Younss Ait Mou

PhD Cyril Reboul

Lucas André

PhD; Alain Lacampagne

PhD Olivier Cazorla
email: olivier.cazorla@inserm.fr

MsD Younss Ait Mou

MsD Younss Ait Mou

MsD Lucas Andre

Younss Aït Mou

Younss Aït Mou

transmural myocardial function in rats with ischaemic heart failure

Keywords: end-stage heart failure, myocardial contractility, contraction, exercise, myocardial infarction

INTRODUCTION

After myocardial infarction (MI), the heart is characterized by reduced contractility and impaired filling resulting from changes in cardiac structure (hypertrophy, dilatation), cell death, maladaptative remodeling of the extracellular matrix, abnormal energy metabolism and cellular dysfunction. The underlying cellular mechanisms that have been suggested include altered calcium homeostasis, 1 impaired myofilament calcium responsiveness, [2][3][4] and reduced crossbridge cycling rate. 5 A hierarchy of cellular events during the development of heart failure (HF) has been proposed, starting with electrical remodeling and altered Ca 2+ homeostasis during the early phase followed by myofilament dysfunction. 6 Moreover, transmural myocardial contractile performance is non-uniform across the different layers of the LV wall. 7,8 This non-uniformity of cardiac function plays a fundamental role in cardiac mechanical work. Its loss has been previously reported to be a sensitive index to discriminate physiological from pathological left ventricular (LV) remodeling. 8 We previously reported that HF affects preferentially the contractile machinery of the sub-endocardial cells (ENDO), leading to loss of transmural heterogeneity. 3 In clinic, pharmacological treatments are used to maintain as much as possible the pump function of the failing heart. However non-pharmacological approaches may be attempted. For example, many clinical studies have shown that exercise training performed by patients with HF is beneficial for heart performance and quality of life. 9,[START_REF] Joshi | Exercise training in the management of cardiac failure and ischaemic heart disease[END_REF] In the normal heart of small [START_REF] Palmer | Shortening and [Ca2+] dynamics of left ventricular myocytes isolated from exercise-trained rats[END_REF] and large [START_REF] Hinken | Porcine cardiac myocyte power output is increased after chronic exercise training[END_REF] animals, exercise training increases cellular contractile function such as cardiomyocyte shortening, Ca 2+ dynamics and myocyte power-generating capacity, and myocardial perfusion capacity. [START_REF] Cohen | Coronary and collateral blood flows during exercise and myocardial vascular adaptations to training[END_REF] There are also clinical evidences that exercise after MI has a beneficial effect on disease progression and survival. 9,[START_REF] Joshi | Exercise training in the management of cardiac failure and ischaemic heart disease[END_REF] A recent study has shown that early

CVR-2008-687R1 3
exercise in MI mice had no effect on LV remodeling but attenuates global LV dysfunction, which can be essentially explained by the exercise-induced improvement of myofilament function. [START_REF] De Waard | Early exercise training normalizes myofilament function and attenuates left ventricular pump dysfunction in mice with a large myocardial infarction[END_REF] However after a small MI, exercise has either no effect or improved LV function independently of the starting point of exercise (early or late after MI). [START_REF] De Waard | Early exercise training normalizes myofilament function and attenuates left ventricular pump dysfunction in mice with a large myocardial infarction[END_REF] For large infarct, exercise can have detrimental effects when performed at an early stage while beneficial effects on LV remodeling and function appear when exercise is started late after MI. [START_REF] Gaudron | Effect of endurance training early or late after coronary artery occlusion on left ventricular remodeling, hemodynamics, and survival in rats with chronic transmural myocardial infarction[END_REF][START_REF] Kubo | Exercise at ventilatory threshold aggravates left ventricular remodeling in patients with extensive anterior acute myocardial infarction[END_REF] In animal models, most of the studies tested the effect of exercise on LV global and/or cellular functions, 4 weeks after MI, when LV remodeling is still ongoing. [START_REF] Wisloff | Aerobic exercise reduces cardiomyocyte hypertrophy and increases contractility, Ca2+ sensitivity and SERCA-2 in rat after myocardial infarction[END_REF][START_REF] Krenz | Impact of beta-myosin heavy chain expression on cardiac function during stress[END_REF] Moreover, it has been shown in rat models that 3-5 weeks after MI exercise capacity is still high and close to shamoperated animals while it decreased significantly from 10 weeks after MI. [START_REF] Trueblood | Biphasic temporal pattern in exercise capacity after myocardial infarction in the rat: relationship to left ventricular remodeling[END_REF] Thus the cellular adaptations between early and late exercise after MI may be different.

Moreover

Animals and exercise training

Myocardial Infarction was produced by permanent ligation of the left coronary artery in male Wistar rats as previously described. 3 Thirteen weeks post-MI, rats were randomly assigned to the sedentary (MI) or 5 weeks treadmill exercised (MI-Ex, 40 min/day, 5 days/week, 16 m/min) groups. At the end of the training protocol, morphological and functional parameters were determined by echocardiography and compared to sham-operated animals. Animals were then sacrificed for cellular investigations.

Echocardiography and noninvasive hemodynamics

Doppler-echocardiography was performed with a MyLab 30 (ESAOTE, Italy).

Wall Thicknesses and Left Ventricular End Diameters (LVED) were obtained at the level of the papillary muscles. LV shortening fraction and End Systolic strain of the posterior wall, measured as deformation from end-diastole to end systole [(PWTs -PWTd)/ PWTd]*100 were calculated. PWTs and PWTd are respectively end systolic and end diastolic Posterior Wall Thickness. [START_REF] Turschner | The sequential changes in myocardial thickness and thickening which occur during acute transmural infarction, infarct reperfusion and the resultant expression of reperfusion injury[END_REF] Ascending aortic blood flow was recorded, as previously described, [START_REF] Reboul | Cardiac remodeling and functional adaptations consecutive to altitude training in rats: implications for sea level aerobic performance[END_REF] via pulsed-wave Doppler permitting measurements of the aortic blood flow velocity time integrals (VTI Ao ). LV sub-endocardial (ENDO) and sub-epicardial (EPI) posterior wall displacements were measured offline at the level of the papillary muscles. Tissue Doppler imaging (TDI) was performed after each conventional echocardiography as previously described. [START_REF] Borenstein | An ovine model of chronic heart failure: echocardiographic and tissue Doppler imaging characterization[END_REF]

Contractile properties in intact cardiomyocytes

Single LV cardiomyocytes were isolated by enzymatic dissociation from the remaining inner free wall (ENDO) and from the outer free wall (EPI) as previously described (n=5 rats per group). [START_REF] Cazorla | SR33805, a Ca2+ antagonist with length-dependent Ca2+ -sensitizing properties in cardiac myocytes[END_REF] Unloaded cell shortening and calcium concentration [Ca 2+ ] (indo 1 dye) were studied using field stimulation (0.5Hz, 22°C, 1.8 mM external Ca 2+ ). Sarcomere length (SL) and fluorescence (405 nm and 480 nm) were simultaneously recorded (IonOptix system, Hilton, USA).

Force measurements in permeabilized cardiomyocytes

Isometric force was measured in single permeabilized cardiomyocytes at different [Ca 2+ ], at 1.9 then 2.3 m SL as described previously (n=5 rats per group). 3,[START_REF] Cazorla | SR33805, a Ca2+ antagonist with length-dependent Ca2+ -sensitizing properties in cardiac myocytes[END_REF] Force was normalized by the cross-sectional area measured from the imaged cross-section. Force-pCa relations were fitted to a Hill equation. To prevent degradation, all solutions contained protease inhibitors (PMSF: 0.5 mmol/L; leupeptin: 0.04 mmol/L and E64: 0.01 mmol/L).

Myosin-Heavy chain composition

MyHC isoforms were separated on a 6% SDS-PAGE and silver stained as previously described. [START_REF] Cazorla | Effects of highaltitude exercise training on contractile function of rat skinned cardiomyocyte[END_REF] -MyHC content was expressed relative to the total amount of MyHC protein.

Western Immunoblotting

SERCA2a and phospholamban (PLB) were separated on a gradient SDS-PAGE (2-20%) and then blotted onto nitrocellulose membrane. Proteins were revealed with specific antibodies and were expressed relative to Calsequestrin content.

Troponin I (TnI) was separated by 15% SDS-PAGE and the protein kinase A phosphorylated form of cardiac TnI was normalized by the total TnI form as previously described. 3 Myosin light chain 2 (MLC-2) phosphorylated and nonphosphorylated forms were separated by a 10% urea gel and were specifically detected with a cardiac MLC-2 antibody. 3 Immunodetection was revealed with ECL Plus system.

Statistical analysis

Data were analyzed using one-way or 2-way ANOVA between groups. When significant interactions were found, a Bonferroni t-test was applied with adjusted P < 0.05 (Sigmastat 3.5). Data are presented as mean±SEM.

Results

In vivo cardiac morphological and functional parameters

MI animals had large antero-lateral infarcts detected by visual inspection. In a preliminary longitudinal study (data not shown), we observed that in our model, most of the morphological and functional alterations occurred over 11-13 weeks. Minimal changes will occur after this period. Thirteen weeks after MI, LV diameters were increased in MI at the end-diastolic and end-systolic phases without hypertrophy of the posterior wall thickness (Figure 1A). The posterior wall end systolic strain during a cardiac cycle was largely reduced (-80%) in MI animals (Figure 1B). In sham animals, the LV sub-endocardial layer contracted faster (+50% of the section motion)

than the sub-epicardial layers (Figure 1C). Heart failure significantly decreased contraction velocities of both EPI and ENDO layers by 15 and 32%, respectively, homogenizing the transmural velocities of contraction toward the slowest values. The global cardiac systolic function was also altered as shown by the reduction in fractional shortening (FS) (Figure 1D). Since the calculation of FS, measured at the level of papillary muscle, is highly affected by the akinetic infarct zone, we measured the aortic blood-flow velocity time integral (VTI Ao ) as another index of systolic function. VTI Ao was also decreased after MI (Figure 1D). Additional weeks after MI corresponding to the training duration had little impact on these parameters (see below).

Effect of exercise on in vivo cardiac function

We then investigated the effect of exercise training while most of the remodeling and dysfunction of the heart had already occurred. Eighteen weeks post-MI, hearts were hypertrophied as shown by the increase in the heart weight/body weight ratio between Sham and MI animals and further increased after exercise training (Table 1). Part of this exercise-induced hypertrophy may be explained by an increase of the posterior wall thickness although the difference did not reach significance (p= 0.069) (Table 1). The main effect of exercise on LV morphologies was a decrease of both end-systolic and end-diastolic left ventricular diameters (Table 1). Eighteen weeks after MI, we observed a large decrease in the end systolic strain (from 83±4% to 24±11% in sham and MI, respectively) and in the absolute ENDO posterior wall displacement between diastole and systole (from 2.7±0.2 mm to 1.3±0.2 mm in sham and MI, respectively). The values obtained were similar to the one measured 13 weeks post-MI confirming the stability of these indexes during this phase of the disease (Figure 2B). Exercise had a significant beneficial effect on the end systolic strain (63±7%) and on the ENDO posterior wall displacement (1.8±0.4 mm). Sm was similarly decreased between 13 and 18 weeks after MI in both regions.

