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Cardiovascular Research 2006 71(4):652-660; doi:10.1016/j.cardiores.2006.06.020
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Copyright © 2006, European Society of Cardiology

Effects of high-altitude exercise training on contractile function of rat skinned cardiomyocyte

O. Cazorlaa,*, Y. Aït Moua, L. Goretb, G. Vassorta, M. Dauzatc, A. Lacampagnea, S. Tanguyb and P. Obertb

aINSERM, U 637, CHU Arnaud de Villeneuve, F-34295 Montpellier, France. Université MONTPELLIER1, UFR de Médecine, F-34295 Montpellier, France
bJE 2426, Physiologie des Adaptations Cardiovasculaires à l'Exercice, Université Avignon et des Pays de Vaucluse, UFR Sciences, F-84000 Avignon, France
cEA 2992, Dynamique des Incohérences Cardio-Vasculaires, Université MONTPELLIER1, UFR de Médecine, F-30908 Nîmes, France

* Corresponding author. Tel.: +33 467 41 52 44; fax: +33 467 41 52 42. Email address: cazorla{at}montp.inserm.fr

Received 22 February 2006; revised 9 June 2006; accepted 14 June 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective Previous studies have questioned whether there is an improved cardiac function after high-altitude training. Accordingly, the present study was designed specifically to test whether this apparent blunted response of the whole heart to training can be accounted for by altered mechanical properties at the cellular level.

Methods Adult rats were trained for 5 weeks under normoxic (N, NT for sedentary and trained animals, respectively) or hypobaric hypoxic (H, HT) conditions. Cardiac morphology and function were evaluated by echocardiography. Calcium Ca2+ sensitivity of the contractile machinery was estimated in skinned cardiomyocytes isolated from the left ventricular (LV) sub-epicardium (Epi) and sub-endocardium (Endo) at short and long sarcomere lengths (SL).

Results Cardiac remodelling was harmonious (increase in wall thickness with chamber dilatation) in NT rats and disharmonious (hypertrophy without chamber dilatation) in HT rats. Contrary to NT rats, HT rats did not exhibit enhancement in global cardiac performance evaluated by echocardiography. Stretch- dependent Ca2+ sensitization of the myofilaments (cellular index of the Frank-Starling mechanism) increased from Epi to Endo in N rats. Training in normoxic conditions further increased this stretch-dependent Ca2+ sensitization. Chronic hypoxia did not significantly affect myofibrilar Ca2+ sensitivity. In contrast, high-altitude training decreased Ca2+ sensitivity of the myofilaments at both SL, mostly in Endo cells, resulting in a loss of the transmural gradient of the stretch-dependent Ca2+ sensitization. Expression of myosin heavy chain isoforms was affected both by training and chronic hypoxia but did not correlate with mechanical data.

Conclusions Training at sea level increased the transmural gradient of stretch-dependent Ca2+ sensitization of the myofilaments, accounting for an improved Frank-Starling mechanism. High-altitude training depressed myofilament response to Ca2+, especially in the Endo layer. This led to a reduction in this transmural gradient that may contribute to the lack of improvement in LV function via the Frank-Starling mechanism.

KEYWORDS Contractile function; Hypertrophy; Hypoxia/anoxia; Myocytes; Mechanotransduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Aerobic exercise induces cardiac morphological remodelling (increase in myocardial mass and ventricular chamber dimensions) and functional adaptations (for review see [1]) leading to increased maximal stroke volume and subsequently cardiac output. Part of the training effect on the cardiac performances is due to increased myocyte contractile function possibly through changes in their intrinsic active properties [1]. Various cellular parameters have been investigated to account for the beneficial effect of exercise on contraction. Training does not appear to modify intracellular Ca2+ transient [2–4]. However, it increased Ca2+ sensitivity of the myofilaments in intact [5] and skinned cardiomyocytes [6]. The aforementioned experiments used various training and/or experimental conditions that could lead to some heterogeneity in the results. Cardiomyocyte properties are not uniform within the myocardium. In the rat heart, there are regional differences in electrical [7] as well as in mechanical properties. We have described previously that intact [8], and skinned [9] myocytes isolated from the sub-endocardium (Endo) are more responsive to stretch than those from the sub-epicardium (Epi), with higher passive and active tensions developed in Endo at the same sarcomere length (SL). Natali et al. [10] showed that voluntary exercise increases the steepness of the active tension–SL relationship of attached intact cardiomyocytes exclusively in Endo but not in Epi. However, Diffee et al. reported that training increases Ca2+ sensitivity in both Endo and Epi skinned myocytes [11]. The latter experiments were performed at 2.35 µm SL, while the intact cells in the former work could be stretched experimentally to 2.05 µm SL at the best.

