José Magalhães

Rita Ferreira

Maria J Neuparth

Paulo J Oliveira

Franklim Marques

António Ascensão

Vitamin E prevents hypobaric hypoxia-induced mitochondrial dysfunction in skeletal muscle

Keywords: high altitude, antioxidants, α-tocopherol, membrane integrity, mitochondrial respiration, apoptotic signaling

The effect of vitamin E on mice skeletal muscle mitochondrial dysfunction and oxidative damage induced by an in vivo acute and severe hypobaric hypoxic insult (48h at a barometric pressure equivalent to 8500m) was analyzed.

Twenty-four male mice were randomly divided into the following 4 groups (n=6): control (C), hypoxia (H), vitamin E (VE; 60mg.kg -1 i.p., 3 times/wk for 3 wks) and hypoxia+VE (HVE).

Significant increase in mitochondrial protein carbonyls groups (CGs) was found in H compared to C. Confirming previous observations from our group, hypoxia induced mitochondrial dysfunction, as identified by altered respiratory parameters. Hypoxia exposure increased Bax content and decreased Bcl-2/Bax ratio, although Bcl-2 content remained unchanged. Inner and outer mitochondrial membrane integrity were significantly affected by hypoxia exposure. However, vitamin E treatment attenuated the effect of hypoxia on mitochondrial oxidative phosphorylation and on the levels of CGs.

Vitamin E supplementation also re-established the Bax and Bcl-2/Bax ratio impairments caused by hypoxia, as well as the decreased inner and outer mitochondrial membrane integrity.

In conclusion, data suggest that vitamin E prevents the loss in mitochondrial integrity and function, as well as the increase in Bax content that suggests that mitochondria are involved on increased cell death induced by severe hypobaric hypoxia in mice skeletal muscle.

Introduction

It has been known for a long time that high-altitude exposure is an aggressive physiological stress which induces several cellular deleterious effects. Recent data evidenced that severe high-altitude hypoxia triggers increased oxidative stress with consequent damage inflicted to lipids, proteins and DNA [START_REF] Bailey | Intermittent hypoxic training: implications for lipid peroxidation induced by acute normoxic exercise in active men[END_REF][START_REF] Moller | Acute hypoxia and hypoxic exercise induce DNA strand breaks and oxidative DNA damage in humans[END_REF][START_REF] Singh | Effect of high altitude (7,620 m) exposure on glutathione and related metabolism in rats[END_REF][START_REF] Magalhaes | Oxidative stress in humans during and after 4 hours of hypoxia at a simulated altitude of 5500 m[END_REF][START_REF] Magalhaes | Acute and severe hypobaric hypoxia-induced muscle oxidative stress in mice: the role of glutathione against oxidative damage[END_REF]. Increased cellular oxidative stress is a favorable condition for the activation of apoptotic cellular death pathways [START_REF] Kowaltowski | Mitochondrial damage induced by conditions of oxidative stress[END_REF]. Apoptosis is a systematic and programmed dismantling of the cell into discrete packages, being a phenomenon that can involve signal transduction pathways originating from mitochondria. In fact, mitochondria are intimately involved in apoptosis as important pro-apoptotic proteins are located in the inter-membrane space; furthermore, mitochondrial are an active source of reactive oxygen and nitrogen species (RONS), which can also modulate the induction of cell death. Some mitochondrial proteins with an active role in the apoptotic process are cytochrome c and apoptosis inducing factor (AIF), which can be both released into the cytosol upon death stimuli, leading to cell death through distinct pathways [START_REF] Kroemer | The mitochondrial death/life regulator in apoptosis and necrosis[END_REF][START_REF] Crompton | The mitochondrial permeability transition pore and its role in cell death[END_REF]. The release of either AIF or cytochrome c from mitochondria is thought to be partially dependent on the formation of a specific channel within the inner mitochondrial membrane, usually known as permeability transition pore (PTP), leading to outer membrane rupture [START_REF] Crompton | The mitochondrial permeability transition pore and its role in cell death[END_REF]. Although some controversy surrounds the role of PTP in the control of mitochondria-mediated apoptosis, consistent data indicates that the formation of PTP complexes (formed by the adenine nucleotide translocator, the voltage-dependent anion channel and cyclophilin-D, among other proteins) on the inner mitochondrial membrane is involved in the release of pro-apoptotic proteins during apoptosis [see [START_REF] Crompton | The mitochondrial permeability transition pore and its role in cell death[END_REF][START_REF] Armstrong | The role of the mitochondrial permeability transition in cell death[END_REF]. It is also known that the conformational status of mitochondrial PTP is regulated by the content of Bcl-2 family proteins, which comprises both pro (i.e. Bax, Bad, tBid, Bak, Bok) and anti (Bcl-2, Bcl-XL, Bcl-w) apoptotic elements; the relative levels of each group (viz Bax/Bcl-2 ratio) determines the response to death stimuli [START_REF] Gross | BCL-2 family members and the mitochondria in apoptosis[END_REF]. Moreover, it is known that the enhanced production of RONS and the consequent oxidation of protein thiol groups, can contribute to PTP opening and determine the pore conductance and reversibility [START_REF] Kowaltowski | Mitochondrial permeability transition and oxidative stress[END_REF][START_REF] Vercesi | The role of reactive oxygen species in mitochondrial permeability transition[END_REF][START_REF] Castilho | The irreversibility of inner mitochondrial membrane permeabilization by Ca2+ plus prooxidants is determined by the extent of membrane protein thiol cross-linking[END_REF].

In a recent study from our laboratory [START_REF] Magalhaes | Acute and severe hypobaric hypoxia increases oxidative stress and impairs mitochondrial function in mouse skeletal muscle[END_REF], we reported that 48 hours of severe hypobaric hypoxia at a barometric pressure equivalent to 8500m induced mice skeletal muscle mitochondrial oxidative stress and damage, as well as respiratory deficit using substrates for both complex I and complex II. We also observed that vitamin E, a Hypobaric hypoxia-induced mitochondrial dysfunction 4 hypobaric hypoxia. However, to the best of our knowledge, the impact of severe hypobaric hypoxia at a barometric pressure equivalent to 8500m on the activation of the intrinsic, mitochondrial-dependent, pathway of apoptosis, in skeletal muscle was not described so far. Most particularly, one of the aims of the present work was to investigate if hypobaric hypoxia damages mitochondrial membranes. Based on previous studies in which mitochondrial oxidative stress and dysfunction elicited by other stimuli resulted in the increased susceptibility to apoptosis [START_REF] Childs | Doxorubicin treatment in vivo causes cytochrome C release and cardiomyocyte apoptosis, as well as increased mitochondrial efficiency, superoxide dismutase activity, and Bcl-2:Bax ratio[END_REF][START_REF] Ascensao | Moderate endurance training prevents doxorubicininduced in vivo mitochondriopathy and reduces the development of cardiac apoptosis[END_REF], it seems reasonable to hypothesize that skeletal muscle mitochondrial oxidative damage and respiratory dysfunction induced by hypobaric hypoxia plays a key role in tissue damage and is correlated with the activation of apoptosis. Given that vitamin E is one of the most important fat-soluble chain-breaking antioxidant in the body, another purpose of the present study was to analyze whether in vivo vitamin E treatment prevents or attenuates the impairment of mitochondrial respiration, membrane integrity and apoptosis induced by severe high-altitude hypobaric hypoxia in mice skeletal muscle.

Methods

Experimental Design

Twenty-four male Charles River CD 1, 8 wk aged mice were randomly divided into four groups (n=6) as follows: control (C), hypoxia (H), vitamin E and hypoxia+VE (HVE).

Two groups (VE and HVE) were intra-peritoneally (i.p.) treated with cumulative doses (three times a week during a course of three weeks, 60mg/Kg) of α-tocopherol acetate (vitamin E). The other two groups (C and H) received the correspondent vehicle solution (sterilized soybean oil) during the same period [17].