Exercise restored Sm completely in EPI and only partially in the ENDO layers (Figure 2C). The gradient of velocity across the LV defined as the difference of velocity between ENDO and EPI, observed in normal conditions (1.66±0.11 cm.s -1 ), disappeared almost completely in MI animals (0.28±0.05 cm.s -1 ) and was partially restored in MI-Ex (0.76±0.05 cm.s -1 ) (Figure 2C). Finally, VTI Ao , used as an accurate index of LV stroke volume, decreased by 37% in MI animals and was partially restored by exercise in MI-EX remaining 14% lower than sham (Figure 2D). Exercise had no impact on the mortality of the animals (Figure 2E).

Effect of exercise on myocyte shortening and Ca 2+ transient

The effect of exercise on the excitation-contraction coupling of long-term MI was tested in intact LV unloaded cardiomyocytes. For this purpose, sarcomere length shortening and intracellular calcium content were simultaneously measured on field stimulated cardiomyocytes. Unloaded contraction decreased significantly with HF only in ENDO cells and was restored by exercise (Figure 3). The duration of unloaded shortening (data not shown) and relaxation (Figure 3B) were not different between the various groups. However, the speed of contraction and relaxation were significantly reduced in ENDO MI cells and were restored by exercise (Figure 3C). The amplitude of Ca 2+ transient decreased significantly only in ENDO MI cells and was restored by exercise (Figure 4B). In addition, the calcium transient decay in both EPI and ENDO MI myocytes was significantly slowed (increase in tau), reflecting an altered calcium reuptake. All parameters were normal in MI-Ex. The calcium transient decay in MI is known to be altered due to changes in SERCA2a expression or in the inhibition of SERCA2a/PLB activity. We found that SERCA2a expression was significantly decreased in MI animals, both in ENDO and EPI layers. PLB expression was unchanged. Levels of protein expression in MI-Ex were similar to sham samples (Figure 4C).

Force development in single permeabilized myocytes

Force development of intact myocytes depends on the amount of calcium released by the sarcoplasmic reticulum and the myofilament Ca 2+ sensitivity. Thus, we measured the myofilament Ca 2+ sensitivity (pCa 50 ) at short length (1.9 m SL) and at long length (2.3 m SL). Passive force and maximal isometric tension measured at both SL were similar between Sham, MI, and MI-Ex (Table 2). Neither pathology nor exercise had any effect on myofilament Ca 2+ sensitivity at short SL (Figure 5, Table 2). Stretching the cells to 2.3 m SL induced a leftward shift of tension in all conditions reflecting an increase in myofilament Ca 2+ sensitivity.

However, pCa 50 was significantly lower in the ENDO cells isolated from MI rats.

Exercise restored Ca 2+ sensitivity at 2.3 m SL (Figure 5A). The difference between pCa 50 at the long and short SL (pCa 50 ), used an index of the length-dependent activation of contractile machinery, was significantly smaller in ENDO MI cells compared to the Sham and MI-Ex cells. EPI myocytes were affected neither by the pathology nor by the exercise at both lengths (Figure 5B).

We have previously shown that pCa 50 is closely related to passive tension rather than to SL. 3 The differences in passive and active properties across the left free wall described a positive relationship in which ENDO cells develop more passive tension after a stretch to 2.3 µm SL associated with higher stretch-induced myofilament Ca 2+ sensitivity than did EPI cells (Figure 5C).This correlation disappeared in MI rats, mostly due to a decrease in pCa 50 of ENDO cells.

Interestingly, values obtained in MI-Ex myocytes were similar to those found in sham rats.

Contractile protein isoforms and phosphorylation

Rat heart expresses two isoforms of MyHC (α-or β-MyHC). Variation in α/β-MyHC expression influences cardiac function. In our conditions β-MyHC content increased after MI in both ENDO and EPI cells. Exercise had no effect on β-MyHC content in either ENDO or EPI cells of MI rats (Figure 6A).

TnI and MLC-2 phosphorylations are known to shift the tension-pCa curves in cardiac muscle. Western blot analysis was performed on non-stretched and stretched skinned muscle strips dissected from the sub-endocardial layer or the sub-epicardial layer. Phosphorylation of TnI on the PKA sites was similar between regions, before and after stretch, in sham and MI animals (Figure 6B). We have previously shown that the linear relationship between pCa 50 and passive tension was associated with changes in the phosphorylation level of MLC-2. 3 As previously reported, stretch increased by ≈10% the amount of phosphorylated MLC-2 in the ENDO myocardium of sham animals and not in MI animals (Figure 6C). Exercise restored the stretchinduced increase in MLC-2 phosphorylation in MI animals. MLC-2 phosphorylation was not affected by pathology, stretch and exercise in EPI myocardium.

Discussion

The present study tested whether a remodeled end-stage failing heart following myocardial infarction could benefit from 5 weeks of endurance exercise training. To this end, we evaluated the effects of exercise on LV remodeling, regional in vivo function, cardiomyocyte contractility, Ca 2+ handling, and myofilament Ca 2+ sensitivity. The main findings were that: (1) normal myocardial function is nonuniform due to heterogeneous cellular properties across the wall (2) transmural nonuniformity of myocardial function was lost in MI rats due to ENDO cellular dysfunction

(3) exercise recovered a transmural heterogeneity by improving LV function mostly in sub-endocardial layer, in relation with a normalization of MI-induced dysfunctions of both Ca 2+ handling and myofilaments Ca 2+ sensitivity in ENDO cardiomyocytes.

Regional pathophysiology of MI-induced LV dysfunction

It is now established that cardiac function is non-uniform across the wall due to larger and faster contraction of the sub-endocardial layer as compared with the sub-epicardial tissue one. 7, A recent study using transmural bead markers under biplane cineradiography showed that the onset of myofiber shortening occurred earlier in endocardium than epicardium while the onset of myofiber relaxation occurred earlier in epicardium than endocardium. [START_REF] Ashikaga | Transmural Dispersion of Myofiber Mechanics: Implications for Electrical Heterogeneity In Vivo[END_REF] These differences in fiber strains may be explained by telediastolic and telesystolic transmural differences in wall stress, increasing toward the endocardium. [START_REF] Buchi | Left ventricular wall stress distribution in chronic pressure and volume overload: effect of normal and depressed contractility on regional stressvelocity relations[END_REF] Our data showed that the higher subendocardial fiber shortening during systole was likely due to higher basal cardiomyocyte contractility and a higher stretch-induced increase in myofilament Ca 2+ sensitivity.

As previously reported, [START_REF] Litwin | Serial echocardiographic assessment of left ventricular geometry and function after large myocardial infarction in the rat[END_REF][START_REF] Konstam | Prevention and reversal of left ventricular remodeling: Summation[END_REF] the remodeling of MI heart was characterized by LV dilatation, hypertrophy, and dysfunction. The cardiac pump dysfunction was characterized here by a reduction in LV fractional shortening and other indexes such as the aortic velocity time integral, and the amplitudes and velocities of contraction of the myocardial layers. We also observed that the pathology did not affect uniformly the heart. Following MI, wall contractility decreased mostly in the sub-endocardial layer with a higher decrease in the amplitude of displacement (-52% vs -28%, Endo vs Epi) and in the velocity of contraction (-36% vs -13%, Endo vs Epi). These changes resulted in a homogenization in regional myocardial function and the complete loss of transmural nonuniformity.

Beneficial effects of exercise training on MI cardiac function

In our MI model, most parameters of the LV remodeling and in vivo dysfunctions were achieved 13 weeks after MI and were stable 18 weeks after MI (Figures 1 and2). We thus assessed whether exercise could improve myocardial function and restore the transmural heterogeneity in a remodeled MI heart. Studies in humans with large MI reported that exercise had either no [START_REF] Sullivan | Exercise training in patients with severe left ventricular dysfunction. Hemodynamic and metabolic effects[END_REF] , or a beneficial [START_REF] Giannuzzi | Attenuation of unfavorable remodeling by exercise training in postinfarction patients with left ventricular dysfunction: results of the Exercise in Left Ventricular Dysfunction (ELVD) trial[END_REF] effect on ejection fraction and LV volumes. Previous studies in animal models reported that exercise had no effect on LV function parameters such as LV dP/dtmax or fractional shortening irrespective of whether exercise was started early or late after MI, despite an improvement in cell function. [START_REF] Gaudron | Effect of endurance training early or late after coronary artery occlusion on left ventricular remodeling, hemodynamics, and survival in rats with chronic transmural myocardial infarction[END_REF][START_REF] Wisloff | Aerobic exercise reduces cardiomyocyte hypertrophy and increases contractility, Ca2+ sensitivity and SERCA-2 in rat after myocardial infarction[END_REF] Neither was fractional shortening in MI improved by exercise in our study. However, other indexes of cardiac function such as aortic blood flow (VTIAo) and regional amplitude and velocities of contraction were improved after exercise. In several clinical studies, alterations in wall motion (including wall thickening) measured by echocardiography were shown to be good predictors of subsequent cardiac events of morbidity and mortality [START_REF] Carluccio | Usefulness of the severity and extent of wall motion abnormalities as prognostic markers of an adverse outcome after a first myocardial infarction treated with thrombolytic therapy[END_REF][START_REF] Moller | Prognostic importance of systolic and diastolic function after acute myocardial infarction[END_REF] and interventions that halt, slow or reverse these ventricular dysfunctions should markedly improve clinical outcomes. [START_REF] Haykowsky | A meta-analysis of the effect of exercise training on left ventricular remodeling in heart failure patients: the benefit depends on the type of training performed[END_REF] The higher exercise-induced improvement of amplitude and velocity of contraction in ENDO allowed to partially recovering a transmural nonuniformity. This may in turn contribute in the exercise-induced beneficial effect on cardiac function.

In view of the concern that late exercise may aggravate LV remodeling after a large MI and life expectancy, we investigated LV remodeling with or without exercise.

Five weeks of exercise decreased LV end-diastolic and end-systolic diameters, and did not aggravate LV remodeling. Our exercise protocol was a moderate endurance protocol, confirmed by the fact that sham animals exercised at the same intensity did not show any change in any parameters investigated (data not shown). These observations are in agreement with a recent study reporting that 8 weeks of moderate exercise in mice with MI cardiomyopathy [START_REF] De Waard | Early exercise training normalizes myofilament function and attenuates left ventricular pump dysfunction in mice with a large myocardial infarction[END_REF] and hypertrophic cardiomyopathy [START_REF] Konhilas | Exercise can prevent and reverse the severity of hypertrophic cardiomyopathy[END_REF] reversed collagen content with little effect on cardiac hypertrophy.

