that pyruvate and lactate had different effects on the bioenergetic system in perfused heart. However, again, those authors used the paced heart system.

A similar [PCr] (estimated from the ATP/ADP ratio) and RPP (but a significantly higher respiration rate) in hearts perfused with pyruvate in comparison with hearts perfused with glucose were encountered in [START_REF] Jeffrey | Respiratory control and substrate effects in the working rat heart[END_REF]. Thus, those findings contradict our data. The reason for this discrepancy is unclear. It may be the different [Ca 2+ ] in the perfusate: 1.2 mM in [START_REF] Korzeniewski | Proportional activation coefficients during stimulation of oxidative phosphorylation by lactate and pyruvate or by vasopressin[END_REF] and 2.0 mM in our study. In our previous study [START_REF] Diolez | Modular regulation analysis of heart contraction: application to in situ demonstration of a direct mitochondrial activation by calcium in beating heart[END_REF] an increase in [Ca 2+ ] in the perfusate from 2.0 mM to 3.5 mM changed to some extend the quantitative properties of the system. This is demonstrated in Fig. 5 where data concerning the G → P transition for [Ca 2+ ] = 3.5 mM extracted from [START_REF] Diolez | Modular regulation analysis of heart contraction: application to in situ demonstration of a direct mitochondrial activation by calcium in beating heart[END_REF] are presented. This figure constitutes only a crude approximation, because the data for glucose and pyruvate were obtained for different hearts. Nevertheless, one can see that in this case RPP slightly increases, which means that the relative inhibition of the consumer is here smaller than for [Ca 2+ ] = 2.0 mM (compare Fig. 1). Another possibility can be the fact that the ventricular pressure was imposed by balloon in our experiment and by afterload in [START_REF] Jeffrey | Respiratory control and substrate effects in the working rat heart[END_REF].

The above comparison of the results of different studies performed on perfused heart in different experimental systems testifies that the properties of the bioenergetic system in heart depend significantly on the experimental conditions. Therefore, the extrapolation of such studies to physiological conditions must be done with caution.

Linearity of response

For the quantitative analysis performed using the Proportional Activation Approach (PAA) it must be assumed that the response of the PCr producer and consumer to PCr is linear. In our previous study [START_REF] Korzeniewski | Physiological heart activation by adrenaline involves parallel activation of ATP usage and supply[END_REF] we demonstrated that the response of the producer is linear over a very wide range of PCr concentrations by using an increase in the balloon volume in the left ventricle that activates the consumer, and an administration of BDM that inhibits the consumer. The calculated elasticities of the producer to PCr were similar in both cases. In the present work we demonstrated the linearity of the response of the consumer to PCr by using three concentrations of NaCN, an inhibitor of the producer (see Figs. 2, 3 and4). Therefore, the discussed assumption is correct. The inhibitory effect of pyruvate on the PCr consumer may seem puzzling, but it is a result of our experimental data obtained in our system, and not of the theoretical method (PAA) used for the analysis of the data (as mentioned above, PAA applied to the data obtained in [START_REF] Zweier | Substrate-induced alterations of high energy phosphate metabolism and contractile function in the perfused heart[END_REF] shows no inhibition of the PCr producer by pyruvate in that system).

Mechanism of insulin effect

In our recent study [START_REF] Korzeniewski | Physiological heart activation by adrenaline involves parallel activation of ATP usage and supply[END_REF] we demonstrated that adrenaline activates both ATP production and ATP consumption to a similar extent. We hypothesized that this parallel activation was related to an increase in the calcium transient. However, this mechanism cannot work for insulin, because in this case only the ATP/PCr producer is activated (see Fig. 4). An increase in the calcium transient should cause an activation of both the producer and consumer. Therefore, it seems that the mechanisms of the action of adrenaline and insulin are completely different. This is not surprising, because the role of these hormones also seems to be different: adrenaline elevates the energy metabolism and heart work, while insulin only selectively increases the uptake by cardiomyocytes of one of the respiratory substrates: glucose. A similar reasoning holds for the action of pyruvate/lactate: the inhibition of the ATP/PCr consumer testifies that an increase in the calcium transient is not involved in the activation of the producer in this case.