Immediately after the last injection, the animals of the C and VE groups were maintained at an atmospheric pressure of 101.3 kPa (760 mmHg) equivalent to sea level. Again immediately after the last injection and one at a time, the animals of the hypoxic groups (H and HVE) were acutely exposed during 48 hours inside a hypobaric chamber to a simulated atmospheric pressure of 35.2 kPa (265 mmHg) equivalent to an altitude of 8500m. The depressurization period to reach the simulated altitude of 8500m and the pressurization period back to sea level conditions lasted for 30 minutes each. All animals were kept at constant temperature (21-25ºC)

Preparation of skeletal muscle mitochondria

The animals were sacrificed by cervical dislocation and hindlimb muscles (soleus, gastrocnemius, tibialis anterior and quadriceps) were extracted for the preparation of isolated mitochondria. Skeletal muscle mitochondria were prepared by conventional methods involving differential centrifugation, as previously described by Tonkonogi and Sahlin [START_REF] Tonkonogi | Rate of oxidative phosphorylation in isolated mitochondria from human skeletal muscle: effect of training status[END_REF]. Briefly, muscles were immediately excised and minced in ice-cold isolation medium containing 100mM sucrose, 0.1mM EGTA, 50mM Tris-HCl, 100mM KCl, 1mM KH 2 PO 4 and 0.2% BSA, pH 7.4. Minced blood-free tissue was rinsed and suspended in 10 ml of fresh medium containing 0.2 mg/ml bacterial proteinase (Nagarse E.C.3.4.21.62, Type XXVII; Sigma) and stirred for 2 minutes. The sample was then carefully homogenized with a tightly fitted Potter-Elvehjen homogenizer and a Teflon pestle. After homogenization, three volumes of Nagarse-free isolation medium were added to the homogenate, which was then fractionated by centrifugation at 700xg for 10 minutes. The resulting pellet was removed and the supernatant suspension centrifuged at 10,000xg during 10 minutes. The supernatant was decanted and the pellet was gently resuspended in isolation medium (1.3mL per 100mg of the initial tissue) and centrifuged at 7,000xg for 3 minutes. The supernatant was discarded and the final pellet, containing the mitochondrial fraction, was gently resuspended (0.4µl.mg -1 initial tissue) in a medium containing 225mM mannitol, 75mM sucrose, 10mM Tris and 0.1mM EDTA, pH 7.4. All mitochondrial isolation procedures were performed at 0-4ºC. Mitochondrial protein concentration was spectrophotometrically estimated with the biuret method using bovine serum albumin as standard. The mitochondrial suspensions were used within 4 hours after the excision of the muscles and were maintained on ice (0-4ºC) throughout this period. There was no significant alteration of the mitochondrial RCR between the first and the last measurements of the same animal.

Aliquots of the mitochondrial suspension were separated and rapidly frozen at -80ºC for later biochemical analysis of vitamin E, protein carbonyl derivatives (CGs), Bax and Bcl-2.

Clinical

Measurement of mitochondrial respiratory activity

Mitochondrial respiratory function was polarographically measured using a Clark-type oxygen electrode (Hansatech DW 1, Norfork, UK). Reactions were conducted in a 0.75ml closed thermostated (25 ºC) and magnetically stirred glass chamber containing 0.5mg of mitochondrial protein in a reaction buffer containing 225mM mannitol 75mM sucrose, 10mM Tris, 10mM KCl, 10mM K 2 HPO 4 and 0.1mM EDTA, pH 7.5 in accordance to Tonkonogi et al [START_REF] Tonkonogi | Mitochondrial function and antioxidative defence in human muscle: effects of endurance training and oxidative stress[END_REF]. After 1-minute equilibration period, mitochondrial respiration was initiated by adding pyruvate (5mM) plus malate (2mM) or succinate (10mM) plus rotenone (4µM). State 3 respiration was determined after adding ADP to a final concentration of 200µM; state 4 respiration was measured as the rate of oxygen consumption which follows total ADP phosphorylation. The respiratory control ratio (RCR, the ratio between state 3 and state 4) and the ratio between the amount of ADP phosphorylated per oxygen consumed (ADP/O) were calculated according to Estabrook [START_REF] Estabrook | Mitochondrial respiratory control and the polarographic measurement of ADP/O ratios[END_REF] and by using 235 nmolO 2 /ml as the value for the solubility of oxygen at 25ºC. The lag phase, determined as the time in seconds reflecting the capacity of mitochondrial to depolarize and repolarize after ADP addition, was also measured for both malate+pyruvate (M-P) and succinate+rotenone (S-R) as substrates.

Oligomycin

(final concentration 1.5µg.ml -1 ) and carbonyl cyanide mchlorophenylhydrazone (CCCP, 2µM) were added during state 3 respiration with saturated amounts of ADP (final concentration 1mM).

Inner and outer mitochondrial membrane integrity

An estimate of the level of inner membrane integrity was made by measuring the ratio of uncoupled respiration induced by CCCP addition versus the rate in state oligomycin (CCCP/oligomycin) using M-P as substrates, according to Benamar et al. [START_REF] Benamar | Membrane integrity and oxidative properties of mitochondria isolated from imbibing pea seeds after priming or accelerated ageing[END_REF] with adaptations. It has been described that the ratio increases as the inner membrane impermeability to protons increases [START_REF] Benamar | Membrane integrity and oxidative properties of mitochondria isolated from imbibing pea seeds after priming or accelerated ageing[END_REF].

Outer-membrane integrity was also assayed according to Benamar et al. [START_REF] Benamar | Membrane integrity and oxidative properties of mitochondria isolated from imbibing pea seeds after priming or accelerated ageing[END_REF] (see Fig. according to Vatassery et al. [START_REF] Vatassery | Iron uncouples oxidative phosphorylation in brain mitochondria isolated from vitamin Edeficient rats[END_REF]. Briefly, 2ml ethanol containing 0.025% (w/v) butylated hydroxytoluene (BHT) and 0.1ml of 30% (w/v) ascorbic acid was added to an aliquot of 100µl mitochondrial extract. Tubes were cooled and 2ml of water were added followed by 2ml of hexane containing 0.025% (w/v) BHT. The hexane phase was separated out and evaporated down under a stream of nitrogen at 40ºC. The residue was re-dissolved in the mobile phase and analyzed by HPLC using the following conditions:

Column=Inertsil, 5 Si, 250x3mm (Varian); mobile phase=n-hexane with 3% dioxane; flow rate=0.7ml/min. α-tocopherol content was fluorimetrically detected (λ emission : 290nm, λ absortion : 330nm).

Analysis of Bax, Bcl-2 and protein carbonylation,

Equivalent amounts of proteins were separated by electrophoresis on a 15% SDS-PAGE gel, followed by blotting on a nitrocellulose membrane (Hybond-ECL; Amersham Pharmacia Biotech). After blotting, non-specific binding was blocked with 5% nonfat dry For protein carbonyl derivatives assay, a certain isolated mitochondrial volume (V) containing 20µg of protein was derivatized with dinitrophenylhydrazine (DNPH). Briefly, the sample was mixed with 1 V of 12% SDS plus 2 V of 20mM DNPH 10% TFA, followed by a 30 minutes of dark incubation, after which 1,5 V of 2M Tris / 18.3% of βmercaptoethanol were added. A negative control was simultaneously prepared for each sample. After diluting the derivatized proteins in TBS to obtain a final concentration of 0.001µg/µL, a 100ml volume was slot-blotted into a Hybond-PVDF membrane.

Immunodetection of carbonyls was then performed using rabbit anti-DNP (DAKO) as the first antibody (1:2000 dilution), and anti-rabbit IgG-Peroxidase (Amersham Pharmacia)

as the second antibody (1:2000 dilution).

For both referred assays, the bands were visualized by treating immunoblotts with ECL chemiluminescence reagents (Amersham, Pharmacia Biotech, Buckinghamshine, UK), according to the supplier's instructions, followed by exposure to X-ray films (Sigma, Kodak Biomax Light Film, St. Louis, USA). The films were analyzed with QuantityOne Software (Bio Rad). Optical density results are expressed as percentage variation of control values.

Statistical analysis

Mean and standard deviation were calculated for all variables in all groups. Two-way ANOVA was used to analyze the effect of severe hypobaric hypoxia and of vitamin E supplementation. A Bonferroni post-hoc test was done to further evaluate differences between group pairs. The Statistical Package for the Social Sciences (SPSS Inc. version 10.0) was used for all analysis. The significant level was set at 5%.

Results

Mitochondrial Vitamin E content

Skeletal muscle mitochondria from VE group showed a significant higher content of vitamin E than the C group (1.24±0.21 vs. 0.65±0.13 nmol.mg -1 of mitochondrial protein).

Mitochondria from both hypoxic groups (H and HVE) had a significant decrease in vitamin E levels when compared to their non-hypoxic counterparts (C and VE) (0.12±0.06 vs. 0.65±0.13 and 0.46±0.12 vs.

Mitochondrial inner and outer membrane integrity

As can be depicted from figure 2, mitochondria energized with M-P from the H group showed a significantly lower CCCP/oligomycin respiratory ratio when compared with all other groups (H vs. C, VE and HVE groups) indicating a higher inner membrane permeability to protons. However, vitamin E treatment attenuated the increased inner membrane permeability induced by hypoxia (H vs. HVE groups).

