Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Numerous studies have reported respiratory muscle weakness during chronic congestive heart failure in both humans (De Troyer et al., 1980; Hammond et al., 1990; Mancini et al., 1992; McParland et al., 1992) and animals (Supinski et al., 1994; Howell et al., 1995; Lecarpentier et al., 1998). This weakness, likely due to structural (Sullivan et al., 1990; Lindsay et al., 1996; Tikunov et al., 1997), metabolic (Weiner et al., 1986; Mancini et al., 1989; Wilson et al., 1992), and biochemical changes (Massie et al., 1988; Sullivan et al., 1990; Drexler et al., 1992), may contribute, at least in part, to exercise intolerance and excessive ventilatory response. Alterations in limb skeletal muscle metabolism have also been reported in experimental cardiac volume overload without congestive heart failure (Chati et al., 1994), raising the possibility that reduced intrinsic muscle performance may develop in the early stage of heart failure.

Effective therapies to improve prognosis and exercise tolerance have been established for chronic heart failure. Angiotensin-converting enzyme (ACE) inhibitors prolong life (The SOLVD Investigators, 1991) and also improve skeletal muscle flow and peak oxygen consumption during exercise (Mancini et al., 1987; Drexler et al., 1992) in patients with chronic heart failure. Peripheral improvements are associated with a gradual reversal of chronic structural alterations in skeletal muscle (Drexler et al., 1992; Schaufelberger et al., 1996). Whether these effects are associated with improved intrinsic muscle performance and whether beneficial effects are observed at an early stage of cardiac hypertrophy remain to be determined.

In the present study, skeletal muscle performance was studied in a rabbit model of chronic cardiac hypertrophy before the occurrence of congestive heart failure (CHF). The first aim of the study was to determine to what extent intrinsic diaphragm weakness occurred in early stages of pressure cardiac overload. Given that cardiac diseases may not affect respiratory and limb skeletal muscles in the same way (Hammond et al., 1990; Howell et al., 1995; Tikunov et al., 1997), we also examined the intrinsic performance of soleus muscle. The second aim of our study was to determine whether a preventive therapy with the ACE inhibitor perindopril (PE) had any beneficial effects on intrinsic skeletal muscle performance during moderate pressure cardiac overload. The third aim was to determine whether modifications in molecular cross-bridge (CB) properties were involved in the potential changes in skeletal muscles during chronic cardiac overload. We therefore investigated the number, kinetics, and single force of CBs (Lecarpentier et al., 1997, 1998; Coirault et al., 1997) and the MHC composition of diaphragm and soleus muscles.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Preparation of Animals and Surgical Procedure. Animal care conformed to international guidelines. Surgical procedures were performed after induction and maintenance of anesthesia with midazolam (0.5 mg, i.v.) and etomidate (4.5 mg, then 20 mg/h, i.v.). Subtotal constriction of the suprarenal abdominal aorta was carried out on female adult New Zealand rabbits. The abdominal aorta was surgically isolated just below the diaphragm and a piece of polyethylene catheter (external diameter of 2.4 mm; Biotrol, Paris) was positioned along it. The catheter and the aorta were ligated together just above the right renal artery and then the catheter was gently removed. This procedure reduced the abdominal aortic lumen by 45% (Gilson et al., 1990). This model constantly induces moderate cardiac hypertrophy with preserved systolic function. Thereafter, the rabbits were randomly treated by p.o. gavage with either PE (1 mg/kg/day; n = 9) or a placebo (PL; n = 15). A third group of sham-operated animals received PL (n = 10). Sham-operated animals were subjected to the same operation without inducement of aortic constriction. Treatment was administered 7 days a week for 10 weeks.