In our study, exercise did not induce a higher mortality consistent with previous studies with human patients. [START_REF] Kubo | Exercise at ventilatory threshold aggravates left ventricular remodeling in patients with extensive anterior acute myocardial infarction[END_REF]

Exercise-induced effects on contractile cellular properties

The mechanisms for LV dysfunction after MI remain incompletely understood but have been proposed to be a consequence of cellular alterations and extracellular matrix remodeling. 1 Recent studies have shown that early exercise in mice after MI had no effect on LV remodeling but slightly attenuates global LV dysfunction mostly by improving myofilament function without Ca 2+ signaling. [START_REF] De Waard | Early exercise training normalizes myofilament function and attenuates left ventricular pump dysfunction in mice with a large myocardial infarction[END_REF] However, similar protocols performed in female Sprague-Dawley rat model showed a recovery of contraction function, Ca 2+ handling and some indexes of improved myofilament Ca 2+ sensitivity following early exercise after MI. 17 sensitivity. Finally the origin of heart failure (ischaemic, pressure overload) and the stage of HF may also affect the results. 6 Heart failure in small rodents is associated with a shift in isomyosin synthesis from predominantly α-myosin toward β-myosin. [START_REF] Swynghedauw | Developmental and functional adaptation of contractile proteins in cardiac and skeletal muscles[END_REF] This shift observed during the LV remodeling has been proposed to improve myocardial work efficiency by generating cross-bridge force with a higher economy of energy consumption and thus maintaining contractility. 6 Similar shift can be observed in exercised animals. [START_REF] Cazorla | Effects of highaltitude exercise training on contractile function of rat skinned cardiomyocyte[END_REF] In our study, β-MyHC content was increased by HF but was not affected by exercise. Thus the beneficial effect of exercise on MI myocardial function is independent on myosin expression.

The level of MLC-2 phosphorylation affects myofilament Ca 2+

sensitivity. [START_REF] Morano | Tuning the human heart molecular motors by myosin light chains[END_REF] MLCK and a protein phosphatase (PP). [START_REF] Rajashree | Modulation of myosin phosphatase targeting subunit and protein phosphatase 1 in the heart[END_REF] MLCK activity is modulated by PKA and PKC phosphorylations, and Ca 2+ -calmodulin interaction offering potential regulators for the exercise-induced effect on MLC-2 phosphorylation. [START_REF] Rajashree | Modulation of myosin phosphatase targeting subunit and protein phosphatase 1 in the heart[END_REF][START_REF] Grimm | The MLCK-mediated alpha1-adrenergic inotropic effect in atrial myocardium is negatively modulated by PKCepsilon signaling[END_REF] The normalization of Ca 2+ transient after exercise in MI may contribute to increasing MLCK activity and thus MLC-2 phosphorylation. MLC-2 dephosphorylation is classically attributed to PP1. Studies suggest that PP1 activity and expression is increased in end-stage CHF in dog [START_REF] Gupta | Cardiac SR-coupled PP1 activity and expression are increased and inhibitor 1 protein expression is decreased in failing hearts[END_REF] but PP1 expression was found unchanged in rat with CHF. [START_REF] Belin | Augmented protein kinase C-alpha-induced myofilament protein phosphorylation contributes to myofilament dysfunction in experimental congestive heart failure[END_REF] In our study, alteration in PP1 activity after MI and/or exercise, could account for the changes in MLC-2 phosphorylation and the lack of effect on TnI between MI and MI-Ex.

However, more experiments are required to explore the kinase/phosphatase alteration after MI and exercise. The present study indicates that exercise training started late after a large MI improved regional LV function and molecular phenotype, without any adverse effects on LV remodeling and survival during the protocol. The beneficial effects of exercise on cellular function have been proposed to restore β1-adrenergic signaling. [START_REF] De Waard | Early exercise training normalizes myofilament function and attenuates left ventricular pump dysfunction in mice with a large myocardial infarction[END_REF] However, there is no sign here of such improvement that should affect the phosphorylation status of various proteins (TnI, PLB, titin-based passive tension).

Limitations of the study

Future studies should be aimed at investigating whether this difference is due to the fact that exercise was performed in organisms in which the β-adrenergic signaling has been already extensively stimulated, suggesting other signaling pathways for exercise-induced beneficial effects. This is of particular interest since most patients with heart failure are treated with β-blockers. Another positive aspect of this study is that exercise preferentially restored the contractile properties of the tissue altered by the pathology (ENDO), allowing physiological cardiac contractile heterogeneity to be restored. Because of its relatively low cost, high availability and ease of use, exercise training is an intervention that could be accessible to most patients with heart failure.

Legends

  , part of the discrepancy on the effect of exercise on MI in the literature may be due to use of swimming training 15 , which is known to have different responses from those to treadmill running, complicated by factors such as the diving reflex, mental stress, and episodes of hypoxia associated with diving. The present study evaluated in a rat model the effect of exercise training, started at a late stage of HF (13 weeks post-MI), on LV remodeling and function of the global and cellular contractile properties in various regions of the LV free wall. The results indicated that in vivo systolic function (Fractional Shortening and aortic Velocity Time Integral (VTI Ao )) was reduced in MI rats in association with a reduction of the amplitude and speed of contraction of the ENDO layer and to a lesser extent of the sub-epicardial layer (EPI). These alterations led to a uniformity of the transmural myocardial function, which were partially reversed by exercise. Exercise improved part of the systolic parameters and slightly reduced LV dilatation. The in vivo beneficial effect of exercise was associated with a restoration of the ENDO cellular properties, by reversing the MI-induced abnormalities in Ca 2+ -handling function and proteins, phosphorylation status of contractile proteins, and myofilament function. METHODS For a detailed description, see expanded Materials and Methods in the online data supplement. Experiments complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publications No. 85-23, revised 1996) and with the approval of the French Ministry of Agriculture.

  In our study, cell shortening decreased after MI exclusively in ENDO cells, most probably due to both reduced Ca 2+ release and decreased myofilament function. Exercise fully restored the ENDO cellular properties without affecting the EPI cells. The recovery of calcium transient after exercise was associated with changes in SERCA2a 17,present study and Na + /Ca 2+ exchanger[START_REF] Wisloff | Aerobic exercise reduces cardiomyocyte hypertrophy and increases contractility, Ca2+ sensitivity and SERCA-2 in rat after myocardial infarction[END_REF] expression suggesting a recovery of the loading conditions of the sarcoplasmic reticulum. Further studies are needed to determine whether the exercise-induced improvement in systolic Ca 2+ levels are due to changes in Ca 2+ release properties through the ryanodine receptors.Myofilament Ca 2+ sensitivity was depressed at end-stage HF in the present study. Previous studies in single skinned myocytes have reported either increases[START_REF] Van Der Velden | Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins[END_REF] or decreases3,4 in myofilament Ca 2+ sensitivity both in human and experimental HF. The reasons for these different findings are not entirely clear. One explanation for these different findings may relate to the different experimental preparations that were studied (multicellular vs. single myocyte skinned myocardium).Another explanation might be the level of neurohormonal stimulation present at the time of tissue preparation that will affect the balance between kinases and phosphatases. Indeed, most of the studies reported alterations of the level of phosphorylation of sarcomeric regulatory proteins that correlated with the changes in myofilament Ca2+

  Increasing MLC-2 phosphorylation by incubating myofilaments with exogenous MLC Kinase (MLCK) increases myofilament Ca 2+ sensitivity. 38- 40 A decrease in MLC-2 phosphorylation has been described in failing human 41 and animal 3 hearts and has been associated with the observed decrease in myofilament Ca 2+ sensitivity; similar results were obtained in Endo MI myocytes of the present study. Thus, the increase in MLC-2 phosphorylation in the sub-endocardial tissue exclusively, observed in the present study after exercise may by itself explain the improved myofilament Ca 2+ sensitivity. The phosphorylation level of MLC-2 depends on the balance of activities between the

From

  the present study, we cannot draw any conclusions as to the effect of this protocol on life expectancy since the animals were sacrificed at the end of the exercise protocol. In particular, future studies should determine how the improvement of the global and regional cardiac function and the normalization of the cellular dysfunctions post-MI at the end of the training could affect the occurrence of sudden cardiac death because of arrhythmic events. It would have been also interesting to obtain intraventricular pressure measurements that would have given more in vivo functional index, allowing to further explore the effect of exercise training on diastolic (dys)function in our model.

Figure 1 .

 1 Figure 1. Left ventricular morphological and functional characteristics 13 weeks post-MI. End-diastolic and end-systolic diameters and diastolic posterior wall thickness (PWTd) (A), end systolic strain (B), Systolic velocity of the LV Epicardial and Endocardial layers (C), global systolic function (D) as indexed by fractional

Figure 2 .

 2 Figure 2. Left ventricular morphological and functional characteristics in MI and MI-Ex rats. A) Echocardiography images with an enlargement of the posterior wall. B) End systolic strain (left) and displacement of Epicardial and Endocardial layers during a cardiac cycle (right). C) Systolic velocity (left) and Gradient of velocity Endo-Epi (right). D) Aortic velocity time integral (VTI Ao ). E) Survival curve following the beginning of exercise program. * adjusted p<0.05 (n=12-17 hearts/group).

Figure 3 .Figure 4 .Figure 5 .Figure 6 .

 3456 Figure 3. Sarcomere length shortening in isolated MI and MI-Ex cardiomyocytes. A) SL shortening examples of endocardial (left trace) and epicardial (right trace) sham, MI, and MI-Ex myocytes. B) Averaged data of SL shortening (in % on baseline) and time to 50% of relaxation (TR50). C) Speeds of contraction (left) and relaxation (right) (n=55-106 cells/5 hearts) * adjusted p<0.05

  Figure 3

  Figure 5

Figure 6

 6 Figure 6

Table 1 : Cardiac morphological and functional parameters :

 1 LVEDs, left ventricular end-systolic diameter; LVEDd, left ventricular end-diastolic diameter; AWTd, enddiastolic anterior wall thickness; PWTd, end-diastolic posterior wall thickness; FS, Fractional shortening. VTI AO , aortic Velocity Time Integral. HR, heart rate. Values are means ± SEM (n= number of cells).

	Sham	MI	MI-Ex
	(n=12)	(n=17)	(n=14)

*

Sham vs MI.

† MI vs MI-Ex ; p<0.05.

Table ( s)

 (

Table 2 : Regional mechanical properties of cardiomyocytes isolated from sham, myocardial infarcted (MI), and MI exercised (MI-Ex) rats :

 2 Tpass) and maximal active (Tmax) tensions in mN/mm 2 were measured at pCa 9 and 4.5 and at 1.9 and 2.3 mm sarcomere length, respectively, on sub-epicardial (EPI) and sub-endocardial (ENDO) cells isolated from sham, MI, and MI-Ex hearts. pCa 50 (pCa for half-maximal activation) and nH (Hill coefficient), were calculated by fitting the force-pCa relation (see materials and methods). Values are means ± SEM (n= number of cells; 5 rats per group).