Bax and Bcl-2 mitochondrial content

In what concerns to the expression of proapoptotic protein Bax (Fig. 4), the results showed a significant increase in animals submitted to acute and severe hypoxia when compared with both the C (+32.02%) and the HVE group (+22.54%). There was no significant difference in the levels of Bax between C, VE and HVE groups. On the other hand, the levels of the antiapoptotic protein Bcl-2 did not differed significantly within all groups independently of the conditions. Therefore, the Bcl-2/Bax ratio (Fig. 4) revealed a significant decrease in the H compared with the C group. Additionally, our study also demonstrated that vitamin E treatment was able to attenuate the impact of this hypoxic oxidant insult on mitochondrial function and on apoptotic signalling of mice skeletal muscle. Nevertheless, considering that mitochondria were isolated from different hindlimb muscles (soleus, gastrocnemius, tibialis anterior, and quadriceps) characterized by distinct biochemical features, data should not be extrapolated to each muscle individually. It is also important to note that in vitro studies performed after isolation of mitochondria from animal models have the ability to identify pre-existent defects on mitochondrial bioenergetics that can provide valuable information on the effects observed in the living organism. The experimental protocol, that can involve the assessment of mitochondrial respiratory parameters, calcium loading capacity or alterations in protein expression, represents a powerful tool for generating convincing hypotheses regarding how mitochondria are affected by xenobiotics, pathologies or environmental alterations to which the intact system is exposed to.

Nevertheless, further complementary in vivo studies (viz. NMR) of mitochondrial metabolism would allow to take the next step of linking in vitro results with metabolic function in the intact organism [START_REF] Marcinek | Mitochondrial dysfunction measured in vivo[END_REF][START_REF] Conley | Mitochondrial dysfunction: impact on exercise performance and cellular aging[END_REF].

Regarding respiratory measurements, the decreased state 3 respiration and the increased phosphorylative lag phase obtained with energized mitochondria using both complex I and II substrates, as well as the increased rate of oxygen consumption during state 4 are consistent with data previously published by our group [START_REF] Magalhaes | Acute and severe hypobaric hypoxia increases oxidative stress and impairs mitochondrial function in mouse skeletal muscle[END_REF], and confirms that hypobaric hypoxic conditions induces mitochondrial dysfunction. Accordingly, the results also showed a decrease in RCR and ADP/O values, which are indicative of decreased coupling between respiration and ATP synthesis and decreased phosphorylation efficiency, respectively. The results from this and our previous work [START_REF] Magalhaes | Acute and severe hypobaric hypoxia increases oxidative stress and impairs mitochondrial function in mouse skeletal muscle[END_REF] indicate that hypoxia jeopardizes the ability of mitochondria to synthesize ATP. Moreover, both inner and outer membrane integrity assays revealed that the severe hypoxic exposure caused a decrease in the impermeability of these mitochondrial membranes. Altogether, these data suggest that severe hypobaric hypoxia may have inflicted mitochondrial functional and structural impairments through several distinct mechanisms. For example, the hypoxia-related reduced oxygen availability as electron acceptor seems to result in the accumulation of reducing equivalents throughout the components of the mitochondria electron transport chain. The end result elicits the impairment of the respiratory chain activity, the increase in the so-called electron leakage to oxygen, and the consequent enhanced production of RONS [START_REF] Kehrer | Cellular reducing equivalents and oxidative stress[END_REF][START_REF] Clanton | Hypoxia induced reactive oxygen formation in skeletal muscle[END_REF] resulting in increased mitochondrial oxidative damage to lipids and proteins, as demonstrated in ours and in other studies [START_REF] Magalhaes | Acute and severe hypobaric hypoxia increases oxidative stress and impairs mitochondrial function in mouse skeletal muscle[END_REF], which ultimately might compromise mitochondrial function. On the other hand, hypoxia may also increase calcium content in mitochondria, which accompanied with RONS and phosphate (Pi) accumulation may predispose to mitochondrial swelling and eventual PTP opening, thereby inducing membrane permeabilization, collapse of the transmembrane osmotic gradient and mitochondrial dysfunction [27]. Moreover, as suggested above, Pi tends to increase in hypoxic cells due to phosphocreatine cleavage, which may also had contributed to swelling and consequent increased permeability of both mitochondrial membranes. Nevertheless, another possibility that can not be excluded in the present study is that, due to swelling, hypoxic mitochondria might have become more prone to damage during the isolation procedure, which would also compromise mitochondrial integrity and function.

Clearly confirming that, at least in part, there is an oxidant-based aetiology for the referred mitochondrial dysfunction and membranes permeabilization, Vitamin E was considerably depleted in hypoxic mitochondria, and its supplementation was able to attenuate the mitochondrial impairments caused by hypoxia exposure, probably through an antioxidant-mediated effect. These data are in accordance with our previous observations [START_REF] Magalhaes | Acute and severe hypobaric hypoxia increases oxidative stress and impairs mitochondrial function in mouse skeletal muscle[END_REF].

Because inner and outer membrane permeation with or without physiological dependence of PTP is also suggested to be regulated by the pro and the anti-apoptotic members of Bcl-2 family [START_REF] Armstrong | The role of the mitochondrial permeability transition in cell death[END_REF][START_REF] Orrenius | Mitochondrial regulation of apoptotic cell death[END_REF], and mitochondrial dysfunction has been considered an earliest step of the intrinsic pathway of apoptosis [START_REF] Skulachev | Membrane-linked systems preventing superoxide formation[END_REF], we also studied that effect of the hypoxic insult on the expression of mitochondrial pro-and anti-apoptotic Bcl-2 family proteins (Bax and Bcl-2, respectively). In fact, the relative expression of these proteins in the mitochondrial outer membrane is thought to decide the apoptotic fate of the cell by regulating membrane integrity [START_REF] Crompton | The mitochondrial permeability transition pore and its role in cell death[END_REF][START_REF] Sreedhar | Heat shock proteins in the regulation of apoptosis: new strategies in tumor therapy: a comprehensive review[END_REF][START_REF] Reed | Bcl-2 family proteins and mitochondria[END_REF]. Data from our study demonstrated a significant increase in Bax content from non-supplemented hypoxic mice suggesting that, in addition to the mechanisms referred above, severe hypoxic insult can also increase Hypobaric hypoxia-induced mitochondrial dysfunction 13 channels [START_REF] Scorrano | Mechanisms of cytochrome c release by proapoptotic BCL-2 family members[END_REF]. Considering the formation of Bcl-2/Bax heterodimers as a mechanism to inhibit apoptosis [START_REF] Reed | Bcl-2 family proteins and mitochondria[END_REF], the imbalance between the expression of Bax and Bcl-2 in nonsupplemented hypoxic animals, expressed by the reduced Bcl-2/Bax ratio, suggests that severe hypobaric hypoxia can predispose cells to apoptosis. In clear contrast with our data, and probably reflecting an hypoxia severity-dependent phenomenon and/or the protective effect of the acclimatization process against skeletal muscle oxidative stress already demonstrated in our lab [START_REF] Magalhaes | Acute and chronic exposition of mice to severe hypoxia: the role of acclimatization against skeletal muscle oxidative stress[END_REF], Riva and coworkers [START_REF] Riva | Bcl-2/Bax protein expression in heart, slow-twitch and fast-twitch muscles in young rats growing under chronic hypoxia conditions[END_REF] showed an overexpression of Bax and Bcl-2 in skeletal muscle mitochondria of young rats growing under moderate chronic hypoxia conditions equivalent to ~6000m. In this case, the graduate and less severe level of hypoxia exposure was translated into an increase of the Bcl-2/Bax ratio allowing a better protection against apoptosis. In the present study, the expression of Bax was significantly depressed in vitamin E supplemented animals, when compared with non-supplemented mice, which corroborates the data from mitochondrial respiratory function.

In conclusion, it was demonstrated for the first time that severe hypoxic conditions resulted in decreased inner and outer mitochondrial membrane integrity with a presumable consequent triggering of the intrinsic pathway to apoptotic cell death. The fact that hypoxia increases the Bax/Bcl-2 ratio is worth of attention as it corroborates that hypoxia is able to trigger several phenomena conducing to cell death. Moreover, vitamin E attenuates the deleterious impact of this pro oxidant and pro apoptotic hypoxic insult in skeletal muscle mitochondria.

Considering the extreme severity of the hypoxic insult, the present results may have many implications in distinct areas of biology and medicine, particularly in chronic heart and lung disease, as this level of hypoxia may represent an insult causing systemic and tissue impairments characteristic of the referred pathophysiological conditions [START_REF] Clanton | Yet another oxygen paradox[END_REF].

Studies regarding the impact of less severe hypobaric hypoxic conditions equivalent to those that most of the humans face on mitochondrial function are still lacking, although data regarding the impact of such hypoxic conditions on whole muscle tissue, blood or plasma oxidative stress and damage markers suggest that less intense alterations would probably occur at the mitochondrial level.