Mounting Procedure and Mechanical Analysis. After anesthesia with sodium pentobarbital (30 mg/kg, i.p.), the animals were thoracotomized and then laparotomized. Two muscle strips were carefully excised per animal from the ventral part of the costal diaphragm. A strip was also dissected from the soleus muscle. Each muscle strip was rapidly mounted in a tissue chamber containing a Krebs-Henseleit solution: 118 mmol/l NaCl, 24 mmol/l NaHCO₃, 4.7 mmol/l KCl, 1.2 mmol/l Mg SO₄7H₂O, 1.1 mmol/l KH₂PO₄, 2.5 mmol/l CaCl₂6H₂O, and 4.5 mmol/l glucose. The solution was bubbled with a gas mixture of 95% O₂-5% CO₂ and maintained at 22°C and pH 7.4. Tendinous extremities were held in spring clips and attached to an electromagnetic force transducer. The electromagnetic lever system used has been described previously (Lecarpentier et al., 1997). Muscles were electrically stimulated by means of two platinum electrodes delivering 1-ms rectangular pulses at 0.17 Hz. Experiments were carried out at the optimal initial resting length (Lo), which corresponds to the apex of the length-active tension curve. The muscle cross-sectional area (in mm²) was calculated from the ratio of fresh muscle weight to muscle length at Lo.

Study Protocol. Tension-frequency curves were determined by stimulating muscle strips at 25, 33, 50, 75, 100, 200, and 400 Hz (train duration: 400 ms, 10/min). The peak isometric tension; i.e., peak force normalized per cross-sectional area, was measured from the fully isometric contraction. Tension-frequency relationships were expressed in terms of absolute tension (in mN · mm²) and of percentage of maximum isometric tension (Po) at the optimal tetanic frequency.

1

CB Number and Kinetics. According to the most widely accepted theory of contraction (Huxley, 1957), CBs act as independent force generators. Therefore, muscle force depends on the elementary force produced per CB and the total number of CBs formed. A. F. Huxley's equations (Huxley, 1957) were used to calculate the rate of total energy release (E, in mW · mm²), the isotonic tension (P_Hux, in mN · mm²), and the rate of mechanical energy (W_M, in mW · mm²) as a function of velocity (V). In these equations, f₁ is the maximum value of the rate constant for CB attachment and g₁ and g₂ are the peak values of the rate constants for CB detachment (Huxley, 1957). The instantaneous movement x of the myosin head relative to actin varies from h to 0, where h is the step size of the CB; h is defined by the translocation distance of the actin filament per ATP hydrolysis and produced by the swing of the myosin head (Huxley, 1969); f₁ and g₁ correspond to x = h, and g₂ corresponds to x  h (Huxley, 1957); s is the resting sarcomere length at Lo;  = (f₁ + g₁) h/2 = b (Huxley, 1957); for reasons of equation dimensions,  was multiplied by s/2 compared with the initial hypothesis (Huxley, 1957). Consequently, calculations of f_1, g₁, and g₂ were divided by s/2 compared with those previously detailed (Lecarpentier et al., 1997, 1998; Coirault et al., 1997) and are given by the following equations:
where w is the mechanical work of a single CB, and e is the free energy required to split one ATP molecule (Huxley 1969; Eisenberg et al., 1980, Huxley and Simmons, 1971);

2

1

Values of Huxley's Equation Constants. A stroke size of 11 nm has been determined by means of optical tweezers (Finer et al., 1994) and corresponds to the three-dimensional structure of the myosin head (Rayment et al., 1993). The distance  is equal to 36 nm (Woledge et al., 1985). The free energy required to split one ATP molecule per contraction site is e = 5.1 × 10²⁰ J. The mechanical work (w) of a single CB is equal to 0.75 e (Huxley, 1957), so that w = 3.8 ×10²⁰ J.