			Sarcomere length, 1.9 µm			Sarcomere length, 2.3 µm
		(n)	Tpass Tmax	pCa 50	n H	Tpass	Tmax	pCa 50	n H	pCa 50
	Endocardium
	Sham	(15) 0.9±0.2 47±4 5.75±0.02 4.1±0.3	11.5±0.9*	50±3	5.98±0.02*	4.7±0.3	0.23±0.01*
	MI	(10) 0.9±0.3 40±3 5.74±0.03 4.6±0.5	9.4±1.2	47±3	5.88±0.03* † ‡ 5.2±0.5	0.13±0.02 † ‡
	MI-Ex	(10) 1.0±0.2 45±5 5.74±0.03 4.7±0.6	11.1±1.9*	46±5	5.98±0.02	5.1±0.4	0.24±0.02
	Epicardium
	Sham	(12) 0.8±0.1 36±4 5.74±0.01 4.4±0.3	8.3±0.8	38±5	5.93±0.01	4.6±0.3	0.19±0.01
	MI	(8) 0.8±0.3 35±4 5.80±0.01 5.0±0.5	7.3±1.5	40±4	5.95±0.01	5.4±0.5	0.15±0.02
	MI-Ex	(13) 0.9±0.3 38±3 5.77±0.01 4.8±0.2	7.1±0.5	40±3	5.94±0.01	5.2±0.6	0.18±0.02
	Passive (

* EPI vs ENDO, † sham vs MI, ‡ MI vs MI-Ex ; p<0.05, two-way ANOVA.

Acknowledgements

Thanks are due to Patrice Bideaux, Guillermo Salazar and Christine Bernard-Crozier for technical assistance.

Funding

This work was supported by the « Association Française contre les Myopathies » and the Leducq Foundation. OC and AL are established investigators of CNRS.

Conflict of interest None

Aït Mou et al, LATE EXERCISE IN HEART FAILURE

DATA SUPPLEMENT

Expanded methods

Animals and exercise training:

Male Wistar rats (5 weeks old) were subjected to coronary artery ligature to produce MI (Myocardial Infarcted) rats as previously described. [1][2][3] Thirteen weeks after myocardial infarction, rats were subdivided into two groups: sedentary (MI) and exercised (MI-Ex) animals. The exercise program consisted of 5 weeks exercise on a treadmill (40 min/day, 5 days/week). The speed of running was gradually increased from 10 m/min to 16 m/min. At the end of the training protocol, morphological and functional parameters were determined by echocardiography and compared to sham-operated animals that were subjected to the same surgical operation without tightening the coronary ligature.

Noninvasive hemodynamics

Ascending aortic blood flow was recorded, as previously described

CVR-2008-687R1

was preferentially used in this study as an accurate index of LV stroke volume and therefore of LV global function because this index is less sensitive to error measurement from the aortic annulus than the stroke volume. 6 LV sub-endocardial (ENDO) and sub-epicardial (EPI) posterior wall displacement was measured offline on the short axis view at the level of the papillary muscles. Tissue Doppler imaging (TDI) was performed after each conventional echocardiography as previously described.

7

Measurement of myocardial velocities resulting from the LV radial contraction was performed on the short axis view at the level of the papillary muscles. TDI analysis was performed offline with the MyLabDesk software (ESAOTE, Italy). The angle of interrogation of the myocardial velocities was carefully aligned to be perpendicular to LV walls. The TDI sample was manually positioned along the posterior wall within the Endo and Epi layers to obtain the ENDO and EPI velocities. Intra-and interobserver Doppler echocardiography and TDI variabilities are evaluated during a previous study in Wistar rats (n=6) by means of variation coefficients. Coefficients were all equal to or lower than 6.5 % (aortic VTI) with a minimal value for LVEDd (1.5 %).

Contractile properties in intact cardiomyocytes

Eighteen weeks post-infarctus, single ventricular myocytes were isolated by enzymatic digestion as previously described (n=5 rats par group). 8 Briefly, hearts were quickly removed and mounted on a Langendorff apparatus. The heart was perfused 10 min with a Ca 2+ -free Hanks-HEPES solution followed by perfusion with an enzymatic solution for 10-20 min at 37°C (see below for solution compositions). Small pieces of ventricular tissues from the inner free wall Endocardium and from the outer free wall Epicardium were dissected and gently dissociated with a pipette in Ca 2+ free solution. Calcium concentration was gradually increased to 1 mmol/L Ca 2+ . To prevent protein degradation after collagenase treatment, all solutions contained protease inhibitors (PMSF: 0.5 mmol/L and E64d [transepoxysuccinyl-1-leucine-guanidobutylamide]: 0.01 mmol/L).

Cells were loaded for 30 min at room temperature with Indo-1 AM (10mol/L Invitrogen inc., France). Experiments were performed in 1.8 mM Ca 2+ containing solution. Cells were electrically stimulated at a frequency of 0.5 Hz (20V, 1 ms), and simultaneously illuminated at 305 nm using a xenon arc bulb light. Sarcomere length (SL) and fluorescences emitted at 405 nm and 480 nm were simultaneously recorded using IonOptix acquisition software (IonOptix system, Hilton, USA).

Force measurements in permeabilized cardiomyocytes

Ventricular myocytes were isolated by mechanical dissociation as described previously. 1,8 The heart was pre-skinned by perfusing relaxing solution (for composition see below) containing 1% Triton X-100 and protease inhibitors (see above) for 10 min. Right ventricle and PMI fibrotic tissue were discarded. Several left sub-epicardial and sub-endocardial skinned strips (8-mm long, 2-mm width, 1mm thick) were dissected under a microscope following the orientation of the fibers in the skinning solution, and were further skinned 10 min at 4°C in relaxing solution containing 1% Triton X-100.

Strips were frozen in liquid nitrogen at slack length or after stretching by ~20-30% of initial slack length to 2.30±0.05 µm SL in relaxing solution for biochemistry or prepared for mechanical experiments as follow. The strips were mechanically disrupted at 11,000 rpm for 2-3 sec (Polytron PT45-80 with a PTA 10TS shaft, KINEMATICA AG, Switzerland) in fresh ice-cold relaxing solution, resulting in a suspension of small clumps of myocyte-sized preparations. The homogenized tissue was filtered and centrifuged at 1.000 rpm for 1 min at 4°C. The pellet containing the myocytes was further skinned in 0.3% Triton X-100 solution for 6 min to remove remaining sarcolemma membranes, and then extensively washed in relaxing solution. To prevent degradation, all solutions contained protease inhibitors (PMSF: 0.5 mmol/L; leupeptin: 0.04 mmol/L and E64: 0.01 mmol/L). Cells were kept on ice and used within the day. Cells were used immediately after cell isolation within one day for mechanical experiments.

Active and passive forces were measured as described previously. 1 Myocytes were attached to a piezoresistive strain gauge (AE801 sensor, Memscap, Crolle, France) and to a stepper motor driven micromanipulator (MP-285, Sutter instrument company, Novato CA, USA) with thin needles and optical glue (NOA 63, Norland products Inc, North Brunswick, NJ) that polymerized by 2 min UV illumination. SL was determined online throughout the experiment at 50 Hz by using a Fast Fourier Transform algorithm on the video images of the cell. Force was normalized by the cross-sectional area measured from the imaged cross-section. After a test-activation at pCa 4.5, the cell was stretched to various SL in relaxing solution using a stepper motor driven micromanipulator at a speed of 0.1 length/sec to evaluate passive tension. Steady state passive tension (after the rapid phase of stress relaxation) was sequentially measured at 1.9, and 2.3 µm SL. Then pCa-force relationships were established at two SL, 1.9 then 2.3 µm at 22°C. The cell was kept five minutes at slack length in relaxing solution between each phase of the protocol for complete refolding of titin. Active tension at

CVR-2008-687R1

each pCa was the difference between total tension and relaxed tension. Cells that did not maintain 80% of the first maximal tension or a visible striation pattern were discarded. The SL change varied somewhat from cell to cell, and we continued with cells that were well attached with minimal SL changes (< 0.1 µm). When required, cell length was varied during contraction in order to keep SL constant. Active tensions at submaximal activations were normalized to maximal isometric tension (classically obtained at pCa 5) at the same SL. The relation between force and pCa was fitted to the following equation:

, where n H is the Hill coefficient and pCa 50 , pCa for halfmaximal activation equals -(log K)/n H .

Experimental solutions

For isolating intact cells, three different solutions were used. Ca 2+ -activating solutions were prepared daily by mixing relaxing (pCa 9.0) and maximal activating (pCa 4.5) solutions. 8 The relaxing and activating buffers contained (in mmol/L): phosphocreatine 12, imidazole 30, free Mg 2+ 1, EGTA 10, Na 2 ATP 3.3, and dithiothreitol 0.3 with pCa 9.0 (relaxing solution) and pCa 4.5 (maximal activating solution), protease inhibitors (PMSF 0.5 mmol/L, leupeptin 0.04 mmol/L, and E64 0.01 mmol/L) pH 7.1 adjusted with acetic acid. Sufficient potassium acetate was added to adjust ionic strength to 180 mmol/L.

Protein analysis:

MyHC isoforms were separated on a 6% SDS-PAGE as previously described 9 . Intact myocytes were dissolved in an SDS lysis buffer (Tris-HCl 50 mM, 2% (w/v) SDS, urea 8 mol/L, EGTA 1 mmol/L, EDTA 1 mmol/L, DTT 80 mmol/L, 10% (v/v) glycerol, pH 6.8, protease inhibitors) and heated 6 min at 50°C. Gels were silver stained and analyzed with an imaging system (Kodak Image Station 2000R). -MyHC content was expressed relative to total amount of the MyHC protein.

CVR-2008-687R1

For the SERCA and phospholamban (PLB) intact myocytes were dissolved 15 Proteins (20 µg) were separated either on 15% SDS-PAGE (for TnI) or 10% urea gel (for MLC-2). For both studies, all samples from one animal in the various conditions (ENDO/EPI, relax/stretch) were always loaded on the same gel. MLC-2 phosphorylated and non-phosphorylated forms were separated by a 10% urea gel and were specifically detected with a cardiac MLC-2 antibody (Coger SA, Paris, France). Total TnI content was determined on the first wells of the membrane with a total cardiac TnI antibody (Cat#4T21, Hytest, Turku, Finland) and the protein kinase A phosphorylated form of cardiac TnI antibody on the other wells (Cat#4T45, Hytest). Immunodetection was revealed with ECL Plus system (Amersham Pharmacia, Little Chalfont Buckinghamshire, England).

Statistical analysis

One-way or 2-way ANOVA was applied for comparison between groups. When significant interactions were found, a Bonferroni t-test was applied with P < 0.05 (Sigmastat 3.5). Data are presented as mean±SEM.