Clinical

  lipophilic antioxidant, was able to attenuate mitochondrial dysfunction induced by Clinical Science Immediate Publication. Published on 18 Jun 2007 as manuscript CS20070075 © 2007 The Authors Journal compilation © 2007 Portland Press Ltd

  on a daily light schedule of 12 hours of light vs. dark with normal activity. Mice were provided with food and water ad libitum during the experimental protocol and were sacrificed 49 hours after the last injection. The Clinical Science Immediate Publication. Published on 18 Jun 2007 as manuscript CS20070075 © 2007 The Authors Journal compilation © 2007 Portland Press Ltd Hypobaric hypoxia-induced mitochondrial dysfunction 5 Ethics Committee of the Scientific Board of Faculty of Sport Sciences approved the study.

  Science Immediate Publication. Published on 18 Jun 2007 as manuscript CS20070075 © 2007 The Authors Journal compilation © 2007 Portland Press Ltd Hypobaric hypoxia-induced mitochondrial dysfunction 6

4 )

 4 . Briefly, 30 µM cytochrome c reduced by 10 mM ascorbate originated an initial oxygen consumption corresponding to the activity of a fraction of mitochondria with damaged outer membranes. The complete rupture of the outer membrane in mitochondria was achieved by the addition of 0.05 % (v/v) Triton X100. The final addition of 0.5 mM Clinical Science Immediate Publication. Published on 18 Jun 2007 as manuscript CS20070075 © 2007 The Authors Journal compilation © 2007 Portland Press Ltd Hypobaric hypoxia-induced mitochondrial dysfunction 7 cyanide made possible the measurement of the non-enzymatic oxidation of cytochrome c. The percentage of mitochondrial outer membrane integrity was calculated according to the following formula: Outer membrane integrity = 100% -% of outer membrane rupture, where: 100% integrity = (respiration after cytochrome c + triton) -(respiration after cyanide) % of outer membrane rupture = respiration after cytochrome c -respiration after cyanide Biochemical assays in mitochondria extracted fraction Vitamin E concentration α-tocopherol was determined by high performance liquid chromatography (HPLC)

  milk in TBS-T [Tris-buffered saline (TBS) with Tween 20] and the membrane was incubated with either anti-Bcl-2 (1:500; sc-7382 mouse monoclonal IgG; Santa Cruz Biotechnology) or anti-Bax (1:500; sc-493 rabbit polyclonal IgG; Santa Cruz Biotechnology) antibodies for 2 hours at room temperature, washed and incubated with Clinical Science Immediate Publication. Published on 18 Jun 2007 as manuscript CS20070075 © 2007 The Authors Journal compilation © 2007 Portland Press Ltd Hypobaric hypoxia-induced mitochondrial dysfunction 8 secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (1:1000; Amersham Pharmacia Biotech) for 2 hours.

  The intactness of the mitochondrial outer membrane was quantified by measuring the cytocrome c-dependent oxygen consumption (Fig 3). If mitochondria have intact outer membranes, exogenously added reduced cytochrome c cannot be oxidized by cytocrome c oxidase on the inner membrane as it cannot cross the outer membrane. Clinical Science Immediate Publication. Published on 18 Jun 2007 as manuscript CS20070075 © 2007 The Authors Journal compilation © 2007 Portland Press Ltd Hypobaric hypoxia-induced mitochondrial dysfunction 10However, when the outer membrane is damaged, the oxidation can occur with an increased rate of oxygen consumption by mitochondria. Thus, the higher rate of oxygen consumption upon cytochrome c addition to mitochondrial suspension in H compared to other groups is indicative of the elevated permeability induced by severe hypobaric hypoxia on skeletal muscle mitochondria. In agreement with protection observed for other parameters, vitamin E attenuated the increased outer membrane permeability caused by hypobaric hypoxia (H vs. HVE).

  Vitamin E supplementation was able to counteract the triggering of the apoptotic pathway induced by acute and severe hypobaric hypoxia. ** INSERT FIG. 4 **DiscussionThe results of the present study provided additional insights into the effects of vitamin E treatment against the impact of an in vivo acute and severe hypobaric hypoxic insult on mice skeletal muscle mitochondria. Data confirms the mitochondrial respiratory derangements and increased oxidative damage which was previously described by our own group[START_REF] Magalhaes | Acute and severe hypobaric hypoxia increases oxidative stress and impairs mitochondrial function in mouse skeletal muscle[END_REF]. Nevertheless, the present study is the first to show the relationship between impaired skeletal muscle mitochondrial function, increased inner and outer mitochondrial membrane permeability and also increased Bax expression caused by 48 hours of in vivo severe hypobaric hypoxia exposure. Data showed that the hypoxic insult Clinical Science Immediate Publication. Published on 18 Jun 2007 as manuscript CS20070075 © 2007 The Authors Journal compilation © 2007 Portland Press Ltd Hypobaric hypoxia-induced mitochondrial dysfunction 11 induced depletion on mitochondrial vitamin E content, increased protein oxidation, impaired mitochondrial respiratory function and decreased inner and outer mitochondrial membrane integrity, while decreased Bcl-2/Bax ratio due to increased Bax expression.

Clinical

  Science Immediate Publication. Published on 18 Jun 2007 as manuscript CS20070075 © 2007 The Authors Journal compilation © 2007 Portland Press Ltd Hypobaric hypoxia-induced mitochondrial dysfunction 12

  outer membrane permeability due to Bax translocation from cytosol to mitochondrial membranes and subsequent homodimeryzation into high molecular weight complexes which can cause cytochrome c release[START_REF] Reed | Bcl-2 family proteins and mitochondria[END_REF][START_REF] Wei | Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death[END_REF][START_REF] Mikhailov | Association of Bax and Bak homo-oligomers in mitochondria. Bax requirement for Bak reorganization and cytochrome c release[END_REF] through the formation of pores or Clinical Science Immediate Publication. Published on 18 Jun 2007 as manuscript CS20070075 © 2007 The Authors Journal compilation © 2007 Portland Press Ltd

  Science Immediate Publication. Published on 18 Jun 2007 as manuscript CS20070075 © 2007 The Authors Journal compilation © 2007 Portland Press Ltd

Figure 1 .

 1 Figure 1. Hypoxia and vitamin E treatment effects on mice skeletal muscle mitochondrial protein carbonyl groups. Control plus placebo solution of sterile soybean oil (C); hypobaric hypoxia plus placebo solution of sterile soybean oil (H); control plus vitamin E treatment (VE) and hypoxia plus vitamin E treatment (HVE). Data represent mean ± SD and are expressed percentage of the control value. *p<0.05, H vs. both C and HVE; **p<0.05, HVE vs. VE.

Figure 2 .

 2 Figure 2. Hypoxia and vitamin E treatment effects on mice skeletal muscle outer (black bars) and inner (white bars) mitochondrial membrane integrity. Control plus placebo solution of sterile soybean oil (C); hypobaric hypoxia plus placebo solution of sterile soybean oil (H); control plus vitamin E treatment (VE) and hypoxia plus vitamin E treatment (HVE). Data represent mean ± SD. *p<0.05, H vs C, VE and HVE; **p<0.05, HVE vs H.

Figure 3 .

 3 Figure 3. Oxygraphic analysis of outer membrane integrity (cytocrome c permeation assay) of mitochondria from control plus placebo solution of sterile soybean oil (C); hypobaric hypoxia plus placebo solution of sterile soybean oil (H); control plus vitamin E treatment (VE) and hypoxia plus vitamin E treatment (HVE). The arrows show the addition of compounds with their final reaction medium concentrations. Numbers under the traces indicate the rates of oxygen consumption in nmol O 2 . min -1 . mg protein -1 .

Figure 4 .

 4 Figure 4. Hypoxia and vitamin E treatment effects on mice skeletal muscle mitochondrial Bax (white bars), Bcl-2 (black bars) and Bcl-2/Bax ratio (gray bars). Control plus placebo solution of sterile soybean oil (C); hypobaric hypoxia plus placebo solution of sterile soybean oil (H); control plus vitamin E treatment (VE) and hypoxia plus vitamin E treatment (HVE). Data represent mean ± SD and are expressed percentage of the control value. A scan of a representative Bax and Bcl-2 western blotting for each group (n=4) is immediately below the histogram. *p<0.05, H vs. both C and HVE.

Science Immediate Publication. Published on 18 Jun 2007 as manuscript CS20070075 © 2007 The Authors Journal compilation © 2007 Portland Press Ltd

	Hypobaric hypoxia-induced mitochondrial dysfunction 9
	protein). However, the vitamin E content of the H group was significantly lower than the
	HVE group.
	Mitochondrial protein oxidative damage
	Hypoxia exposure inflicted significant protein oxidation, as observed by the oxidation of
	protein carbonyls (Fig 1). A significant increase in mitochondrial CGs was found in the H
	group when compared to the C group. Vitamin E protected mitochondria from CGs
	formation induced by hypobaric hypoxia, although the values found on the HVE group
	were still higher that on the VE group.
	Hypoxia significantly increased the phosphorylation lag-phase with both M-P
	(87.0±11.03 seconds) and S-R (70.20±10.22 seconds) when compared to control
	(60.24±8.01 and 45.80±6.76 seconds, respectively). Vitamin E treatment significantly
	reduced the augmented phosphorylation lag phase caused by hypoxia (to 73.20±8.21
	with M-P and 51.0±6.01 seconds with S-R).