Myosin Electrophoresis. Preparations of crude myosin were obtained from the ventral part of the costal diaphragm and from soleus muscles, as previously described (Coirault et al., 1997). Electrophoresis was performed in a Bio-Rad Mini-Protean II Dual Slab Cell electrophoresis system (Bio-Rad, Hercules, CA) for 24 h at 4°C and 70 V (constant voltage). MHCs were separated in dissociating conditions with 0.75 mmol SDS-polyacrylaminde gel minigel electrophoresis (Talmage and Roy, 1993; Mortola and Naso, 1995). Stacking gel was composed of 4% acrylamide (2.67% bis-acrylamide), 70 mmol Tris (pH 6.8), 30% glycerol, 4 mmol EDTA, and 0.1% SDS. The composition of separating gel was 8% acrylamide (1% bis-acrylamide), 0.2 mol Tris pH 8.8, 0.1 mol glycine, and 0.4% SDS. Separate upper and lower running buffers were used. The upper running buffer consisted of 0.1 mol Tris (base), 150 mmol glycine, and 0.1% SDS. The lower running buffer consisted of 50 mmol Tris (base), 75 mmol glycine, and 0.05% SDS. Both buffers were prepared shortly before use and cooled at 4°C. Gels were stained with 0.2% Coomassie blue, 50% ethanol, and 10% acetic acid, and destained with 5% ethanol and 5% acetic acid. The different MHC isoforms were quantified by one-dimensional densitometry (GS-690; BioRad). The amount of each isoform was determined by the area of each peak. Data were expressed as percentages of the area of each peak over the sum of the areas of all peaks.

Plasma ACE Assay. Blood samples were taken after catheterization of the right ventricle. The samples were centrifuged for 10 min at 10,000/rpm at 4°C, and plasma was stored at 80°C. The tripeptide (¹⁴C)-hippuryl-histidyl-leucine was used as a substrate for ACE determination. The (¹⁴C)-hippuric acid generated was extracted with ethyl acetate and counted in a Packard liquid scintillation spectrometer. ACE activity was expressed in mU/ml (1 mU/ml = 1 nmol hippuric acid generated per minute at 37°C).

Statistical Analysis

Data are expressed as means ± S.E. Comparison of several means was performed using ANOVA and the Scheffé test. Linear regression was based on the least-squares method. The asymptotes a and b of the Hill hyperbola were calculated by means of multilinear regression and the least-squares method. A p value of <.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Cardiac Hypertrophy. In both PE and PL groups, there were no physical signs of CHF (i.e., s.c. edema, ascites, pericarditis, pleurisy, and/or pulmonary and hepatic congestion). The degree of cardiac hypertrophy was assessed in terms of the heart weight/body weight ratio. This ratio was increased by approximately 27% in PL compared with shams (p < .05) but did not differ significantly between PE and sham-operated rabbits (Table 1).

Plasma ACE Activity. In PE-treated rabbits, plasma ACE activity was 3.8 ± 0.8 mU/ml. Plasma ACE activity was inhibited by 95%, compared with placebo-treated rabbits (71.4 ± 5.9 mU/ml), and was inhibited by 93%, compared with shams (56.6 ± 5.9 mU/ml).

Diaphragm Contractile Properties. When absolute values of tension were considered at different tetanic frequencies, diaphragm strength was significantly reduced in PL, with the entire tension-frequency curve of PL shifted downward, compared with shams (Fig. 1A). However, the overall shape of tension-frequency curves were similar in shams and PL, as shown by the fact that when tension was expressed as a percentage of Po, tension-frequency curves for shams and PL groups were similar (Fig. 1B). Finally, there was no significant difference between shams and PE with regard to the tension-frequency curves.

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Effects of increasing stimulation frequency on tension development in diaphragm and soleus muscles at 22°C. A, absolute peak isometric tension normalized per cross-sectional area. B, relative peak isometric tension. Values are means ± S.E.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Main mechanical parameters. A, peak isometric tension at the optimal stimulation frequency (33 Hz). B, maximum unloaded shortening velocity. In each panel, means ± S.E. are presented for diaphragm and soleus muscles. Open columns, sham-operated rabbits; solid columns, PL-treated rabbits; cross-hatched columns, PE-treated rabbits. Values are means ± S.E.; *p < .05, **p < .01.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   CB properties. A, total number of CBs. B, Elementary force per CB. In each panel, means ± S.E. are presented for diaphragm and soleus muscles. Open columns, sham-operated rabbits; solid columns, PL-treated rabbits; cross-hatched columns, PE-treated rabbits. *p < .05, **p < .01.