Olivier Cazorla

Szabolcs Szilagyi

Jean-Yves Le Guennec

Guy Vassort
email: vassort@montp.inserm.fr

Alain Lacampagne

its alteration after myocardial infarction

Keywords: Ca 2+ sensitization, Frank-Starling law, ventricular myocytes, myosin light chain 2, heart failure

eart muscle has the property to develop positive inotropism in response to an increase in end-diastolic ventricular volume. This phenomenon, known as the Frank-Starling law, originates from cell adaptation to stretch involving a sarcomere length (SL) dependence of the Ca 2+ sensitivity of myofilament activation [START_REF] Lakatta | Length modulation of muscle performance. Frank-Starling law of the heart[END_REF]. The mechanisms that underlie this intrinsic property may involve a length-dependent increase in the affinity of the regulatory site of troponin H C for Ca 2+ . There is also substantial evidence that stretch reduces interfilament lattice spacing, increasing the probability of a myosin head to bind actin [START_REF] Fuchs | Sarcomere length versus interfilament spacing as determinants of cardiac myofilament Ca2+ sensitivity and Ca2+ binding[END_REF]. However, we previously observed in intact isolated cardiomyocytes that active tension correlated to diastolic properties rather than to SL per se [START_REF] Cazorla | Resting tension participates in the modulation of active tension in isolated guinea pig ventricular myocytes[END_REF]. We showed on isolated cardiomyocytes that reducing passive tension by degrading titin with a mild trypsin digestion [START_REF] Cazorla | Length modulation of active force in rat cardiac myocytes: is titin the sensor?[END_REF] or by modulating the prehistory of stretch [START_REF] Cazorla | Titin-based modulation of calcium sensitivity of active tension in mouse skinned cardiac myocytes[END_REF] decreases Ca 2+ sensitivity in proportion to titin-based passive tension. Combining the differences in titin isoform expression in bovine atrium and ventricular strips with variation of the stretch prehistory revealed that the SL-dependent increases in Ca 2+ sensitivity of force and maximal active force were most pronounced in myocardium with high titin-based passive force [START_REF] Fukuda | Titin isoform variance and length dependence of activation in skinned bovine cardiac muscle[END_REF]. Recent studies proposed that titin-based passive tension could promote actomyosin interaction by reducing the interfilament lattice spacing [START_REF] Cazorla | Titin-based modulation of calcium sensitivity of active tension in mouse skinned cardiac myocytes[END_REF][START_REF] Fukuda | Titin isoform variance and length dependence of activation in skinned bovine cardiac muscle[END_REF]. Differential coexpression of titin isoforms influences cellular stiffness but also the degree of interfilament lattice spacing changes. conferring a modulator role in the Frank-Starling mechanism on titin. Other studies concluded that length-dependent activation in the heart cannot be entirely explained by alterations in interfilament lattice spacing and preferred a central role of troponin I (TnI) in cardiac myofilament activation [START_REF] Konhilas | Myofilament calcium sensitivity in skinned rat cardiac trabeculae: role of interfilament spacing[END_REF][START_REF] Konhilas | Troponin I in the murine myocardium: influence on lengthdependent activation and interfilament spacing[END_REF].

Although cardiac contractility is altered under various pathological conditions, changes of the Frank-Starling properties are still controversial. In human heart failure, the Frank-Starling relationship has been reported to be normal [START_REF] Holubarsch | Existence of the Frank-Starling mechanism in the failing human heart. Investigations on the organ, tissue, and sarcomere levels[END_REF] or lost in accordance with a smaller increase in the Ca 2+ sensitivity of the myofibrils after stretch [START_REF] Schwinger | The failing human heart is unable to use the Frank-Starling mechanism[END_REF]. Similar alterations of contractility, as a consequence of remodeling of the whole heart were described in a rat model of postmyocardial infarction (PMI) [START_REF] Anand | Isolated myocyte contractile function is normal in postinfarct remodeled rat heart with systolic dysfunction[END_REF]. However, the latter authors did not observe any perturbation during shortening measurements on unloaded isolated PMI cardiomyocytes. Others reported variable effects of heart failure on myofibrillar Ca 2+ sensitivity depending on the study, that is, either a decrease in PMI porcine preparations, PMI and spontaneous hypertensive cardiomyocyte rat models of heart failure, or no change in failing rats, or an increase was observed in human dilated cardiomyopathy and its canine model (for review, see ref [START_REF] De Tombe | Altered contractile function in heart failure[END_REF]. Note that these studies were performed at only one SL. Heart failure was also associated with changes in the distribution and content of myocardial contractile proteins, including a decrease in myosin light chain (MLC) content in human chronic heart failure [START_REF] Margossian | Light chain 2 profile and activity of human ventricular myosin during dilated cardiomyopathy. Identification of a causal agent for impaired myocardial function[END_REF]. Furthermore, several experimental works [START_REF] Morano | Chronic hypertension changes myosin isoenzyme pattern and decreases myosin phosphorylation in the rat heart[END_REF][START_REF] Van Der Velden | The effect of myosin light chain 2 dephosphorylation on Ca2+ -sensitivity of force is enhanced in failing human hearts[END_REF] but not all ( 16) reported a reduced level of MLC2 phosphorylation in heart failure known to be associated with reduced Ca 2+ sensitivity of the contractile proteins.

Cardiomyocytes do not present homogeneous behavior within the whole heart, and their alterations during pathologies differ with their localization. This has been particularly documented for the electrical properties of cardiomyocytes. Sub-endocardial (ENDO) cells exhibit a longer action potential than sub-epicardial (EPI) cells. This gradient, abolished in rat models of hypertrophy induced by aortic stenosis or of heart failure after myocardial infarction, is mostly due to a reduction of the transient outward K + current (Ito) in the epicardium [START_REF] Aimond | Ionic basis of ventricular arrhythmias in remodeled rat heart during long-term myocardial infarction[END_REF]. Regarding the contractile properties, we previously reported in intact ventricular myocytes of various species a gradient of cellular stiffness across the ventricular wall [START_REF] Cazorla | Length-tension relationships of subepicardial and sub-endocardial single ventricular myocytes from rat and ferret hearts[END_REF]. In addition, according to their location in the wall, the cells function over different ranges of SL-active tension relationships despite similar calcium transients, suggesting structural differences within the contractile machinery and/or different length-signaling pathways [START_REF] Cazorla | Length-tension relationships of subepicardial and sub-endocardial single ventricular myocytes from rat and ferret hearts[END_REF].

The aim of the present study was to determine whether in skinned cardiomyocytes such a gradient of passive mechanical properties across the ventricular wall was observable and whether it was concomitant with a gradient of active properties. We also sought to determine whether such a gradient would be altered in a postmyocardial infarction model of cardiomyopathy.

MATERIALS AND METHODS

Experimental myocardial infarction

Male Wistar rats (180-220 g) were subjected to coronary artery ligature to produce PMI rats [START_REF] Aimond | Ionic basis of ventricular arrhythmias in remodeled rat heart during long-term myocardial infarction[END_REF]. In brief, rats were anesthetized (with pentobarbital, 60 mg/kg) and ventilated. Shamoperated animals were subjected to the same surgical operation without tightening the coronary ligature. Animals were killed 14 wk postligature. Scar size was ~25% of the left ventricular free wall area, and necrosis was transmural. Heart weight/body weight ratio (mg/g) was significantly increased from 2.9 ± 0.5 in sham (n=7) to 3.8 ± 0.4 in PMI (n=16) rats. Cells isolated from PMI rats were hypertrophied (cell width: 25±1 and 28±1 µm, n=40 and 30 cells in sham and PMI, respectively). Pressure/volume relationships established on perfused hearts showed a 41% decrease in peak systolic pressure in PMI rats relative to the sham group (data not shown). Hemodynamic measurements performed on another series of animals demonstrated a significant increase in end-diastolic pressure from 2.5 ± 0.5 to 17.1 ± 13.5 mm Hg (in 9 sham and 9 PMI, respectively).

Myocyte preparation

Ventricular myocytes were isolated by mechanical dissociation as described previously with some modifications [START_REF] Cazorla | Differential expression of cardiac titin isoforms and modulation of cellular stiffness[END_REF]. In brief, rats were anaesthetized by intraperitoneal injection of pentobarbital (0.2 g/100 g). The heart was perfused retrogradely (3 ml.min -1 ) with a Ca 2+ -free Hanks-HEPES buffered solution for 5 min at room temperature containing in mM: NaCl 117, KCl 5.7, NaHCO 3 4.4, KH 2 PO 4 1.5, MgCl 2 1.7, HEPES 21, glucose 11, taurine 20, pH 7.2, adjusted with NaOH and bubbled with 100% O 2 . 2,3-Butanedione monoxime (15 mM) was also added to the buffer to inhibit contraction and to preserve energetic stocks. Then, the heart was preskinned by perfusing relaxing solution (for composition, see below) containing 1% Triton X-100 and protease inhibitors (0.5 mM PMSF, 0.04 mM leupeptin, and 0.01 mM E64) for 7-10 min. Right ventricle and PMI fibrotic tissue were discarded. Several left EPI and ENDO strips (8 mm long, 2 mm wide, 1 mm thick) were dissected from the same heart and frozen in liquid nitrogen for biochemistry or further skinned 10 min at 4°C in relaxing solution containing 1% Triton X-100 for mechanical experiments. The strips were then placed in fresh ice-cold relaxing solution and mechanically disrupted at 11,000 rpm for 2-3 s (Polytron PT45-80 with a PTA 10TS shaft, KINEMATICA AG, Switzerland), resulting in a suspension of small clumps of myocyte-sized preparations and cell fragments. The homogenized tissue was filtered and centrifuged at 1000 rpm for 1 min at 4°C. The pellet containing the myocytes was skinned in 0.3% Triton X-100 solution for 6 min to remove remaining sarcolemma membranes and then was extensively washed in relaxing solution. To prevent degradation, all solutions contained protease inhibitors (see above). Cells were kept on ice and used within the day. Some cells were used immediately after cell isolation for mechanical experiments; others were solubilized for gel electrophoresis.

Experimental solutions

Ca 2+ -activating solutions were prepared daily by mixing relaxing (pCa 9.0) and maximal activating (pCa 4.5) solutions [START_REF] Cazorla | SR33805, a Ca2+ antagonist with length-dependent Ca2+ -sensitizing properties in cardiac myocytes[END_REF]. The relaxing and activating buffers contained (in mmol/l): phosphocreatine 12, imidazole 30, free Mg 2+ 1, EGTA 10, Na 2 ATP 3.3, and dithiothreitol 0.3 with pCa 9.0 (relaxing solution) and pCa 4.5 (maximal activating solution), pH 7.1 adjusted with acetic acid (osmolarity 280 mOsm). Sufficient potassium acetate was added to adjust ionic strength to 180 mmol/l.

Single cell SL and force measurements

The procedure of cell attachment was previously reported [START_REF] Cazorla | SR33805, a Ca2+ antagonist with length-dependent Ca2+ -sensitizing properties in cardiac myocytes[END_REF]. The skinned myocyte was attached at either end to a piezoresistive strain gauge (model AE 801, SensoNor a.s., Horten, Norway; 500 Hz unloaded resonant frequency) and a stepper motor driven micromanipulator (MP-285, Sutter Instrument Company, Novato, CA) with thin glass needles and optical glue (NOA 63, Norland Products, North Brunswick, NJ) that was polymerized by UV illumination for 3 min. SL was determined online throughout the experiment at 50 Hz by using a fast Fourier transform algorithm (FFT) on the video images of the attached cell. The cell was positioned at the tip of a conical microcapillary that received the outlet of ten microcapillaries connected to 5ml syringes. Solutions were selected with clips allowing flowing or not of the different solutions (flow rate 200 µl.min -1 ). Force was normalized to cell cross-section area using the width of the cell measured on the video monitor and an estimated value of 2/3 of cell width for the thickness. This ratio was determined on a population of myocytes loaded with membrane fluorescent indicator di-4-ANEPPS (5 µM for 5 min, Molecular Probes, Eugene, OR) to visualize the surface membrane with a laser scanning confocal microscope (Zeiss LSM510). Cell thickness was measured by Z-axis scanning through the upper and lower surfaces of the cell at a central point. Thus, an averaged value for the ratio between the width and the thickness was determined in sham ENDO (0.67±0.02), sham EPI (0.68±0.01), PMI ENDO (0.66±0.02), and PMI EPI (0.67±0.02) (n=40 cells in each group).