1.24±0.21 nmol.mg -1 of mitochondrial Clinical ** INSERT FIG. 1 **

Standard mitochondrial respiratory parameters

Mitochondrial respiration was significantly impaired in H as demonstrated by decreased state 3, respiratory control ratio (RCR) and ADP/O, as well as by the enhanced state 4 using both complex I and II-linked substrates. However, vitamin E treatment attenuated the effect of hypoxia on state 3, RCR, ADP/O and abolished hypoxia-induced changes in state 4 with M-P as substrates (data not shown).


MD Giovanni Di Salvo

PhD, MAHA, FESC Giuseppe Pacileo

MD Giuseppe Limongelli

PhD, FESC Marina Verrengia

MD Alessandra Rea

MD Giuseppe Santoro

MD Simona Gala

MD Biagio Castaldi

Pio Antonello D'andrea

Caso

MD Antonello D'andrea

PhD, FESC, # Pio Caso

MD, FESC Maria Giovanna Russo

MD Raffaele Calabro'

Abnormal regional myocardial deformation properties and increased aortic stiffness in normotensive patients with aortic coarctation despite successful correction: an ABPM, standard echocardiography and strain rate imaging study

Keywords: Aortic Coarctation and strain rate imaging Aortic coarctation, ventricular function, strain rate imaging. 2

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Introduction. The long-term follow-up data subsequent to a successful repair of aortic coarctation (AoC) show that life expectancy remains reduced in AoC patients [START_REF] Cohen | Coarctation of the aorta: long-term follow-up and prediction of outcome after surgical correction[END_REF][START_REF] Clarkson | Results after repair of coarctation of the aorta beyond infancy: a 10 to 28 years follow-up with particular reference to late systemic hypertension[END_REF]. Late arterial hypertension, more often systolic, which occurs in nearly one third of patients after repair, and atherosclerosis are the main determinants of cardiovascular events [START_REF] Cohen | Coarctation of the aorta: long-term follow-up and prediction of outcome after surgical correction[END_REF][START_REF] Clarkson | Results after repair of coarctation of the aorta beyond infancy: a 10 to 28 years follow-up with particular reference to late systemic hypertension[END_REF][START_REF] Seirafi | Repair of aortic Coarctation during infancy minimizes the risk of late hypertension[END_REF]. Coronary heart disease, stroke, sudden cardiac death, and heart failure account for the majority of premature deaths [START_REF] Cohen | Coarctation of the aorta: long-term follow-up and prediction of outcome after surgical correction[END_REF][START_REF] Clarkson | Results after repair of coarctation of the aorta beyond infancy: a 10 to 28 years follow-up with particular reference to late systemic hypertension[END_REF][START_REF] Seirafi | Repair of aortic Coarctation during infancy minimizes the risk of late hypertension[END_REF] , and prognosis is related to the age at intervention [START_REF] Cohen | Coarctation of the aorta: long-term follow-up and prediction of outcome after surgical correction[END_REF][START_REF] Clarkson | Results after repair of coarctation of the aorta beyond infancy: a 10 to 28 years follow-up with particular reference to late systemic hypertension[END_REF][START_REF] Seirafi | Repair of aortic Coarctation during infancy minimizes the risk of late hypertension[END_REF] . Other studies suggest that AoC might not be simply a mechanical obstruction of the aorta, but more likely a generalized disease of the cardiovascular system [START_REF] Meyer | Predicting the risk of early atherosclerotic disease development in children after repair of aortic Coarctation[END_REF].

Despite this, previous studies based on standard echocardiography demonstrated normal or increased systolic cardiac function in patients after successful repair of AoC [START_REF] Kimball | Persistent hyperdynamic cardiovascular state at rest and during exercise in children after successful repair of Coarctation of the aorta[END_REF][START_REF] Kimball | Persistent ventricular adaptations in postoperative Coarctation of the aorta[END_REF][START_REF] Moskowitz | Altered systolic and diastolic function in children after "successful" repair of Coarctation of the aorta[END_REF][START_REF] Carpenter | Left ventricular hyperkinesias at rest and during exercise in normotensive patients 2 to 27 years after Coarctation repair[END_REF][START_REF] Leandro | Ambulatory blood pressure monitoring and left ventricular mass and function after successful surgical repair of Coarctation of the aorta[END_REF] . New tools, such as the ultrasonically derived strain rate (SR) imaging, have been added to our capabilities [START_REF] Sutherland | Strain and strain rate imaging: a new clinical approach to quantifying regional myocardial function[END_REF][START_REF] D'hooge | Regional strain and strain rate measurements by cardiac ultrasound: principles, implementation and limitations[END_REF] and it is now possible to detect early subclinical myocardial abnormalities [START_REF] Weidemann | The variation of morphological and functional cardiac manifestation in Fabry disease: potential implications for the time course of the disease[END_REF][START_REF] Wong | Alterations of left ventricular myocardial characteristics associated with obesity[END_REF][START_REF] Poulsen | Relation between plasma amino-terminal propeptide of procollagen type III and left ventricular longitudinal strain in essential hypertension[END_REF][START_REF] Mottram | Usefulness of B-type natriuretic peptide in hypertensive patients with exertional dyspnea and normal left ventricular ejection fraction and correlation with new echocardiographic indexes of systolic and diastolic function[END_REF][START_REF] Poulsen | Doppler tissue imaging reveals systolic dysfunction in patients with hypertension and apparent "isolated" diastolic dysfunction[END_REF][START_REF] Yuda | Myocardial abnormalities in hypertensive patients with normal and abnormal left ventricular filling: a study of ultrasound tissue characterization and strain[END_REF].The myocardial deformation properties in patients after AoC repair have not yet been described. We have sought to evaluate the myocardial deformation properties in normotensive AoC children late after successful repair and to assess their relationship with arterial stiffness.

Methods

Study Sample. We studied consecutive AoC patients regularly followed at our outpatient clinic from June 2003 to September 2006 without: 1. major associated cardiovascular abnormalities (such as ventricular septal defect and aortic and mitral valve functional abnormalities); 2. evidence of recoarctation (>20 mmHg pressure gradient at continuous Doppler in the aortic arch and the presence of a diastolic tail) [START_REF] De Divitiis | Ambulatory blood pressure, left ventricular mass, and conduit artery function late after successful repair of Coarctation of the aorta[END_REF][START_REF] De Divitiis | Vascular dysfunction after reapir of Coarctation of the aorta: imact of early surgery[END_REF]; 3. evidence of aortic aneurysm at the last outpatient visit, and 4. hypertension.

A patient was assumed to be "normotensive", if: 1. he was without anti-hypertensive drug treatment, 2. showed systolic (SBP) and diastolic blood pressure (DBP) < 90° percentile for age, sex, and height at office measurements; 3. 24h-SBP and DBP were < 90° percentile for age, sex, and height, and 4. showed no hypertension during exercise.

If at least one of these signs were positive, the patient was classified to be "hypertensive" and excluded from the study.

Control group. We also studied, by identical protocol, 83 age-and sexcomparable subjects without detectable cardiovascular risk factor. Volunteer controls were all recruited in Naples (Italy), were selected from our departments of pediatric cardiology among children investigated for dizziness, or minor orthostatic complaints, or for sport eligibility. None of the control subjects had family history of cardiomyopathy, cardiovascular structural or functional abnormalities or received any medication.

All studies were performed in accordance with the rules of the Ethical Committee, Second University of Naples. All parents gave their written informed consent to participate in the study.

Clinical Assessment. During a one-day stay, anamnestic and anthropometric data were reviewed. Resting BP was measured at all extremities by an automatic oscillometric cuff device (Dinamap, Critikon Inc., Tampa, FL, USA).

Ambulatory Blood Pressure Measurements. The 24-h ambulatory blood pressure (BP) was measured on the right arm (Space Labs Inc., Issaquah, WA, USA). BP measurements were recorded automatically every 15 min from 8:00 a.m. to 20:00 p.m. and every 30 min from 20:00 p.m. to 8:00 a.m.. BP studies were excluded if there was an interval of 2 h of invalid or absent measurements. Hypertension at ABPM was defined as 24 hSBP or 24h-DBP over the 90th percentile of the reference values provided by Soergel et al. [START_REF] Soergel | Oscillometric 24-h blood pressure values in healthy children and adolescents: a multicenter trial including 1141 subjects[END_REF]. To allow interpretation of data, by ABPM normative data [START_REF] Soergel | Oscillometric 24-h blood pressure values in healthy children and adolescents: a multicenter trial including 1141 subjects[END_REF] only patients with a height between 115 and 185 cm were included.