In their constant search to improve performance, many athletes train at high altitude because hypoxia exposure enhances erythropoiesis and subsequently oxygen carrying capacity. However, there is now clear evidence in both animal models and human that training at high altitude does not provide advantages over training at sea level as regards maximal oxygen uptake and aerobic performance [12,13]. This altitude-training paradox is in part due to changes in cardiac morphological and functional parameters determining stroke volume [13]. We showed previously that rats trained under hypobaric hypoxic conditions presented a specific cardiac remodelling characterised by increased left ventricular wall thickness without chamber enlargement. Moreover, contrary to sea-level training, cardiac function was not improved when training was conducted under these experimental conditions. In particular resting and maximal stroke volumes were not improved, leading to the assumption that high-altitude training depressed heart and especially myocyte contractile properties.

This study was specifically designed to test the hypothesis that high-altitude training limits the beneficial effects classically observed after training at sea level. Considering that cardiomyocyte properties, including the response to exercise training, are not uniform within the myocardium, we investigated the effects of training under normoxic and hypobaric hypoxic conditions on myofilament Ca2+ sensitivity and its stretch-dependency in single skinned cardiomyocytes, taking into account their localization in the sub-endocardium and sub-epicardium.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Training procedure
Male Wistar rats (300–350 g) were randomly assigned to one of the four groups (7 animals per group): rats living continuously either in normoxic conditions without or with aerobic training sessions (N and NT, respectively) and rats living continuously in hypoxic conditions without or with aerobic training sessions (H and HT, respectively). High altitude was simulated by a hypobaric hypoxia environment obtained with a vacuum pump as described previously [13]. Rats were maintained for 5 weeks at a barometric pressure of 760 mm Hg (PIO2{approx}159 mm Hg, altitude{approx}80 m) for N and NT, or at a pressure of 475 mm Hg (PIO2{approx}90 mm Hg, altitude{approx}4000 m) for H and HT. Some were trained under sea-level (NT) or high-altitude (HT) conditions for 5 weeks in a driven wheel as described previously [13]. Briefly, the training program consisted of 5 sessions (45-min) per week at 80% of the maximal aerobic velocity (MAV) determined in each environmental condition. MAV was evaluated using a driven wheel during a maximal exercise test [14] in each environment and for each rat before the study, during the 3rd week, and at the end of the protocol. All procedures were performed in agreement 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.

2.2. Echocardiography and heart weighing
Rats were anesthetized with intraperitoneal ketamine HCl (75 mg/kg) and xylazine (10 mg/kg). A two-dimensional short-axis view of the left ventricle was obtained at the level of the papillary muscles using an echocardiograph (5–8 MHz micro-convex transducer HDI 3000, ATL, Phillips Company, Bothell, USA) as reported previously [13]. End-diastolic anterior and posterior wall thicknesses (AWT and PWT, respectively), and left ventricular end-diastolic (LVEDd) and end-systolic (LVESd) diameters were measured. The diastolic relative wall thickness (RWT=AWT+PWT/LVEDd) was calculated as an index of cardiac remodelling. Left ventricular stroke volume (LVSV) and heart mass were evaluated as described previously [13]. Measurements from at least three consecutive cardiac cycles were averaged.

2.3. Single cardiomyocyte sarcomere length and force measurements
The Ca2+-activated force of single skinned myocyte was measured as described previously [9,15]. Myocytes were isolated by mechanical dissociation from pre-skinned left ventricular strips. Some cells were kept on ice and used within a day for mechanical experiments; the others were solubilized for gel electrophoresis.