After a test activation at pCa 4.5, the cell was sequentially stretched in relaxing solution to 1.9, 2.1, and 2.3 µm SL using a stepper motor driven micromanipulator at a speed of 0.1 length.sec -1 to evaluate steady-state passive tension. Force-pCa relationships were established at two SL, 1.9 and 2.3 µm. Active forces at submaximal activations were normalized to maximal tension (pCa 4.5) at the same SL. The relation between force and pCa was fitted to the following equation: force

= [Ca 2+ ] nH /(K + [Ca 2+ ] nH )
, where n H is the Hill coefficient and pCa 50 , pCa for half-maximal activation, equals -(log K)/n H .

Biochemical analysis of titin

Skinned myocardial strips were pulverized in liquid nitrogen and solubilized for 3 min at 60°C in Laemmli buffer. A batch of cells used for mechanical experiments was also solubilized at the end of the day. The samples were analyzed with SDS-PAGE (2.5-7% acrylamide gradient gels) [START_REF] Cazorla | Differential expression of cardiac titin isoforms and modulation of cellular stiffness[END_REF]. After electrophoretic separation gels were stained with 0.1% Coomassie blue G 250. Wet gels were acquired with an imaging system (Kodak Image Station 2000R). Images were analyzed using Kodak 1D image analysis software.

Immunoblots

Freshly dissected skinned strips, maintained at slack length or stretched by ~20-30% of initial slack length to 2.30 ± 0.05 µm SL for 1 min, were quick-frozen, then homogenized in ice-cold trichloroacetate (10%). This allows for keeping the phosphorylation status of proteins. Samples were then washed three times in acetone /10 mM DTT. The pellet was mixed with urea buffer (in M: urea 8, DTT 0.5, Tris 0.22, glycin 0.25, saturated glucose, EDTA 0.4, PMSF 5.10 -4 , NaF 0.35) [START_REF] Hathaway | A radioimmunoblotting method for measuring myosin light chain phosphorylation levels in smooth muscle[END_REF]. Twenty micrograms of extracted proteins were separated either on 15% SDS-PAGE for TnI analysis or 10% urea gel for MLC2 isoforms [START_REF] Hathaway | A radioimmunoblotting method for measuring myosin light chain phosphorylation levels in smooth muscle[END_REF] and blotted onto nitrocellulose membrane (Protran, Schleichen and Schuele, Dassel, Germany). For both studies, all samples from one animal were always loaded on the same gel. MLC2 isoforms were specifically detected with a cardiac MLC2 antibody (Coger SA, Paris, France). Total TnI content was determined on the first wells of the membrane with a total cardiac TnI antibody (clone 6F9, Hytest, Turku, Finland) and the protein kinase A phosphorylated form of cardiac TnI antibody on the other wells (clone 5E6, Hytest). Immunodetection was revealed with ECL Plus system (Amersham Pharmacia, Little Chalfont Buckinghamshire, England). Results were expressed relative to the total amount of the detected protein.

Data analysis and statistical methods

Data are presented as mean ± SE (n=number of cells studied). One-way or two-way ANOVA was applied for comparison between different groups (as appropriate). When significant interactions were found, a Holm-Sidak t test was applied (Sigmastat3 software), with P < 0.05.

RESULTS

Length-dependent contractile properties across the left ventricular wall

Skinned cells isolated from sham and PMI EPI and ENDO heart layers were maintained in isometric conditions. Active force was generated by superfusing cells with various Ca 2+containing solutions (Fig. 1). Myofilament sensitivity to Ca 2+ (pCa 50 ) established at 1.9 µm SL was not significantly different between cell layers and between sham and PMI animals (Table 1). Stretching sham cells induced a leftward shift of the tension-pCa curve (∆pCa 50 ) that was more pronounced in the ENDO cells (0.24±0.02 pCa unit, n=10) compared with EPI cells (0.11±0.01, n=10). However, this difference disappeared in PMI hearts where ∆pCa 50 values obtained in EPI and ENDO cells were identical (0.14±0.01 pCa unit, n=8 and 11 cells, respectively). The reduced ENDO-PMI stretch-sensitization compared with ENDO-sham indicates that myocardial infarction affects the regulation of cellular contractility predominantly in the endocardial layer. Maximal active tension and Hill coefficient were not significantly different between the various groups.

Passive properties of control and PMI cardiomyocytes

Cellular stiffness of each cell was measured by gradual stretch from slack length (measured before attachment) to 1.9, 2.1, and 2.3 µm SL. In sham hearts, ENDO cells developed significantly higher passive tension when stretched to 2.3 µm SL than EPI cells (Fig. 2A). This gradient of stiffness was maintained in PMI tissues. Over the physiological operating range of SL, titin is essentially responsible for the cellular passive properties [START_REF] Granzier | Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments[END_REF]. Rat ventricles express mostly the short titin isoform [START_REF] Cazorla | Differential expression of cardiac titin isoforms and modulation of cellular stiffness[END_REF]. However, muscle stiffness can be modulated by differential expression of titin isoforms in physiological [START_REF] Cazorla | Differential expression of cardiac titin isoforms and modulation of cellular stiffness[END_REF] and pathological conditions [START_REF] Neagoe | Titin isoform switch in ischemic human heart disease[END_REF][START_REF] Granzier | The giant protein titin: a major player in myocardial mechanics, signaling, and disease[END_REF] but also by varying titin content within the sarcomere [START_REF] Toursel | Passive tension of rat skeletal soleus muscle fibers: effects of unloading conditions[END_REF]. Variation of titin content and isoforms with localization and disease was investigated. In control conditions, the migration of titin revealed a major T1 band corresponding to full-length titin associated with a lower band, T2 resulting from an alteration of titin filament (Fig. 2) as reported previously [START_REF] Granzier | Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments[END_REF]. No change in titin isoform was observed across the sham and PMI ventricles (Fig. 2B, 2C inset). However, the amount of titin relative to myosin varied with localization (Fig. 2C). The ratio was lower in EPI (0.22±0.02) than in ENDO (0.31±0.02) sham cells. A similar trend was observed in PMI rats between EPI (0.26 ±0.02) and ENDO (0.34±0.03) tissues.

Transmural gradient of passive tension-dependent contractile properties

It has been proposed that ∆pCa 50 is closely correlated to titin-based passive tension rather than to SL [START_REF] Cazorla | Length modulation of active force in rat cardiac myocytes: is titin the sensor?[END_REF][START_REF] Fukuda | Titin isoform variance and length dependence of activation in skinned bovine cardiac muscle[END_REF]. We thus studied the relationship between ∆pCa 50 and the passive tension developed at 2.3 µm SL. Figure 3 represents the average values of ∆pCa 50 established in cells isolated from sham and PMI rats, taking into account their location in the left ventricular free wall. Passive tension ranged from ~3-9 mN/mm 2 and ∆pCa 50 from 0.08-0.30 pCa unit. We found a positive correlation between titin-based passive tension and Ca 2+ sensitivity of activation. As previously reported in intact cells [START_REF] Cazorla | Length-tension relationships of subepicardial and sub-endocardial single ventricular myocytes from rat and ferret hearts[END_REF], EPI cells were more compliant and the SL-dependent sensitization of activation was less effective than in ENDO cells. Mid-myocardial (MID) cells demonstrated intermediate properties. This representation demonstrates a gradient of passive and active properties across the left ventricular free wall. More interestingly, we observed that this positive correlation was abolished after myocardial infarction due to a diminished ∆pCa 50 of ENDO cells. The gradient of stiffness was unchanged, but the length sensitization of activation of ENDO-PMI cells was significantly reduced compared with ENDO-sham cells. When the SL dependence of Ca 2+ sensitivity was indexed by ∆[Ca 2+ ] 50 (i.e., difference in [Ca 2+ ] i required for half-maximal activation at 1.9 and 2.3 µm SL), similar correlations were obtained (data not shown). Note that passive tension of both EPI and ENDO-PMI cells was much more variable than of sham ones, most probably indicating remodeling of myocardial tissue. However one cannot fully exclude that cell isolation from the two layers was more difficult due to wall thinning. For the latter reason, we were not able to properly isolate MID cells from PMI hearts.

Phosphorylation of thin and thick filament regulatory proteins

Disappearance of the gradient of stretch-induced increase in Ca 2+ sensitivity in PMI cells suggests an alteration in the biomechanical sensing pathway(s) in diseased rats. Taking into account that the gradient of passive tension is maintained in PMI hearts, we hypothesized that stretch-induced physical rearrangement process was not primarily affected. We thus measured before and after 1-min stretch of skinned myocardial strips, the state of phosphorylation of troponin I and of the two ventricular isoforms of MLC2, a thin and thick regulatory protein, respectively. TnI phosphorylation by PKA is well known to affect Ca 2+ sensitivity and was proposed to enhance length-dependent activation [START_REF] Konhilas | Troponin I in the murine myocardium: influence on lengthdependent activation and interfilament spacing[END_REF]. Using a specific antibody that recognized the PKA-phosphorylated TnI, we determined TnI phosphorylation levels in EPI and ENDO layers of sham and PMI hearts. TnI content and its phosphorylation level were similar between regions of both sham and PMI rats and were unaffected by stretch (Fig. 4A).

Phosphorylation of MLC2 has been previously shown to induce a leftward shift of the tension-pCa curves in cardiac muscles [START_REF] Clement | Protein kinase C enhances myosin light-chain kinase effects on force development and ATPase activity in rat single skinned cardiac cells[END_REF]. In addition, MLC2 phosphorylation is not homogeneous within the whole heart [START_REF] Davis | The overall pattern of cardiac contraction depends on a spatial gradient of myosin regulatory light chain phosphorylation[END_REF]. We thus hypothesized that MLC2 phosphorylation could be induced by a stretch-dependent mechanism tuned across the wall, resulting in a gradient of activation. Four bands were detected corresponding to the two ventricular isoforms of VLC2 (VLC2a and VLC2b) and their phosphorylated forms (P-VLC2a and P-VLC2b) as already described using 2-D gel electrophoresis [START_REF] Morano | Chronic hypertension changes myosin isoenzyme pattern and decreases myosin phosphorylation in the rat heart[END_REF][START_REF] Van Der Velden | Calcium sensitivity of force in human ventricular cardiomyocytes from donor and failing hearts[END_REF][START_REF] Van Der Velden | Increased Ca2+sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins[END_REF]. Phosphorylation level of VLC2a was significantly lower in ENDO-PMI compared with ENDO-sham tissues, and was unaffected by stretch in any of the studied groups. VLC2b phosphorylation was similar in EPI-sham and PMI tissues, and independent of stretch. Interestinglyl in ENDO-sham tissues, VLC2b phosphorylation was hardly detectable at rest, but was increased sixfold by stretch. This stretch-dependent phosphorylation of VLC2b was lost in ENDO-PMI cells (Fig. 4B).