Implications for control in health and disease

Our results clearly suggest that heart energetics in vivo is controlled not only by sympathetic/parasympathetic neural system and 'high-energy' hormones such as adrenaline, but also by the substrate composition of blood and the transport of substrates from blood to cardiac myocytes. In particular, pyruvate and lactate are used by heart preferentially in relation to glucose and activate NADH supply to the respiratory chain. The physiological relevance of the inhibition of the ATP consumption by pyruvate/lactate encountered in our unpaced system is not clear, because the heart in vivo is 'paced' by the sympathetic neural system and such inhibition is not seen in electrically paced perfused hearts. An increase in glucose content in blood (taking place e.g. in diabetes) by itself (at high levels of pyruvate/lactate) does not increase the glucose utilization by heart. Glucose uptake from blood is also diminished at low levels of insulin, also taking place in diabetes. This leads to the conclusion that the heart in diabetic individuals cannot use the excess of glucose as a preferential respiratory fuel and therefore contribute to a decrease in its concentration in blood. Perhaps this restriction could be overcome by lowering the pyruvate/lactate concentration in blood and/or increase in the adrenaline level.

Summary

Summing up, in the present study we demonstrate that the substrate/hormone composition of the perfusion medium has a pronounced influence on the kinetic properties of oxidative phosphorylation in perfused mammalian heart. In our system, with no heart pacing, pyruvate and lactate activate the ATP-producing block, but at the same time they significantly inhibit the ATPconsuming block. Glucose exerts essentially no effect in the presence of high pyruvate concentrations. Insulin (in the presence of glucose) activates only the ATP producer, probably through stimulating glucose uptake from the perfusion medium. Generally, our data suggest that various hormones (adrenaline and insulin) have very different mechanisms of action. It appears interesting to study the combined effects of physiological modulators of cardiac energetics in healthy and pathological hearts. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. A c c e p t e d M a n u s c r i p t corresponds to the initial state (before the action of the external effector), while the area surrounding this point represents all possibilities of external action. When the effect overlaps with the elasticity of the consumer to M (ε C ), only the producer is activated/inhibited. When the effect overlaps with the elasticity of the producer to M (ε P ), only the consumer is activated/inhibited. When the effect lies somewhere between ε C and ε P , both producer and consumer are affected (activated or inhibited), as indicated in the figure. [START_REF] From | Regulation of the oxidative phosphorylation rate in the intact cell[END_REF]. Gluc, glucose; Pyr, pyruvate. It can be clearly seen that pyruvate activates ATP producer and inhibits ATP consumer; however, the inhibition of consumer is smaller than for low (2.0 mM) calcium ion concentration in perfusate measured in the present study (compare Fig. 2).

  the consumer and producer to M, respectively, while τ is the response of the system to the factor F, defined as the relative change in the metabolite concentration divided by the relative change in flux: (∆M/M)/(∆J/J).

5 Biochemical

 5 Journal Immediate Publication. Published on 18 Aug 2009 as manuscript BJ20090395 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090395 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

  . Scaling for 100 % is important because it enables to compare different experiments and Proportional Activation Approach deals with relative changes in fluxes and metabolite concentrations. The values of RPP and [PCr] after particular transitions (modifications in the substrate/hormone composition) were scaled appropriately in relation to this reference point. Relative changes (in %) in RPP and [PCr] during particular transitions are shown in first two columns of Table

Fig. 1 .

 1 Fig. 1. Potential possibilities of activation/inhibition by an external effector of producer and consumer of an intermediate metabolite M within Proportional Activation Approach. 100 % for flux and [M]corresponds to the initial state (before the action of the external effector), while the area surrounding this point represents all possibilities of external action. When the effect overlaps with the elasticity of the consumer to M (ε C ), only the producer is activated/inhibited. When the effect overlaps with the elasticity of the producer to M (ε P ), only the consumer is activated/inhibited. When the effect lies somewhere between ε C and ε P , both producer and consumer are affected (activated or inhibited), as indicated in the figure.

Fig. 2 .

 2 Fig. 2. Effect of G → P and G → GP transition. ε C , elasticity of consumer to [PCr] determined by NaCN addition; ε P , elasticity of producer to [PCr] determined by increase in balloon volume; Gluc, glucose; Pyr, pyruvate. It can be clearly seen that pyruvate activates ATP producer, but inhibits ATP consumer.

Fig. 3 .

 3 Fig. 3. Effect of G → GL transition. ε C , elasticity of consumer to [PCr] determined by NaCN addition; ε P , elasticity of producer to [PCr] determined by increase in balloon volume; Gluc, glucose. It can be clearly seen that lactate activates ATP producer, but inhibits ATP consumer.

Fig. 4 .