Skinned myocytes were attached to a piezoresistive strain gauge (AE801 sensor, Memscap, Crolle, France) and to a stepper motor driven micromanipulator with thin needles and UV-polymerized optical glue (NOA 63, Norland products Inc., North Brunswick, NJ). Sarcomere length (SL) was determined online throughout the experiment (Ionoptix Boston, USA). Force was normalized to cross-sectional area measured from the imaged cross-section as described previously [16]. Slack SL was measured before attachment to serve as zero reference and did not differ significantly between myocytes from normoxic (1.87±0.01 µm SL, n=45) and hypoxic (1.86±0.01 µm SL, n=43) rats. The pCa (= – log[Ca2+]i)–force relationships were established at two SL: 1.9 then 2.3 µm, at 22 °C. Cells that did not maintain 80% of the first maximal tension or a visible striation pattern were discarded. We continued with cells that were well attached with minimal SL changes (<0.1 µm) during activation. 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:



Formula

where nH is the Hill coefficient and pCa50, pCa for half-maximal activation equals – (log K)/nH.

2.4. Solutions
Solutions were similar to those used previously [9]. For cell dissociation, the Ca2+-free Hanks–HEPES buffered solution contained (in mM): NaCl 117, KCl 5.7, NaHCO3 4.4, KH2PO4 1.5, MgCl2 1.7, HEPES 21, glucose 11.7, taurine 20, 2,3-butanedione monoxime 15, pH 7.15 adjusted with NaOH, and was bubbled with 100% O2.

For myocyte force measurements, activating solutions were prepared daily by mixing relaxing and maximal activating solutions containing (in mM): Na-acetate 10, phosphocreatine 12, imidazole 30, free Mg2+ 1, EGTA 10, Na2ATP 3.3 and dithiothreitol 0.3 with either pCa 9.0 (relaxing solution) or pCa 4.5 (maximal activating solution), protease inhibitors (PMSF 0.5; leupeptin 0.04 and E64 [trans-epoxysuccinyl-1-leucine-guanidobutylamide] 0.01), pH 7.1 adjusted with KOH. Ionic strength was adjusted to 180 mM with K-acetate.

2.5. Biochemistry
Myosin heavy chain (MHC) isoforms were separated following a procedure adapted from Warren and Greaser [17]. The myocytes were dissolved in a SDS lysis buffer (TRIS–HCl 50 mM, 2% (w/v) SDS, urea 8 M, EGTA 1 mM, EDTA 1 mM, DTT 80 mM, 10% (v/v) glycerol, pH 6.8, protease inhibitors) and heated at 60 °C for 5 min. Proteins (~0.1 µg) were separated on a 6% SDS-PAGE at 16 mA constant current for 20 h at 4 °C (Shelton Scientific gel system). The gels were silver stained and analysed with an imaging system (Kodak Image Station 2000R). β-MHC content was expressed relative to the total amount of the MHC protein.

Citrate synthase (CS) activity was assayed by measuring absorbance over 3 min at 412 nm at 25 °C in soleus muscle homogenate. Muscle was first homogenized in buffer (in mM: 210 sucrose, 2 EGTA, 40 NaCl, 30 HEPES, 5 EDTA, and 2 PMSF, pH 7.4) and then mixed with 15 µM acetylCoA, 0.5 mM oxaloacetate, 0.1 mM DNTB [18].

2.6. Statistics
Data are presented as mean±SEM. The effects of hypoxia and training were examined by using a 2-way analysis of variance (factor 1: hypoxia vs. normoxia, factor 2: sedentary vs. trained) with or without repeated measures (i.e. time or sarcomere length) depending on variables. The effect of the cellular localization on the stretch-dependent Ca2+ sensitization in the different conditions was tested by a 3-way ANOVA (Statview, Abacus concepts, Inc., Berkeley, CA). Post-hoc tests of Fisher's Protected Least Significant Difference were used when appropriate. Linear regression analyses performed on raw data examined relationships between {delta}pCa50 and β-MHC content or passive tension. Comparison of linear regressions (Y intercepts and slopes) was examined using Statgraphics Centurion XV. Statistical significance was defined as P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Training features and cardiac morphological parameters
Efficiency of the training program in NT and HT rats was evaluated from maximal aerobic velocity (MAV) and skeletal muscle citrate synthase activity. Both increased significantly after training, irrespective of environmental conditions (Fig. 1A). LVSV increased significantly in NT but was unchanged in HT (Table 1). No change was reported for N and H rats for all parameters.