DISCUSSION

For a better understanding of the molecular mechanisms that underlie the Frank-Starling relationship, the goal of this study was to characterize the length-dependent contractile properties of cardiomyocytes isolated from control and failing animals, taking into account their localization within the left ventricular wall. The present work confirms the observation that Ca 2+ sensitivity of the contractile machinery is more correlated to stretch-induced modification in passive tension than to SL. Further, it demonstrates for the first time the existence of a gradient of passive tension-dependent Ca 2+ sensitivity of the contractile machinery within the left ventricular wall accompanied by a marked passive tension-dependent increase in VLC2 phosphorylation of ENDO cells. Following myocardial infarction, this transmural gradient of passive tension-dependent Ca 2+ sensitivity is lost together with the passive tension-induced MLC2 phosphorylation. The remaining component of length-dependent Ca 2+ sensitization is similar to that seen in control or diseased epicardium (Fig. 5).

Transmural contractile properties and passive tension rather than length dependence of force

Here, we report that both passive and active contractile properties follow a positive gradient from epicardium toward endocardium. Skinned ENDO cells are stiffer and demonstrate a larger increase in Ca 2+ sensitivity following stretch. This is consistent with our previous observations that passive and active tensions measured in intact attached cells are higher in ENDO than in EPI cells [START_REF] Cazorla | Length-tension relationships of subepicardial and sub-endocardial single ventricular myocytes from rat and ferret hearts[END_REF].

The SL range across the wall and its changes during cardiac cycle are still unknown. It has been proposed however that during filling, the nonuniform transmural SL distribution observed in the unloaded state becomes more uniform, with the inner sarcomeres elongating more than the outer ones [START_REF] Rodriguez | Effect of residual stress on transmural sarcomere length distributions in rat left ventricle[END_REF]. This is in accordance with higher circumferential strains and ejection fraction observed in vivo in the endocardium [START_REF] Bogaert | Regional nonuniformity of normal adult human left ventricle[END_REF]. In line with these observations, our study shows that heterogeneous cellular mechanical properties may contribute to the nonuniform deformation of the wall. It could also be anticipated that the higher stiffness in the endocardium allows for faster relaxation of this layer during the early diastolic refilling due to titin-based restoring force [START_REF] Helmes | Titin develops restoring force in rat cardiac myocytes[END_REF]. Furthermore, ∆pCa 50 is more closely correlated to passive tension than to SL. This has been initially demonstrated by degrading titin with a mild trypsin treatment (4) and confirmed later on in other animal species coexpressing various titin isoforms or by varying prehistory of stretch before activation [START_REF] Cazorla | Titin-based modulation of calcium sensitivity of active tension in mouse skinned cardiac myocytes[END_REF][START_REF] Fukuda | Titin isoform variance and length dependence of activation in skinned bovine cardiac muscle[END_REF]. Note that despite the use of different species, mouse [START_REF] Cazorla | Titin-based modulation of calcium sensitivity of active tension in mouse skinned cardiac myocytes[END_REF] or bovine [START_REF] Fukuda | Titin isoform variance and length dependence of activation in skinned bovine cardiac muscle[END_REF], similar ∆pCa 50 -passive tension relations were found. Such a correlation between ∆pCa 50 and cell stiffness was also observed in this study in the sham rat heart. Differences in cell compliance could not be explained by cross-bridge-dependent passive tension because they were also seen in the presence of 2,3-butadione monoxime (data not shown). Titin isoform switch was not detected across the left ventricular wall to explain the stiffness gradient, but instead we noticed differential titin amount, with stiffer ENDO cells expressing more titin per half-sarcomere than EPI cells. The difference in stiffness across the free wall seems to be essentially a titin-based mechanism, because we observed that degradation of titin with mild trypsin treatment in both sham and PMI homogenizes passive tension across the wall to ~2 mN/mm 2 (data not shown). From this study, we cannot however exclude that other mechanisms may participate in the passive tension modulation across the wall such as PKA phosphorylation, titin-actin interaction, Ca 2+ binding for titin-based modulation [START_REF] Granzier | The giant protein titin: a major player in myocardial mechanics, signaling, and disease[END_REF], or a difference in myofibrillar area between EPI and ENDO cells.

Stretch-induced phosphorylation of the regulatory MLC2

Titin has been proposed to be involved in the Frank-Starling mechanism at least by reducing the interfilament lattice spacing [START_REF] Cazorla | Titin-based modulation of calcium sensitivity of active tension in mouse skinned cardiac myocytes[END_REF][START_REF] Fukuda | Titin isoform variance and length dependence of activation in skinned bovine cardiac muscle[END_REF]. In addition, stretch-dependent structural changes in the thick filaments in response to passive stretch has been proposed for cardiac muscle [START_REF] Cazorla | Length modulation of active force in rat cardiac myocytes: is titin the sensor?[END_REF][START_REF] Fukuda | Length dependence of tension generation in rat skinned cardiac muscle: role of titin in the frank-starling mechanism of the heart[END_REF]. Indeed, stretch-dependent activation of the cardiac sarcomere cannot solely be explained by lattice spacing, implying other modulation pathways [START_REF] Moss | Frank-starling relationship: long on importance, short on mechanism[END_REF]. Therefore, we postulated that in ENDO cells a passive tension-induced signaling pathway is tuned by stretch, on top of the length-induced effect that could be explained by a change in interfilament lattice spacing. It has recently been suggested that the phosphorylation level of cTnI (on PKA sites) regulates length dependence of activation [START_REF] Konhilas | Troponin I in the murine myocardium: influence on lengthdependent activation and interfilament spacing[END_REF]. This hypothesis would imply a dephosphorylation of TnI with stretch to account for increased activation. Under our experimental conditions, cTnI phosphorylation was similar in both EPI and ENDO cells and was not affected by stretch. TnI is known to have specific sites of phosphorylation by PKC that may affect myofibrillar Ca 2+ sensibility [START_REF] Pi | Protein kinase C and A sites on troponin I regulate myofilament Ca2+ sensitivity and ATPase activity in the mouse myocardium[END_REF]. This latter study did not explore specifically this pathway of phosphorylation of TnI nor the effect of acute stretch. However, we showed previously that incubating myocytes with MLC kinase (MLCK) increases myofibrillar Ca 2+ sensitivity. No additional sensitization was observed when myocytes were incubated with both MLCK and PKC simultaneously while TnI is phosphorylated in these conditions [START_REF] Clement | Protein kinase C enhances myosin light-chain kinase effects on force development and ATPase activity in rat single skinned cardiac cells[END_REF]. This suggests that PKC may increase Ca 2+ sensitivity by activating MLCK.

Phosphorylation of the regulatory protein MLC2 by MLCK represents an important and ubiquitous regulatory mechanism to modulate force generation of smooth, skeletal, and cardiac muscles. It has been shown that MLC2 phosphorylation modulates active tension of the whole heart [START_REF] Kopp | Phosphorylation of the 19,000-dalton light chain of myosin in perfused rat heart under the influence of negative and positive inotropic agents[END_REF]. By increasing the population of myosin heads that could interact with actin, MLC2 phosphorylation increases myofibrillar Ca 2+ sensitivity [START_REF] Craig | Structural changes accompanying phosphorylation of tarantula muscle myosin filaments[END_REF][START_REF] Pi | Protein kinase C and A sites on troponin I regulate myofilament Ca2+ sensitivity and ATPase activity in the mouse myocardium[END_REF]. Based on our previous experience that MLC2 phosphorylation by MLCK sensitizes myofilaments [START_REF] Clement | Protein kinase C enhances myosin light-chain kinase effects on force development and ATPase activity in rat single skinned cardiac cells[END_REF], we proposed that stretch could result in MLC2 phosphorylation. We observed here that stretch mostly activates phosphorylation of VLC2b in ENDO cells that have a very low basal phosphorylation level compared with EPI cells [START_REF] Davis | The overall pattern of cardiac contraction depends on a spatial gradient of myosin regulatory light chain phosphorylation[END_REF]. The present study did not explore the stretch-dependent mechanisms leading to the increase in VLC2b phosphorylation that can include an activation of MLCK as well as an inhibition of a sustained phosphatase activity.

Reduction of the stretch-dependent Ca 2+ sensitivity in PMI

The mechanisms underlying the decline in cardiac pump function during cardiac insufficiency are poorly understood. They lead to a gradual increase in left ventricular end-diastolic pressure and a decrease in systolic pressure. The pathological model used in this study was a PMI rat that represents a clinically relevant model, because post-ischemic cardiomyopathy is the major clinical origin of heart failure. Isolated whole heart from this rat model develops a reduced Frank-Starling relationship. At the cellular level, mechanical behavior of the epicardium seems unaffected. ENDO cells exhibit a markedly reduced stretch-dependent activation that occurs without any effect on the Ca 2+ sensitivity at resting length (1.9 µm) or changes in titin content or isoform expression. The normal, unaffected Ca 2+ sensitivity of ENDO-and EPI-PMI cells at 1.9 µm SL is in accordance with the observations of Anand et al. [START_REF] Anand | Isolated myocyte contractile function is normal in postinfarct remodeled rat heart with systolic dysfunction[END_REF] who observed remodeling of the myocardium up to 6 wk postinfarction, but normal contractile function and intracellular Ca 2+ transient in unloaded intact myocytes.

In heart failure, the transition of  α-MHC to β-MHC isoform was associated with a decrease in the Ca 2+ sensitivity of contractile proteins [START_REF] Metzger | Effects of myosin heavy chain isoform switching on Ca2+-activated tension development in single adult cardiac myocytes[END_REF]. In this study, the electrophoretic method performed was not able to discriminate the αand β-isoforms. Considering that the conversion of αto β-MHC in a hypothyroid rat model does not significantly change SL dependence of Ca 2+ sensitivity [START_REF] Akella | Diminished Ca2+ sensitivity of skinned cardiac muscle contractility coincident with troponin T-band shifts in the diabetic rat[END_REF], such a transition is also unlikely to account for our results either. This idea is also supported by the fact that albeit ATPase is known to be faster in the α-MHC than β-MHC protein, the Frank-Starling relationship is not determined by the cross-bridge detachment [START_REF] Wannenburg | The Frank-Starling mechanism is not mediated by changes in rate of cross-bridge detachment[END_REF].

Changes in titin isoform expression have recently been reported in ischemic human and rat tissues [START_REF] Neagoe | Titin isoform switch in ischemic human heart disease[END_REF]. N2BA titin overexpression associates with reduced passive tension in diseased human myofibrils. We could not detect any significant N2BA titin expression in sham or PMI rat hearts despite a high titin extraction efficiency compared with the work cited, nor did we observe significant changes in passive tension either in ENDO or in EPI cells after infarction.