Exercise test.

An exercise test was performed on a bicycle in a sitting position according to international guidelines [START_REF] Gibbons | ACC/AHA 2002 guideline update for exercise testing: summary article: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee to Update the 1997 Exercise Testing Guidelines)[END_REF]. The WHO protocol was used starting with 25 Watt and increasing work load by 25 Watt every 2 minutes [START_REF] Fletcher | Exercise standards for testing and training: a statement for healthcare professionals from the American Heart Association[END_REF] . ECG was monitored continuously; blood pressure was measured manually every 2 minutes.

Hypertension during exercise was present when the peak systolic blood pressure [mmHg] was more than 2 SD above the age and work load dependent reference value In this studies 2 echocardiographers were involved. All the echocardiographic examinations were performed by the same observer (echocardiographer 1). All the echo measurements were performed off-line on digitally stored raw data by another investigator (echocardiographer 2) who was blind to the clinical status of the studied subjects. The ascending aorta and the aortic arch were visualized by means of high, long axis view and suprasternal view. Left ventricular measurements were taken from twodimensional guided M-mode tracings.

Parameters measured by echocardiography were pressure gradient throughout the former coarctation region and the left ventricular and aortic morphology. Left ventricular mass indexed for height 2.7 (LVMI) was calculated [START_REF] De Simone | Left ventricular mass and body size in normotensive children and adults: assessment of allometric relations and impact of overweight[END_REF]. LV end-diastolic and end-systolic volumes and the LVEF at rest were computed from apical 4 and 2 chamber views, using a modified Simpson's biplane method. Each representative value was obtained from the average of three measurements. The endocardial mean velocity of circumferential fiber shortening corrected for the heart rate (VCFc) and end systolic circumferential stress [START_REF] Douglas | Comparison of echocardiographic methods for assessment of left ventricular shortening and wall stress[END_REF] were calculated.

To assess global LV and right ventricular (RV) longitudinal function, from the standard apical view, the atrioventricular ring displacement was measured for the septal and lateral mitral ring (MAPSE) and lateral tricuspid ring (TAPSE) by conventional M-mode methods.

Using pulsed-wave Doppler, mitral inflow velocities, peak early diastolic velocity (E), peak late diastolic velocity (A), E/A ratio, E deceleration time (DT), A duration, and isovolumic relaxation time (IVRT) were measured.

Echocardiography of the Aorta. Thoracic aortic diameters (mm/m 2 ) were measured 3 cm above the aortic valve by two dimensional guided M-mode transthoracic echocardiography of the aortic root at left parasternal long-axis view. Aortic systolic diameter (AoS) was measured at the time of full opening of the aortic valve, and diastolic diameter (AoD) at the peak of the QRS complex at the simultaneous electrocardiogram recording. Aortic stiffness index was calculated as follows:

(ln(SBP/DBP) x (AoS-AoD)/AoD [START_REF] Ikonomidis | Aortic elastic properties and left ventricular diastolic function in patients with adamantiades-Behcet's disease[END_REF].

Color Doppler Myocardial Imaging Study (CDMI). CDMI data to assess longitudinal

function were recorded from the interventricular septal and the left ventricular (LV) lateral walls from standard apical view.

Radial function for the LV posterior wall was recorded using parasternal long axis view. All data were acquired at a frame rate of 200±15 frames/s (GE Vingmed System Seven; 3.5 MHz). This frame rate was necessary to resolve cardiac mechanical events and to average out the influence of any random noise in the Doppler velocity signal. An appropriate velocity scale was chosen in order to avoid CDMI data aliasing. The narrowest image sector angle possible (usually 30°) was used to achieve the maximum color Doppler frame rate possible. For apical views, care was taken to maintain each wall in the center of the ultrasound sector in an attempt to align at as near as possible to the direction of longitudinal motion. For parasternal long axis view, care was paid to keeping the posterior LV wall perpendicular to the ultrasound beam in order to be aligned as near as possible to radial motion.

As breath holding is not feasible in young patients data from 3 consecutive cardiac cycles (to be used for subsequent analysis) were recorded during normal quiet respiration. CDMI data were stored in digital format and transferred to a computer workstation for the off-line analysis of regional SR curves. This was carried out using dedicated software (GE Workstation). Longitudinal SR's were estimated by measuring the spatial velocity gradient over a computation area of 10 mm. LV regional longitudinal function was evaluated on the apical, mid and basal segments of septal and lateral LV walls. We chose those LV walls because we had the best alignment with the ultrasound beam.

LV radial SR's were estimated using a computation area of 5 mm, from the mid segment of the LV posterior wall.

To derive SR profiles from the different segments, the region of interest was maintained in a constant position within the segment being interrogated by using a semi-automatic tracking algorithm.

The timing of ventricular ejection and relaxation was obtained using an anatomic grey scale M-mode cursor positioned visually in the underlying grey scale data set. The timing of these events was measured by placing the cursor at the level of aortic cusps, and mitral valve. From the averaged SR data, peak systolic SR was measured. We decide to measure only SR because this is the less-load dependent contractility index obtained by CDMI data [START_REF] Greenberg | Doppler-derived myocardial systolic strain rate is a strong index of left ventricular contractility[END_REF][START_REF] Weidemann | Myocardial function defined by strain rate and strain during alterations in inotropic states and heart rate[END_REF].

Pulsed-wave tissue Doppler of the septal annulus was used for the measurement of early peak diastolic mitral annular velocity (e'). The E/e' ratio was calculated.

Statistical Analysis. All the analyses were performed using a commercially available package (SPSS , Rel 11.0 2002. Chicago: SPSS inc).

Data are presented as the mean value ± SD. The normality Kolmogorov-Smirnov test was performed to determine whether continuous variables were normally distributed. Age at the time of repair, which presented a skewed distribution, is presented as the median value (range). Qualitative data were compared using Mantel-Haenszel' s test. Continuous variables were compared using paired t-test and Wilcoxon matched pairs test. The correlations were studied by linear regression analysis. In addition, to identify significant predictors of average LV peak systolic SR in AoC patients, their individual association with clinical relevant variables was assessed by multiple regression analyses. The following variables were included into the analysis: clinical data (age, age at repair, 24hsystolic blood pressure) and echocardiographic-Doppler indexes (LVMI, aortic stiffness index, E wave-DT, A wave duration, E\e'). These variables were selected according to results of univariate analysis and to their clinical relevance and potential impact on prognosis, as shown by earlier studies [START_REF] Cohen | Coarctation of the aorta: long-term follow-up and prediction of outcome after surgical correction[END_REF][START_REF] Clarkson | Results after repair of coarctation of the aorta beyond infancy: a 10 to 28 years follow-up with particular reference to late systemic hypertension[END_REF][START_REF] Seirafi | Repair of aortic Coarctation during infancy minimizes the risk of late hypertension[END_REF][START_REF] Meyer | Predicting the risk of early atherosclerotic disease development in children after repair of aortic Coarctation[END_REF][START_REF] Kimball | Persistent hyperdynamic cardiovascular state at rest and during exercise in children after successful repair of Coarctation of the aorta[END_REF][START_REF] Kimball | Persistent ventricular adaptations in postoperative Coarctation of the aorta[END_REF][START_REF] Moskowitz | Altered systolic and diastolic function in children after "successful" repair of Coarctation of the aorta[END_REF][START_REF] Carpenter | Left ventricular hyperkinesias at rest and during exercise in normotensive patients 2 to 27 years after Coarctation repair[END_REF][START_REF] Leandro | Ambulatory blood pressure monitoring and left ventricular mass and function after successful surgical repair of Coarctation of the aorta[END_REF]. The assumption of linearity was checked graphically by studying the smoothed martingal residuals from the null model plotted against the covariate variables. The linearity assumptions were satisfied. The Hosmer-Lemeshow goodness of fit test was used to check that the model adequately fit the data.

Model was cross validated by the bootstrap technique (200 runs) [START_REF] Efron | An Introduction to the Bootstrap[END_REF].

The null hypothesis was rejected for a p value <0.01.

Reproducibility between the 2 echocardiographers was determined in 60 randomly selected subjects (30 patients and 30 controls). Inter-and intraobserver variability was examined by using both Pearson's bivariate two-tailed correlations and Bland-Altman analysis. Relation coefficients, ninety-five percent confidence limits and percent errors were reported.

Results

In this study 83 AoC normotensive patients were included, of these, 45% had a bicuspid aortic valve without significant functional abnormalities (only trivial to mild aortic regurgitation, or peak pressure gradient at continuous Doppler < 20 mmHg). AoC repair had been performed at a median age of 4 months (range 0.1 to 156 months), with 58% of subjects undergoing surgery within the 4 months of life (mean age 12 ± 4 years, male 70%). All were in the typical juxtaductal position. Repair was by end-to-end anastomosis in 48% of patients, by subclavian flap in 13%, by patch angioplasty in 16%, and by primary percutaneous stent implantation in 19%. The median time from coarctation repair to the study was 100 months (range 16 to 216 months).