Figure 1
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  		Fig. 1 Training features and left ventricular morphological characteristics of sedentary and trained rats maintained in normoxic or hypoxic conditions. (A) Maximal aerobic velocity (MAV) is expressed as the relative variation between pre- and post-protocol (left). Citrate synthase activity is expressed as IU g– 1 wet weight (right). (B) Morphological parameters were measured by echocardiography and data are expressed as the relative variation between pre- and post-protocol for the LV end-diastolic diameter (LVEDd), end-diastolic anterior wall thickness (AWT), and relative wall thickness (RWT) (*P<0.05).

 

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  		Table 1 Cardiac morphological and functional parameters

 
Right ventricular mass increased significantly with hypoxic exposure. Left ventricular mass did not differ between the two sedentary groups, but it increased significantly to the same extent in the two groups after exercise training performed at sea level or high altitude (Table 1).

Echocardiographic morphological parameters were affected differently by training depending on environmental conditions (Fig. 1B). Namely, LVEDd increased in NT but decreased in HT rats. AWT and PWT increased significantly with training conducted under normoxic as well as hypoxic conditions. The cardiac remodelling was harmonious in NT rats (unchanged RWT values) but disharmonious (marked increase in RWT highlighting a concentric LV hypertrophy) in HT rats. LV dimensions were not affected throughout the study period in N and H rats.

3.2. Stretch-dependent contractile properties
The Ca2+ sensitivity of myofilament activation (pCa50) at 1.9 µm SL was similar across the ventricular wall in N, H and NT rats. However Ca2+ sensitivity decreased significantly at this SL in HT rats for both Epi and Endo cells (Fig. 2). Stretching cells to 2.3 µm SL induced a leftward shift of the tension–pCa curve in all groups, indicative of increased myofilament sensitivity to Ca2+. For both Epi and Endo cells, Ca2+ sensitivity at 2.3 µm SL increased with training in normoxic conditions but decreased when training was performed in hypoxic conditions (Fig. 2C).


Figure 2
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  		Fig. 2 Effects of training in normoxia or hypoxia on length-dependency of the active tension in isolated skinned cardiomyocytes. Tension (normalized to maximal tension)–pCa curves of left ventricular myocytes isolated from sup-epicardium (close symbols, left) or sub-endocardium (open symbols, right) were measured at 1.9 (continuous line) and 2.3 (dashed line) µm sarcomere length, in sedentary (N, H) and trained (NT, HT) rats maintained for 5-weeks in normoxic (A) or hypoxic (B) environments. (N: {blacksquare} {square}, NT: O •, H: {blacktriangleup} {triangleup}, HT: * *, with Epi (close) and Endo (open)) (C) Ca2+ sensitivities determined at 1.9 (gray bar) and 2.3 (open bar) µm SL by the measurement of pCa50 (pCa for half-maximal activation) in individual Epi (left) and Endo (right) cells. The number of cells in each condition is indicated in Table 1 (*P<0.05).

 
The stretch-dependent modulation of activation or Ca2+ sensitization ({delta}pCa50' difference in pCa50 obtained at 2.3 and 1.9 µm SL) is an important factor accounting for the Frank–Starling mechanism of the heart. It increased significantly with chronic exercise at sea level in Endo and in Epi cells (Fig. 3). Chronic hypoxia exposure did not modify significantly {delta}pCa50 although a slight decrease was observed in Endo H cells compared to Endo N cells (p=0.09). High-altitude endurance training had no beneficial effect on stretch-dependent Ca2+ sensitization. Rather it even decreased in Endo cells from HT when compared to N and NT (Fig. 3 and Table 2). Maximal active tension did not differ between the various groups and myocardium localization (Table 2). The Hill coefficient increased significantly only in Epi from the HT group at both SL, but this effect was not investigated further.


Figure 3
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  		Fig. 3 Stretch-dependent Ca2+ sensitization ({delta}pCa50) in cells isolated from sub-epicardium (left) or sub-endocardium (right) in normoxic (N) or hypoxic (H) sedentary and trained (NT–HT hatched pattern) rats (3-way ANOVA, *P<0.05).