In the ENDO-PMI cells, reduction in the stretch-dependent myofilament activation is associated with a lack of VLC2b phosphorylation (Fig. 5). Phosphorylation of both VLC2 isoforms is reduced in PMI rats and particularly the VLC2b isoform does not get phosphorylated following stretch. Decreased phosphorylation of VLC2 had been associated with depressed responsiveness of the contractile apparatus to the activating Ca 2+ , accounting in part for the severe heart failure of patients in whom both VLC2 isoforms were dephosphorylated [START_REF] Morano | Chronic hypertension changes myosin isoenzyme pattern and decreases myosin phosphorylation in the rat heart[END_REF]. Nevertheless, it was mentioned that incubation in cardioplegic solution used to keep human tissues might have dephosphorylated MLC2 as well. Similar dephosphorylation of MLC2, in particular VLC2a, was also observed in diseased human tissues and was then correlated with increased contractility of myocytes [START_REF] Van Der Velden | The effect of myosin light chain 2 dephosphorylation on Ca2+ -sensitivity of force is enhanced in failing human hearts[END_REF][START_REF] Van Der Velden | Increased Ca2+sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins[END_REF]. As indicated by the authors, this result is rather surprising considering that MLCK increases Ca 2+ sensitivity of myofilaments [START_REF] Clement | Protein kinase C enhances myosin light-chain kinase effects on force development and ATPase activity in rat single skinned cardiac cells[END_REF][START_REF] Pi | Protein kinase C and A sites on troponin I regulate myofilament Ca2+ sensitivity and ATPase activity in the mouse myocardium[END_REF].

The reduced stretch-dependent contractile properties and VLC2 phosphorylation seen in the ENDO-PMI cells supports the hypothesis that MLC2 is actively involved in the stretch-sensing regulation of contractility. This conclusion, however, does not rule out the participation of other contractile proteins such as MHC, TnI, or TnC in the modulation of contractility. To this point, in ENDO-control cells, the regulation of contractility involves a complex cascade in which titin seems to act as a crucial upstream link. This is supported by the observations that after mild trypsination (4) of both EPI and ENDO cells from sham and PMI rats to degrade titin until passive tension reaches ~2 mN/mm 2 , ∆p Ca50 was decreased to ~0.1 U similarly in all four cell types (unpublished data).

We thus demonstrate that increasing passive tension by stretching titin modulates the myofibrillar sensitivity for Ca 2+ by, yet unidentified, mechanisms that lead to VLC2 phosphorylation. It has been proposed for striated and smooth muscles that MLC phosphorylation could move the S1 subfragment of myosin away from the thick-filament backbone and modulate the actomyosin interaction [START_REF] Sweeney | Myosin light chain phosphorylation in vertebrate striated muscle: regulation and function[END_REF]. Such a mechanism could be related to observations, in skeletal muscle, that stretch induces structural changes in the thick filament that lead to an increase in the fraction of cross-bridges in the disordered state [START_REF] Wakabayashi | X-ray diffraction evidence for the extensibility of actin and myosin filaments during muscle contraction[END_REF]. In cardiac tissue, mainly ENDO cells, the signal transduction pathway that determines passive tension-based modulation of actomyosin interaction has yet to be analyzed.

In conclusion, nonuniformity is a major characteristic of the normal adult left ventricle (LV). The heterogeneous myocardial deformation pattern might be caused not only by the morphological heterogeneity of the tissue in the LV wall, but also by the nonuniform contractile properties of the myocytes across the wall. The loss of a contractile transmural gradient after myocardial infarction should contribute to the impaired LV function. In addition to sham and PMI samples from EPI and ENDO, a skeletal muscle (with a long titin isoform) and a trypsin-treated cardiac tissue (with degradation products of titin) were also loaded. C) The upper part is an expanded view of a gel showing the two typical forms of titin present in the various samples: T1 (full-length titin) and T2 (first degradation product). No titin isoform change was observed in these conditions (confirmed by anti-N2B and anti-N2A antibodies; data not shown). Optical density (OD) area of titin forms and MHC were analyzed by Gaussian curve fits. To ensure that results were obtained in the linear range of the SDS-PAGE system, we electrophoresed a range of loadings for each sample on the same gel. The OD of titin (T1+T2) and MHC were determined for each lane and were plotted vs. their loadings. The slope of the linear range of this relation was determined using linear regression analysis, and the slope ratio of titin and MHC were taken as a measure of the relative amount of titin in the samples. In both PMI and sham, the amount of titin is significantly lower (*P<0.05) in EPI than in ENDO (n=10 animals/group). Immunoblots were performed on permeabilized strips of sham (open column) and PMI (hatched column) myocardium dissected from EPI and ENDO layers before (white) and after (gray)1-min stretch. A) Immunoblots with an anti-cardiac TnI and anti-cTnI phosphorylated by PKA showed that TnI phosphorylation was unaffected by stretch (n=6 animals/group). The specificity of the anti-phospho TnI was tested on sham cells before and after PKA treatment (right part of the blots). B) Immunoblots (upper panels) show four bands of MLC2 corresponding to the two ventricular isoforms (VLC2a and VLC2b) and their phosphorylated forms (P-VLC2a and P-VLC2b). For phosphorylation quantification, each isoform was separated and expressed as % of its total amount (phosphorylated + nonphosphorylated forms) for VLC2a and VLC2b. VLC2b is ~30% of the total amount of MLC2. This form did not vary with localization in sham hearts (34±3% in EPI vs. 29±2% in ENDO), whereas it decreased significantly in ENDO (23±1%) compared with EPI-PMI strips (31±1%) (n=7 animals/group).

Fig. 1 Figure 1 .

 11 Fig. 1

Fig. 2

 2 Fig. 2

Figure 2 .

 2 Figure 2. Passive properties of left ventricular isolated skinned myocytes. A) Steady-state passive tension wasmeasured in relaxing solution (pCa 9) by stretching the skinned cardiomyocyte from 1.9 to 2.1 and 2.3 µm sarcomere length (SL). Experiments were performed in ENDO (square) and EPI (circle) cells (same cells as in Fig.1) and in midwall (star) cells. Passive tension decreased significantly (*P<0.05) in both sham (left panel) and PMI (right panel) cardiomyocytes from ENDO to EPI. B) SDS-PAGE (2.5-7%) was performed to analyze high molecular weight proteins (titin and MHC). In addition to sham and PMI samples from EPI and ENDO, a skeletal muscle (with a long titin isoform) and a trypsin-treated cardiac tissue (with degradation products of titin) were also loaded. C) The upper part is an expanded view of a gel showing the two typical forms of titin present in the various samples: T1 (full-length titin) and T2 (first degradation product). No titin isoform change was observed in these conditions (confirmed by anti-N2B and anti-N2A antibodies; data not shown). Optical density (OD) area of titin forms and MHC were analyzed by Gaussian curve fits. To ensure that results were obtained in the linear range of the SDS-PAGE system, we electrophoresed a range of loadings for each sample on the same gel. The OD of titin (T1+T2) and MHC were determined for each lane and were plotted vs. their loadings. The slope of the linear range of this relation was determined using linear regression analysis, and the slope ratio of titin and MHC were taken as a measure of the relative amount of titin in the samples. In both PMI and sham, the amount of titin is significantly lower (*P<0.05) in EPI than in ENDO (n=10 animals/group).

Fig. 3

 3 Fig. 3

Figure 3 .

 3 Figure 3. Correlation between passive tension and stretch-induced Ca 2+ sensitization across the wall. Data were taken from Figure 1 and Table 1. Variations in pCa 50 induced by stretching cells from 1.9 to 2.3 µm SL were plotted against mean passive tension for each tissue area. Symbols and error bars correspond to each group of cell (ENDO, ; EPI, ; MID, star ) in sham (closed circle and square) and PMI (open circle and square). Linear regressions were calculated on a scatter plot, including all individual data in sham (slope 3.2±0.5, r=0.85) and PMI (slope 0.1±0.3, r=0.07).

Fig. 4

 4 Fig. 4

Figure 4 .

 4 Figure 4. Localized stretch-dependent effect on TnI and MLC2 phosphorylations in control and diseased heart.

Fig. 5

 5 Fig. 5

Figure 5 .

 5 Figure 5. Three-dimensional schematic representation of the stretch-induced Ca 2+ sensitization of contractile proteins in sham and post-myocardial infarcted (PMI), sup-epicardial (EPI), and sub-endocardial (ENDO) cells. Note that an EPI to ENDO gradient of Ca 2+ sensitization, associated with phosphorylation (gray), occurs only in sham cells, on top of the ubiquitous component of stretch-dependent Ca 2+ -sensitization (white) seen in both sham and PMI cells.

Table 1 Regional mechanical characteristics and Ca 2+ sensitivity of skinned cardiomyocytes isolated from sham and post-myocardial infarction (PMI) rats a Sarcomere length, 1.9 µm Sarcomere length, 2.3 µm

 1 Passive (Tpass) and maximal active (Tmax) tensions in mN/mm 2 were measured at pCa 9 and 4.5 and at 1.9 and 2.3 µm sarcomere length, respectively, on sub-epicardial (EPI), mid-wall (MID), and sub-endocardial (ENDO) cells isolated from sham and PMI hearts. Resting sarcomere length before attachment was between 1.82 and 1.88 µm, similar in cells isolated from sham and PMI hearts and across the ventricular wall. pCa 50 , pCa for half-maximal activation, and nH, Hill coefficient, were obtained by fitting the force-pCa relations (see Materials and Methods).

		(n)	Tpass	Tmax	pCa 50	n H	Tpass		Tmax	pCa 50			n H	∆pCa 50
	Sham
	EPI	(10) 0.2±0.1	37±5	5.60±0.03	3.9±0.3	4.3±0.2	41±6	5.70±0.04	3.5±0.2	0.11±0.01
	MID	(6)	0.3±0.2	30±2	5.62±0.02	2.9±0.1	6.4±1.3	34±2	5.81±0.02	b	2.9±0.1	0.20±0.01	b
	ENDO	(10) 0.4±0.1	36±3	5.60±0.01	3.8±0.1	8.3±0.4	b	36±5	5.84±0.02	b	3.4±0.2	0.24±0.02	b
	PMI
	EPI	(8)	0.4±0.1	35±3	5.59±0.03	3.8±0.2	4.8±0.5	39±4	5.73±0.03	3.2±0.2	0.14±0.01
	ENDO	(11) 0.5±0.1	38±3	5.62±0.02	4.1±0.3	8.3±0.9	b	43±5	5.76±0.02	c	3.7±0.4	0.14±0.01	c
	a

b EPI vs. ENDO/MID. c ENDO-or EPI-sham vs. ENDO-or EPI-PMI cells.

ACKNOWLEDGMENTS

This work was supported in part by Association Française contre les Myopathies, Fondation pour la Recherche Médicale, Fondation de France, Fondation Simone Del Duca, and Conseil Régional du Languedoc-Roussillon. SS held a Eurogendis Marie Curie grant fellowship. We thank Patrice Bideaux and Guillermo Salazar for technical assistance and Drs. A. Lazou, A. Siamkouri, and H. Viko for preliminary experiments.