Clinical characteristics of the study sample are shown in Table 1.

Standard echocardiographic study (Table 2) demonstrated an increased wall thickness and a significant increase in LVMI in AoC patients. LV ejection fraction, VCFc, were significantly increased in AoC patients. End systolic stress was significantly reduced in AoC patients (Table 2).

Among the studied diastolic parameters, E wave, DT, and A wave duration were significantly prolonged in AoC patients. The E\e' ratio was significantly increased in patients.

The stiffness of the aortic wall as assessed by aortic stiffness index was significantly increased in patients (p<0.0001).

Radial peak systolic SR was significantly increased in AoC patients (Table 3), while regional longitudinal peak systolic SRs were significantly reduced in AoC patients (Table 3). Among AoC patients, 16 (19%) were outside the 95% lower confidence limits in the normal group.

Patients surgically treated showed a LV EF of 72 ± 8%, a MAPSE-septal wall of 13±3mm, a LVMI of 47±17 g/m 2.7 ,and an average peak systolic longitudinal SR of 1.4±0.5 1\s. AoC patients who underwent primary stent implantation showed LV EF of 74 ± 7%, a MAPSE-septal wall of 13±5 mm, a LVMI of 55±16 g/m 2.7 , and an average peak systolic longitudinal SR of 1.5±0.6 1\s.

Determinans of Diastolic Function.

E\A was significantly correlated with age at repair (p:0.004; r: -45), LVMI (p:0.001; r: -36), and 24h-DBP (p:0.002; r: -48). E\e' was significantly correlated with age at surgery (p:0.003; r: -34), LVMI (p:0.004; r: -33), and aortic stiffness index (p:0.001; r: -55).

Determinans of Myocardial Deformation Properties.

At univariate analysis, average peak systolic longitudinal SR showed a significant correlation with age at repair (r 2 : 0.68; p< 0.001), age (r 2 : 0.13; p<0.001), LVMI (r 2 : 0.20; p<0.001), aortic stiffness index (r 2 : 0.61; p<0.001, and 24h-SBP (r 2 : 0.15; p<0.001) (Figure 1).

At multilinear regression analysis, the only independent predictors of LV average peak systolic longitudinal SR were age at correction (p: 0.005; Coeff.: -0.201; SE: 0.027) and aortic stiffness index (p=0.0029; Coeff.: 0.334; SE: 0.423).

Reproducibility. Pearson's correlations: Peak systolic SR: r = 0.88, p = 0.0001; Peak systolic S: r = 0.92, p = 0.00001. Bland-Altman analysis: Peak systolic SR (95% CI+3.1, per cent error 3.5%); Peak systolic S (95% CI+2.5, per cent error 2.3%).

Discussion

To the best of our knowledge, this is the first published study of SR imaging in AoC patients. Regional longitudinal systolic myocardial deformations are significantly reduced in a large sample of AoC young patients, despite a successful correction and in presence of a (super) normal LV ejection fraction and an increased VCFc/stress. The degree of this impairment is significantly related with age at correction and aortic stiffness.

Left Ventricular Function in AoC Patients: Increased systolic function and enhanced standard echocardiographic indexes of contractility have been reported in patients after successful repair of AoC during childhood [START_REF] Kimball | Persistent hyperdynamic cardiovascular state at rest and during exercise in children after successful repair of Coarctation of the aorta[END_REF][START_REF] Kimball | Persistent ventricular adaptations in postoperative Coarctation of the aorta[END_REF][START_REF] Moskowitz | Altered systolic and diastolic function in children after "successful" repair of Coarctation of the aorta[END_REF][START_REF] Carpenter | Left ventricular hyperkinesias at rest and during exercise in normotensive patients 2 to 27 years after Coarctation repair[END_REF], a group known to have an increased incidence of LV hypertrophy, systemic hypertension, and a significant late mortality risk [START_REF] Cohen | Coarctation of the aorta: long-term follow-up and prediction of outcome after surgical correction[END_REF][START_REF] Clarkson | Results after repair of coarctation of the aorta beyond infancy: a 10 to 28 years follow-up with particular reference to late systemic hypertension[END_REF][START_REF] Seirafi | Repair of aortic Coarctation during infancy minimizes the risk of late hypertension[END_REF][START_REF] Meyer | Predicting the risk of early atherosclerotic disease development in children after repair of aortic Coarctation[END_REF]. However, the mechanism underlying this apparent persistent elevation of myocardial contractility is difficult to explain. Chronic ventricular hypertrophy is known to be associated with contractile dysfunction [START_REF] Shimizu | Left ventricular chamber filling and midwall fiber lengthening in patients with left ventricular hypertrophy: overestimation of fiber velocities by conventional midwall measurements[END_REF][START_REF] Shimizu | Left ventricular midwall mechanics in systemic arterial hypertension. Myocardial function is depressed in pressure-overload hypertrophy[END_REF][START_REF] Palmon | Intramural myocardial shortening in hypertensive left ventricular hypertrophy with normal pump function[END_REF][START_REF] Schwartzkopff | Heart failure on the basis of hypertension[END_REF], thus the enhanced myocardial contractility found in AoC patients may be artifactual [START_REF] Gentles | Misrepresentation of left ventricular contractile function by endocardial indexes: Clinical implications after Coarctation repair[END_REF] . Indeed, all those studies [START_REF] Kimball | Persistent hyperdynamic cardiovascular state at rest and during exercise in children after successful repair of Coarctation of the aorta[END_REF][START_REF] Kimball | Persistent ventricular adaptations in postoperative Coarctation of the aorta[END_REF][START_REF] Moskowitz | Altered systolic and diastolic function in children after "successful" repair of Coarctation of the aorta[END_REF][START_REF] Carpenter | Left ventricular hyperkinesias at rest and during exercise in normotensive patients 2 to 27 years after Coarctation repair[END_REF][START_REF] Leandro | Ambulatory blood pressure monitoring and left ventricular mass and function after successful surgical repair of Coarctation of the aorta[END_REF] suggesting elevation of myocardial contractility after successful repair of AoC are in contrast with results of both animal models -with experimentally induced pressure overload [START_REF] Aoyagi | Myocardial function in immature and mature sheep with pressure-overload hypertrophy[END_REF][START_REF] Aoyagi | Transition from compensated hypertrophy to intrinsic myocardial dysfunction during development of left ventricular pressure overloadhypertrophy in conscious sheep. Systolic dysfunction precedes diastolic dysfunction[END_REF][START_REF] Mirsky | The contractile state as a major determinant in the evolution of left ventricular dysfunction in the spontaneously hypertensive rat[END_REF] -and studies on humans with hypertension [START_REF] De Simone | Left ventricular chamber and wall mechanics in the presence of concentric geometry[END_REF][START_REF] Wolf | The midwall stress velocity relationship during manipulation of afterload[END_REF]. In those studies LV hypertrophy is commonly associated with a gradual deterioration of LV function and contractility.

Most of the previous studies assessing myocardial contractility in AoC patients used endocardial stress-velocity indexes which can overestimate myocardial performance and contractility, especially in hypertrophied hearts [27;29;31; 39-40].

This misrepresentation of fiber mechanics by those standard echocardiographic derived indexes has been found to be clinically important in subjects with LV hypertrophy caused by systemic hypertension [30;32;34;39-40] resulting in a failure to recognize depressed fiber shortening.