 

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  		Table 2 Regional mechanical characteristics and Ca2+ sensitivity of skinned cardiomyocytes

 
3.3. Involvement of MHC isoform
The heart co-expresses two isoforms of MHC ({alpha}- or β-MHC). The change in {alpha}/β-MHC ratio influences cardiac function and occurs under various stresses including exercise (see for review [19]). As shown previously rat ventricles used in our study contained mostly {alpha}-MHC (Fig. 4A). After training in normoxic conditions, β-MHC expression was reduced significantly in both Epi and Endo cells. Chronic hypoxia exposure increased β-MHC expression particularly in Endo cells. β-MHC expression was similar in N and HT rats in both Endo and Epi cells. Thus, chronic exercise affected β-MHC expression in both myocardial layers and by chronic hypoxia mostly in Endo cells. Accordingly, we investigated the relationship between the stretch-dependent Ca2+ sensitization and β-MHC expression level. A significant negative correlation was obtained in Endo and Epi when data from the 4 groups were pooled (Fig. 4B). When groups were analysed separately, the correlation between {delta}pCa50 and β-MHC expression levels was still evident in N and NT rats (Fig. 4C). After normoxic training, {delta}pCa50 was higher and associated with lower β-MHC expression in both myocardial layers. However under hypoxic conditions, the correlation was no longer obtained in Endo cells. Values in Epi cells from sedentary and trained rats were mixed and spread similarly from 20 to 40% of β-MHC, resulting in similar average values.


Figure 4
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  		Fig. 4 Myosin heavy chain expression in cardiomyocytes isolated from sedentary and trained rats maintained in normoxic (open bar) or hypoxic (gray bar) conditions. (A) SDS-PAGE for {alpha}- and β-MHC of myocytes isolated from the left ventricle. β-MHC content was expressed relative to the total MHC in Epi and Endo in each group (n=4 hearts per group analysed in duplicate). *P<0.05. (B) Correlation between the {delta}pCa50 and the relative amount of β-MHC. Linear regressions were calculated on a scatter plot including all raw data points from Epi (line, {blacksquare}) and Endo (dash line, •) cells isolated from N, NT, H, and HT rats. A significant negative correlation was found in Epi (y=0.23 – 0.0020x, r2= – 0.44, P=0.005) and Endo (y=0.21 – 0.0025x, r2= – 0.53, P=0.01) cells for the overall population. (C) The relationship between {delta}pCa50 and β-MHC content was analysed in populations of cells obtained from rats exposed and/or trained either under normoxic (left) or hypoxic (right) environments. (N: {blacksquare} {square}, NT: O •, H: {blacktriangleup} {triangleup}, HT: * *, Epi (close symbols, continuous line) and Endo (open symbols, dashed line)). A significant negative correlation was found in normoxic Epi (y=0.22 – 0.0045x, r= – 0.48, P=0.03), in normoxic Endo (y=0.25 – 0.0036x, r= – 0.46, P=0.03), and in hypoxic Epi (y=0.25 – 0.0038x, r= – 0.58, P=0.007) cells. No correlation was found in hypoxic Endo cells (y=0.14 – 0.0003x, r=0.06, P=0.79).

 
3.4. Passive tension-dependent contractile properties
In the relaxing solution Endo cells developed significantly higher passive tension than Epi cells after a stretch from slack length up to 2.3 µm SL (Table 2). Chronic hypoxic exposure or exercise training did not affect the transmural gradient of passive tension.

{delta}pCa50 is closely related to passive tension rather than to sarcomere length [20,21] and there is a transmural gradient of stretch-sensitization [9]. We thus established the relationships between {delta}pCa50 and the passive tension developed at 2.3 µm SL by individual cells (Fig. 5). A positive correlation was found across the free wall in N and NT rats. Higher passive tensions and {delta}pCa50 were obtained in NT when compared to N rat cells but individual values from the 2 groups were distributed along the same regression lines (Fig. 5A). A positive correlation between passive tension and {delta}pCa50 was also observed for H rat cells. The regression line (slope and intercept) of H rats was not significantly different to the ones observed in N and NT rats although its slope was slightly lower (Fig. 5B). When rats were trained under a hypoxic environment, the relationship between passive tension and {delta}pCa50 disappeared, mostly due to a diminished {delta}pCa50 of Endo cells. When the stretch-dependence of Ca2+ sensitivity was indexed by {delta}[Ca2+]50 (i.e.. difference in [Ca2+]i required for half-maximal activation at 1.9 and 2.3 µm SL, Table 2) similar correlations were obtained between the groups (data not shown).