Soleus Contractile Properties. Soleus strength was significantly reduced in PL, with the entire tension-frequency curve of PL shifted downward compared with sham-operated rabbits (Fig. 1A). PE treatment tended to prevent the shift in the tension-frequency curve and no significant difference was observed between PE and shams. However, when expressed as a percentage of Po, tension-frequency curves for sham and PL groups were similar (Fig. 1B). In the three groups, Po was observed at a 33-Hz stimulation frequency (Fig. 1A). At 33 Hz, soleus Po was 33% lower and the total number of CBs was 31% lower in PL, compared with shams (p < .01 for both; Figs. 2A and 3A). Perindopril played a significant role in preventing the decrease in both Po and m (Figs. 2A and 3A). There was no significant difference among the three groups with regard to V_max and  (Figs. 2B and 3B). In sham-operated rabbits, the G curvature was 7.7 ± 0.6 and did not differ among groups (7.9 ± 0.6 and 8.0 ± 0.4 in PL and PE, respectively). Eff_max did not differ significantly between groups (43 ± 1%). Similarly, the rate constant for attachment (f₁) and detachment (g₁ and g₂) and tc did not differ significantly among the three groups (Table 2).

MHC Distributions. Electrophoresis showed the presence of three MHC isoforms in myosin from diaphragm; namely, fast IIA and IIX and slow-type I myosin isoforms (Fig. 4). The sham soleus expressed both type I and IIA MHCs, with type I accounting for nearly 80% of the total MHC pool. In both diaphragm and soleus, there were no significant differences in MHC isoforms among the three groups (Table 3).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Typical electrophoretic pattern of MHC in diaphragm and soleus from sham-operated rabbit. Homogenates prepared were subjected to SDS-glycerol electrophoresis to resolve the MHC isoforms.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We analyzed the intrinsic function of diaphragm and soleus muscles in a rabbit model of moderate cardiac hypertrophy induced by chronic pressure overload. The major findings of our study were as follows: 1) moderate cardiac hypertrophy was associated with a marked impairment in the intrinsic performance of diaphragm and limb skeletal muscles, at a stage when CHF has not yet occurred; 2) single therapy with the ACE inhibitor PE tended to preserve the intrinsic performance of both diaphragm and limb skeletal muscles.

Skeletal Muscle Failure in Placebo-Treated Cardiac Hypertrophic Rabbits. There is evidence that exercise intolerance and fatigue poorly correlate with the degree of left ventricular dysfunction (Franciosa et al., 1981; Szlachcic et al., 1985; Massie et al., 1988) and inadequate skeletal muscle blood flow (Massie et al., 1988). However, observations reported so far support the view that alterations in skeletal muscle in heart failure are, after all, the consequences of impaired cardiac function. For the first time, our results showed that rabbits with moderate cardiac hypertrophy exhibited a marked reduction in diaphragm and soleus muscle performance, indicating that skeletal muscle weakness can occur early in the course of the cardiopathy, i.e., when CHF has not yet occurred. In both PL diaphragm and soleus, lower peak tetanic tensions were associated with a significant decrease in the number of CBs.

ACE Inhibitor Therapy. For the first time, our study showed that early therapy with the ACE inhibitor PE tended to preserve the intrinsic performance of diaphragm and soleus muscles in rabbits with moderate cardiac hypertrophy. Previous studies have reported reversal of ultrastructural abnormalities (including reduced number and size of mitochondrial abnormalities and increased muscle fiber area and lactate dehydrogenase activity) after ACE inhibitor therapy (Drexler et al., 1992; Schaufelberger et al., 1996). In chronic cardiac pressure overload, our study indicated that preserved muscle performance after PE was mainly caused by its preventive effect on the decline in CB number. These beneficial effects of ACE could provide a partial explanation for the increased exercise capacity and higher peak oxygen consumption during exercise observed in patients suffering from CHF and receiving ACE therapy (Mancini et al., 1987; Drexler et al., 1989).

Conclusions. In rabbit, chronic cardiac pressure overload without CHF induced early alterations in diaphragm and soleus muscle performance. Effective therapy with the ACE inhibitor PE tended to prevent a reduction in muscle strength and maximum shortening velocity. Changes in CB number but not in the single force per CB were the probable explanation for modifications in diaphragm and soleus strength before and after ACE therapy.