Other studies using midwall index of contractility, thus providing a physiological correction, still showed evidence of an unexplained hypercontractile state in AoC patients [START_REF] Gentles | Misrepresentation of left ventricular contractile function by endocardial indexes: Clinical implications after Coarctation repair[END_REF]. However, it has been demonstrated that midwall stress-velocity relationship at low afterload has a similar behavior to the endocardial stress-velocity relationship [START_REF] Wolf | The midwall stress velocity relationship during manipulation of afterload[END_REF]. LV contractility [START_REF] Greenberg | Doppler-derived myocardial systolic strain rate is a strong index of left ventricular contractility[END_REF][START_REF] Weidemann | Myocardial function defined by strain rate and strain during alterations in inotropic states and heart rate[END_REF]. In several studies the new ultrasonic derived deformation indexes demonstrated a higher ability than standard echocardiography to unmask preclinical systolic abnormalities [START_REF] Weidemann | The variation of morphological and functional cardiac manifestation in Fabry disease: potential implications for the time course of the disease[END_REF][START_REF] Wong | Alterations of left ventricular myocardial characteristics associated with obesity[END_REF][START_REF] Poulsen | Relation between plasma amino-terminal propeptide of procollagen type III and left ventricular longitudinal strain in essential hypertension[END_REF][START_REF] Mottram | Usefulness of B-type natriuretic peptide in hypertensive patients with exertional dyspnea and normal left ventricular ejection fraction and correlation with new echocardiographic indexes of systolic and diastolic function[END_REF][START_REF] Poulsen | Doppler tissue imaging reveals systolic dysfunction in patients with hypertension and apparent "isolated" diastolic dysfunction[END_REF][START_REF] Yuda | Myocardial abnormalities in hypertensive patients with normal and abnormal left ventricular filling: a study of ultrasound tissue characterization and strain[END_REF]. In hypertensive patients with apparently normal systolic function, and in presence of a normal diastolic function, the application of SR imaging demonstrated the presence of a preclinical LV systolic abnormalities [START_REF] Wong | Alterations of left ventricular myocardial characteristics associated with obesity[END_REF][START_REF] Poulsen | Relation between plasma amino-terminal propeptide of procollagen type III and left ventricular longitudinal strain in essential hypertension[END_REF][START_REF] Mottram | Usefulness of B-type natriuretic peptide in hypertensive patients with exertional dyspnea and normal left ventricular ejection fraction and correlation with new echocardiographic indexes of systolic and diastolic function[END_REF][START_REF] Poulsen | Doppler tissue imaging reveals systolic dysfunction in patients with hypertension and apparent "isolated" diastolic dysfunction[END_REF][START_REF] Yuda | Myocardial abnormalities in hypertensive patients with normal and abnormal left ventricular filling: a study of ultrasound tissue characterization and strain[END_REF].

Myocardial Deformation Properties in

Of note, in our study abnormal systolic function was detected studying myocardial deformation along the longitudinal axis, while myocardial deformation properties along the radial axis were increased. Indeed, peak systolic radial SR is expression of midwall fiber contraction and in presence of low afterload has a similar behavior to the endocardial stress-velocity relationship, showing evidence of an unexplained hypercontractile state in AoC patients [START_REF] Gentles | Midwall shortening after Coarctation repair: the effect of through-plane motion on singleplane indices of left ventricular function[END_REF]. This is in agreement with a recent report demonstrating increased midwall fractional shortening and decreased longitudinal shortening in 15 AoC patients late after successful correction, using magnetic resonance imaging [START_REF] Gentles | Midwall shortening after Coarctation repair: the effect of through-plane motion on singleplane indices of left ventricular function[END_REF].

In our study sample endocardial end systolic stress, index of afterload [START_REF] Meyer | Predicting the risk of early atherosclerotic disease development in children after repair of aortic Coarctation[END_REF], was reduced. This finding is not surprising and is in agreement with previous studies [START_REF] Meyer | Predicting the risk of early atherosclerotic disease development in children after repair of aortic Coarctation[END_REF][START_REF] Moskowitz | Altered systolic and diastolic function in children after "successful" repair of Coarctation of the aorta[END_REF] . In a mathematical model [START_REF] Sutherland | Strain and strain rate imaging: a new clinical approach to quantifying regional myocardial function[END_REF], keeping normal myocardial contractility and reducing afterload, SR increased. Conversely in our study, longitudinal peak systolic SR was significantly reduced even in presence of a reduced end-systolic stress, suggesting an impairment in myocardial longitudinal function. This finding may explain, at least in part, the reduced exercise duration in AoC patients compared to controls (Table 1). These abnormalities are more frequently found in the precoarctation aorta and proximal arteries even in patients after early and successful repair [18; 41-42].

Aortic stiffness in

In our study, longitudinal deformation impairment was significantly correlated with the age at correction and aortic stiffness index .

These findings suggest that although early repair has a beneficial effect, it cannot change the innate structural and functional abnormalities of the proximal aorta and their deleterious effect on myocardial function [START_REF] Vriend | Increased carotid and femoral intima-media thickness in patients after repair of aortic coarctation: influence of early repair[END_REF]. Indeed, despite early repair the estimated survival in postedecoarctectomy patients is still reduced with 80% of the surgical patients alive at 40-50 years after surgery [1; 43].

Study Limitations. This study carries some limitations: 1-Patient sample: We included in our study also AoC patients in medical treatment but this corresponds to the usual clinical situation encountered in hospital practice. However, we excluded patients under beta blockers to avoid any effect of drugs on myocardial contractility. 2-Echo study: The usual limitations inherent in the angle dependency of all echocardiographic Doppler techniques also apply to ultrasound derived SR imaging. In this study, care was taken to always align the ultrasound beam with the direction of myocardial deformation to be interrogated and we included in the study only the walls with the best alignment.

  SD = [111.2 + (0.310 • age [years] )]+ (0.334 • work rate [Watt]) ± 2 • 17.9 Standard Echocardiographic Evaluation. Echocardiography measurements were taken with a System Seven (GE, Norway).

  AoC patients. Using for the first time peak systolic SR we demonstrated the presence of an abnormal longitudinal systolic function in AoC patients. SR is a new parameter recently proposed as a strong index of

  AoC patients. Aortic stiffness index was significantly increased in AoC patients in agreement with previous studies demonstrating abnormalities in vascular structure and function in patients after successful decoarctectomy.

Table 1 .

 1 General Characteristics of Studied Sample.

	AoC Patients Controls	P
	(n=83)	(n=83)	Value

18 Clinical

Science Immediate Publication. Published on 3 May 2007 as manuscript CS20070085 © 2007 The Authors Journal compilation © 2007 Biochemical SocietyTable 2 .

 2 Standard Echocardiographic Characteristics of Studied Sample.

	Clinical
		AoC Patients	Controls	P
		(n=83)	(n=83)	Value
	IVS end-diastole (mm)	11±2	7±2	<0.0001
	LV end-diastole (mm)	40±7	40±7	0.9
	LVPW end-diastole (mm)	9±2	7±2	<0.0001
	LV ejection fraction (%)	68±8	64±7	0.0001
	LVMI (g/m 2.7 )	49± 20	33± 14	<0.0001
	VCFc (circ/sec)	1.3±0.2	1.1±0.2	<0.0001
	End Systolic Stress (g/cm 2 )	38±14	46±12	0.001
	MAPSE-septal wall (mm)	12±5	13±3	0.05
	MAPSE-lateral wall (mm)	12±4	15±6	0.11
	TAPSE (mm)	24±5	22±6	0.14
	E/A	1.8± 0.6	1.7±0.1	0.06
	E-DT (msec)	239 ± 57	150 ± 15	<0.0001
	A duration (msec)	97 ± 22	86 ± 17	0.001
	E/e'	7 ± 2	5 ± 1	<0.0001
	IVRT (msec)	48± 19	50±16	0.36
	Aortic stiffness index	12±9	6±4	<0.0001
				19

Science Immediate Publication. Published on 3 May 2007 as manuscript CS20070085 © 2007 The Authors Journal compilation © 2007 Biochemical Society Table

  2 legend. IVSd: interventricular septum; LV: left ventricle; PW: posterior wall; VCFc: velocity of circumferential fiber shortening corrected for the heart rate. MAPSE: Mitral annulus peak systolic excursion; TAPSE: Tricuspid annulus peak systolic excursion.E-DT: E wave deceleration time; IVRT: Isovolumic relaxation time. E\A: ratio between early (E) and late (A) transmitral peak diastolic velocities. E\e': ratio between peak early transmitral diastolic velocity (E) and peak early diastolic mitral annular velocity (e').

20

Clinical

Science Immediate Publication. Published on 3 May 2007 as manuscript CS20070085 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 3 .

 3 Peak Systolic Strain Rate (1/s) Values of the Studied Sample. Linear regression between: A average peak systolic longitudinal strain rate (SR) and age (years); B average peak systolic longitudinal strain rate (SR) and left ventricular mass/ height 2.7; C average peak systolic longitudinal strain rate (SR) and age at repair (months); D average peak systolic longitudinal strain rate (SR) and aortic stifness; E average peak systolic longitudinal strain rate (SR) and 24h systolic blood pressure (24h-SBP).

	AoC Patients	Controls	P
	(n=83)	(n=83)	Value

Clinical Science Immediate Publication. Published on 3 May 2007 as manuscript CS20070085 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 3 May 2007 as manuscript CS20070085 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 3 May 2007 as manuscript CS20070085 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 3 May 2007 as manuscript CS20070085 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 3 May 2007 as manuscript CS20070085 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 3 May 2007 as manuscript CS20070085 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 3 May 2007 as manuscript CS20070085 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 3 May 2007 as manuscript CS20070085 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 3 May 2007 as manuscript CS20070085 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 3 May 2007 as manuscript CS20070085 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 3 May 2007 as manuscript CS20070085 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 3 May 2007 as manuscript CS20070085 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 3 May 2007 as manuscript CS20070085 © 2007 The Authors Journal compilation © 2007 Biochemical Society