Figure 5
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  		Fig. 5 Correlation between stretch-induced Ca2+ sensitization ({delta}pCa50) and passive tension across the wall. {delta}pCa50 was plotted against mean passive tension for each tissue area. Symbols and error bars correspond to each group of cell (Endo: open symbols, Epi: close symbols) in sedentary (square for N and triangle for H) and trained (circle for NT and star for HT) rats living in normoxic (A) or hypoxic (B) conditions. Linear regressions were calculated on a scatter plot including all raw data points from both myocardial layers in each condition (line for sedentary, dash line for trained). Exercise increased {delta}pCa50 and passive tension but values are distributed over the same linear relationship in N (y=0.036+0.017x, r=0.67 p=0.001) and NT rats (y=0.069+0.016x, r=0.78 p=0.001). Stretch-dependent sensitization was decreased by chronic hypoxia exposure (y=0.071+0.010x, r=0.47 p=0.02). No correlation was found in HT cells (y=0.120+0.003x, r=0.21 p=0.38).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
To gain insight into the effects of exercise training at high altitude on cardiac function, we subjected rats to chronic hypobaric hypoxia and endurance training. Hypoxic stress had a moderate negative impact on the contractile properties of the myofilaments, affecting preferentially cardiomyocytes from the endocardium. The transmural gradient of cardiomyocyte contractile properties, classically observed within the normal ventricular wall, was improved by sea-level training but disappeared after high-altitude training. This depressed cardiac myofilament calcium responsiveness may contribute to the lack of improvement in ventricular function observed after high-altitude training.

4.1. Effect of training at sea level
Sea-level training improved the contractile properties of the myofilaments in both Endo and Epi layers. This is in accordance with previous studies reporting an increase in contraction and Ca2+ sensitivity in cardiomyocytes isolated from endurance-trained rats [5,6]. Diffee and Nagle [22] reported an increase of the length-dependence of maximal and sub-maximal tension in LV cardiomyocytes, mostly due to increased Ca2+ sensitivity at long sarcomere length. Furthermore, we reported recently a gradient of contractile properties across the LV wall. After stretch, Endo cells developed more passive tension than Epi cells and the stretch-dependent Ca2+ sensitization of the myofilaments ({delta}pCa50) was higher [9]. Sea-level training shifted the stretch-induced Ca2+ sensitization–passive tension relationships of Epi and Endo cells to higher values. Cells from NT rats were more responsive to stretch than those from N rats but the reasons for this improvement are still unclear (see below). These results may however account for the steeper slope of the active tension–SL relationship observed by Natali et al. [10] in attached intact cardiac myocytes from trained rats.

The changes in cellular properties observed in the present study might contribute to the enhanced ventricular function (i.e. higher LV stroke volume) reported in NT rats. This is in line with Reboul et al. [13] who observed a greater increase in LVSV following volume overloading in endurance-trained rats compared to sedentary ones, indicative of an exercise-induced improvement of the Frank–Starling mechanism of the whole heart. In accordance with previous results from our group [13] and others [5], sea-level training induced LV hypertrophy, accounted for increased wall thickness and internal dimensions, with also important significance for cardiac performance. Thus, the enhanced LVSV in NT rats was likely due to greater ventricular filling associated with improved contractile properties of the myofibrils across the ventricular wall.

4.2. Effect of training at altitude
High-altitude training did not improve LVSV (Table 1). From the present results, we suggest that this is partly due to specific cardiac remodelling (reduced LVEDd) and deteriorations of the myofilament contractile properties. The marked wall thickening may be a compensatory mechanism for the depressed contractile function, allowing maintenance of the cardiac output and a response to the exercise-induced increase in energetic demand.

Chronic hypoxia exposure alone did not alter significantly Ca2+ sensitivity of the myofilaments. However high-altitude exercise training reduced the Ca2+ sensitivity of the myofilaments in both Epi and Endo cells whatever the sarcomere length. Moreover, the transmural gradient of stretch-induced Ca2+ sensitization of the myofilaments was abolished completely in HT rats. Whether haemodynamic factors and/or hypoxia per se are responsible for this effect cannot be ascertained. On one hand, assuming similar LV end-diastolic pressure in NT and HT rats and considering the cardiac remodelling in these 2 groups, lower end-diastolic wall stress is to be expected in HT when compared to NT rats. On the other hand, we could postulate that episodes of severe myocardium hypoxemia occurred as a result of exercise at high-altitude thus affecting the contractile machinery. In this context, it is interesting to note that similar alterations in cardiomyocyte contractile properties were obtained previously by our group in a rat model of chronic myocardial ischemia [9]. Long-term exposure to high-altitude hypoxemia of foetal sheep has also been reported to decrease contraction of intact papillary muscle [23]. Finally, 4 weeks of chronic hypoxia has been shown to reduce Ca2+ transient in association with impaired Ca2+ release and uptake in rat cardiomyocytes [24]. The loss of the contractile transmural gradient in HT rats should contribute to the lack of improvement in LVSV after training at simulated high altitude. Similarly the present cellular data can account for the lack of improvement after high-altitude training in the Frank–Starling mechanism investigated with a volume overloading protocol [13].