 4 Fig. 4. Effect of G → GI transition. ε C , elasticity of consumer to [PCr] determined by NaCN addition; ε P , elasticity of producer to [PCr] determined by increase in balloon volume. It can be clearly seen that insulin activates only ATP producer.

Fig. 5 .

 5 Fig.5. Effect of G → GP transition for high (3.5 mM) calcium ion concentration in perfusate: data extracted from[START_REF] From | Regulation of the oxidative phosphorylation rate in the intact cell[END_REF]. Gluc, glucose; Pyr, pyruvate. It can be clearly seen that pyruvate activates ATP producer and inhibits ATP consumer; however, the inhibition of consumer is smaller than for low (2.0 mM) calcium ion concentration in perfusate measured in the present study (compare Fig.2).

Fig. 1 16

 1 Fig. 1

Fig. 2 Biochemical

 2 Fig. 2

Fig. 3

 3 Fig. 3

Fig. 5

 5 Fig. 5

Table 1 .

 1 Mechanical performance of hearts (rate-pressure product, RPP) and phosphocreatine concentrations ([PCr]) for control hearts (with glucose) and modified substrates/hormone compositions.Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

	_________________________________________________________________________________ transition RPP (mmHg.beat.min -1 ) [PCr] (mM)
	(number of hearts)	(mean ± SD)		(mean ± SD)
		control	after	control	after
		(glucose)	transition	(glucose)	transition
	_________________________________________________________________________________
	G → P (n = 7)	48031	35683	10.71	17.16
		± 9483	± 3783 **	± 1.00	± 1.27 ***
	G → GP (n = 6)	43879	33753	11.35	16.28
		± 6687	± 1956 ***	± 1.30	± 0.71 ***
	G → L (n = 5)	48075	40195	13.3	17.6
		± 5974	± 3634 **	± 0.6	± 1.0 ***
	G → GI (n = 6)	50734	57347	14.03	16.12
		± 2298	± 1097 **	± 1.17	± 1.35 ***
	_________________________________________________________________________________
	**P < 0.01 between control (glucose) and modified substrates/hormone composition
	***P < 0.001 between control (glucose) and modified substrates/hormone composition

Table 2 :

 2 Heart Rate (HR) and left ventricle pressure (LVP) for control hearts (with glucose) and modified substrates/hormone compositions. Biochemical Journal Immediate Publication. Published on 18 Aug 2009 as manuscript BJ20090395 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090395

	_________________________________________________________________________________ transition HR (beat min -1 ) L V P ( m m H g )
	(number of hearts)	(mean ± SD)		(mean ± SD)
		control	after	control	after
		(glucose)	transition	(glucose)	transition
	_________________________________________________________________________________
	G → P (n = 7)	285	258	172	138
		± 23	± 25 *	± 16	± 19 **
	G → GP (n = 6)	241	222	194	150
		± 39	± 20 *	± 2	± 32 *
	G → L (n = 5)	300	292	166	136
		± 16	± 35	± 15	± 17 ***
	G → GI (n = 6)	256	241	178	238
		± 16	± 7	± 25	± 8 **
	_________________________________________________________________________________
	* P < 0.05 between control (glucose) and modified substrates/hormone composition
	** P < 0.01 between control (glucose) and modified substrates/hormone composition
	*** P < 0.001 between control (glucose) and modified substrates/hormone composition

Table 3 .

 3 Averaged relative changes (in %) in RPP and [PCr] as well as calculated responses and proportional activation coefficients during transition from control (glucose) to modified substrate/hormone composition. Biochemical Journal Immediate Publication. Published on 18 Aug 2009 as manuscript BJ20090395 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090395

	______________________________________________________________________________
	transition	∆RPP (%) ± SD	∆[PCr] (%) ± SD	τ (∆PCr/PCr)	PC F P
				/(∆RPP/RPP)
	______________________________________________________________________________
	G → P G → GP G → L	-25.9 ± 8.4 -21.7 ± 9.0 -16.0 ± 5.0	+ 60.0 ± 2.4 + 45.8 ± 2.7 + 32.9 ± 10.8	-2.32 -2.11 -2.06	-0.296 -0.307 -0.310
	G → GI	+ 13.2 ± 5.1	+ 14.9 ± 1.5	+ 1.13	-0.012
	______________________________________________________________________________

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

ACKNOWLEDGEMENTS

This work was supported by the AFM (Association Française contre les Myopathies) and the Region Aquitaine, France

Biochemical Journal Immediate Publication. Published on 18 Aug 2009 as manuscript BJ20090395 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090395

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.