4.3 Molecular basis of the stretch-dependent Ca2+ sensitivity
The stretch-induced Ca2+ sensitization of the myofilaments relates to passive tension rather than to SL [9,20,21]. This relationship across the wall was enhanced in NT rats but no longer existed in HT rats. Over the physiological range of SL, titin is the major component of the cellular passive properties [20]. No change in titin content or isoform expression was observed in the present study (data not shown). Although not significant, the opposite passive tension changes observed in NT and HT rats could be due to other mechanisms that may have participated in the fine tuning of passive tension-dependent modulation of activation across the wall such as PKA phosphorylation, titin–actin interaction or Ca2+ binding on titin [25] that may directly and/or indirectly affect the properties of the contractile and/or regulatory proteins.

β-MHC is characterized by lower adenosine triphosphatase activity and lower filament sliding velocity, but can generate cross-bridge force with a higher economy of energy consumption than {alpha}-MHC [26]. In accordance with the present data, previous [27–29] but not all [30,31] studies have shown increased {alpha}-MHC isoform expression after sea-level training. Therefore, an increase in {alpha}-MHC isoform expression could be at least in part responsible for the enhanced stretch-dependent Ca2+ sensitization of the myocytes in NT rats. In the present study, we did find a correlation between the level β-MHC and the stretch-dependent mechanism at sea level. However, Endo cells from N and HT rats had similar β-MHC content but different {delta}pCa50. This suggests that other proteins are very likely responsible for the changes in Ca2+ sensitivity observed in our study. In a previous publication [9] we observed in normoxic sedentary rats that Endo cells had a higher {delta}pCa50 than Epi cells due to a higher phosphorylation level of myosin-light chain 2 (MLC2*) after stretch. In post-myocardial infarcted rats, the MLC2* was no longer phosphorylated after stretching and was associated with reduced {delta}pCa50 in Endo cells. It is thus possible that the level of phosphorylation of MLC-2 had been increased in NT rats and decreased in HT rats, contributing thus to the Ca2+ sensitivity results of the present study. The slight decrease in passive tension and the rightward shift of the tension–pCa curves in HT rats may be due to an increase in PKA-dependent stimulation [15]. The levels of phosphorylation of MLC2 and other regulatory proteins such as TnI and cMyBP-C before and after stretch have to be explored.

In conclusion, nonuniformity is a major characteristic of the normal adult left ventricle that is partly due to heterogeneous contractile properties of the myocytes across the wall. The major mechanical adaptations to sea-level training improved the transmural gradient of stretch-dependent Ca2+ sensitization of the myofilaments. This cellular mechanism should improve the Frank–Starling relationship solicited particularly during exercise. High-altitude training induced a reduction of Ca2+ sensitivity of the myofilaments and stretch-dependent Ca2+ sensitization mostly in the Endo layer, which might have contributed to the lack of improvement of the ventricular function observed in vivo.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by the "Association Française contre les Myopathies", "Fondation pour la Recherche Médicale", and "Région Languedoc-Roussillon". OC and AL are established investigators of CNRS. Thanks are due to Patrice Bideaux and Guillermo Salazar for technical assistance. We are thankful to Guillaume Py and Cyril Reboul for helpful expertise in citrate synthase activity and echocardiography, respectively.


   Notes
 
Time for primary review 20 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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Y. A. Mou, C. Reboul, L. Andre, A. Lacampagne, and O. Cazorla
Late exercise training improves non-uniformity of transmural myocardial function in rats with ischaemic heart failure
Cardiovasc Res, September 4, 2008; (2008) cvn229v2.
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