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Skeletal muscle myosin heavy chain expression in rats with monocrotaline-induced cardiac hypertrophy and failure. Relation to blood flow and degree of muscle atrophy

Giorgio Vescovo, Claudio Ceconi, Palmira Bernocchi, Roberto Ferrari, Ugo Carraro, Giovanni Battista Ambrosio, Luciano Dalla Libera
DOI: http://dx.doi.org/10.1016/S0008-6363(98)00041-8 233-241 First published online: 1 July 1998


Background: In congestive heart failure (CHF) the skeletal muscle of the lower limbs develops a myopathy characterised by atrophy and shift from the slow to the fast type fibres. The mechanisms responsible for these changes are not clear yet. Objectives: We investigated the influence of blood flow and degree of muscle atrophy on the myosin heavy chains (MHC) composition of the soleus and extensor digitorum longus (EDL) of rats with right ventricle hypertrophy and failure. Methods: CHF was induced in 16 rats by injecting 30 mg/kg monocrotaline. Eight animals had the same dose of monocrotaline but resulting in compensated right ventricle hypertrophy. Two age- and diet-matched groups of control animals (nine and five respectively) were also studied. The relative percentage of MHC1 (slow isoform), MHC2a (fast oxidative) and MHC2b (fast glycolytic) was determined by densitometric scan after electrophoretic separation. The relative weights of soleus and EDL (muscle weight/body weight) were taken as an index of muscle atrophy. Skeletal muscle blood flow was measured by injecting fluorescent microspheres. Results: CHF and Control (Con) rats showed similar degree of atrophy both in soleus (0.40±0.06 vs. 0.44±0.06 p=NS), and EDL (0.47±0.04 vs. 0.45±0.02, p=0.09). In CHF rats these two muscles showed a statistically significant MHCs redistribution toward the fast type isozymes. In fact in EDL of CHF rats MHC2a was 30.5±6.1% vs. 35.8±8.6% of the Con (p<0.05). MHC2b was however higher (68.5±6.6% vs. 61.0±9.6%, p=0.017). In the soleus of CHF rats MHC1 was decreased (87.6±3.4% vs. 91.9±5.2%, p=0.02), while MHC2a was increased (12.04±3.5% vs. 7.9±5.2%; p=0.028). Similar changes were not found in the muscles of the compensated hypertrophy animals. No correlation was found between MHC pattern and the relative muscle weight in the CHF animals. Soleus blood flow in CHF rats was significantly lower than that of Con (0.11±0.03 ml/min/g vs. 0.22±0.03 p<0.05), while no differences were found in EDL (0.06±0.02 ml/min/g vs. 0.08±0.02, p=NS). Conclusions: In rats with CHF a skeletal muscle myopathy characterised by a shift of the MHCs toward the fast type isoforms occurs. The magnitude of the shift correlates neither with the degree of atrophy, nor with the skeletal muscle blood flow, suggesting that these two factors do not play a pivotal role in the pathogenesis of the myopathy.

  • Heart failure
  • Myosin heavy chains
  • Skeletal muscle
  • Monocrotaline

Time for primary review 30 days.

1 Introduction

Congestive heart failure (CHF) is characterised by a disordered skeletal muscle performance that can in part explain fatigue and shortness of breath [1, 2]. A series of abnormalities possibly contributing to exertional fatigue have been described. These involve loss of muscle bulk [3], changes in muscle fibres with reduction of the type 1 and a shift toward the fast type 2b [3–6], mitochondrial abnormalities [7], altered muscle metabolism [8, 9]. Whether muscle atrophy due to deconditioning plays a role in the genesis of this myopathy is not clear yet. We have recently suggested that the CHF myopathy is specific being not related to detraining. The gastrocnemius of patients with profound disuse atrophy shows in fact a preferential synthesis of the slow myosin heavy chain (MHC) 1 [10], but the debate on the possible causes of this myopathy is still open. It has been hypothesised that cytokine activation and loss of anabolic function [11], ergo–metaboloreceptors dysfunction [12], or changes in blood flow [13, 9]may be of importance. This latter point is still debated and there are conflicting reports on the role of blood flow. Wilson et al. [13]have in fact reported an impaired nutritive flow to skeletal muscle, while Massie et al. [9]showed no relation between severity of CHF and blood flow. The present study was undertaken in the attempt to shed some light on the pathophysiological role of muscle atrophy and nutritive blood flow on skeletal muscle alterations. We used a well characterised and largely accepted animal model of heart failure [14, 15]: the monocrotaline-treated rat. Monocrotaline is a pyrrolizidine alkaloid that induces pulmonary vascular disease with severe hypertension, right ventricle hypertrophy and in some animals overt CHF heart failure [16]. We also compared animals with overt heart failure to rats with compensated right ventricle hypertrophy.

2 Methods

2.1 Experimental model

Four groups of animals were studied:

  • 16 animals with CHF (CHF)

  • 8 animals with right ventricle hypertrophy (Hyp)

  • 9 saline-injected controls for the CHF group (Con)

  • 5 saline-injected controls for the Hyp group (ConHyp)

Sprague–Dawley rats of weight 75 to 100 g were injected intraperitoneally with monocrotaline (30 mg/kg). Treated rats were allowed to eat freely from a supply of standard rat cubes. The control rats were diet-matched to the treated rats by allowing them only the quantity of food consumed on the previous day by the treated rats. The CHF rats are those animals that 4 weeks after monocrotaline injection develop pleural, pericardial and peritoneal effusions, accompanied by severe right ventricle hypertrophy and dilatation. Rats that do not develop signs of decompensation at 4 weeks (Hyp) [16], are kept alive for an extra week. They develop concentric right ventricle hypertrophy, but not CHF. They have age- and diet-matched controls since they grow slightly more, reaching a higher body weight. Rats were kept in 30×30 cm cages. After 4 and 5 weeks respectively, animals were killed, body weight (BW), right ventricle weight (RVW), lung weight measured, together with pleural and peritoneal effusions. soleus (Sol) and extensor digitorum longus (EDL) were excised and weighed. The ratio Sol/BW and EDL/BW (relative muscle weight) was then calculated and taken as an index of muscle atrophy. Right ventricle weight/body weight (RVW/BW) was calculated and taken as an index of right ventricle hypertrophy. Before sacrifice a blood sample was also drawn for determination of β-ANP (Atrial Natriuretic Peptide). The assay was performed with the method previously described by Comini et al. [17].

Experiments were approved by the Ethical Committee of the Interdepartmental Biological Building of the University of Padua, and were carried out according to the Italian laws.

2.2 Effect of monocrotaline on the skeletal muscle

Although selective target organs for monocrotaline are liver and lung [18, 19], we looked at MHCs changes in the EDL and Sol of four rats, 2 weeks after the injection of 30 mg/kg monocrotaline. Four age- and diet-matched controls were also killed. RVW/BW, sol/BW and EDL/BW were measured.

2.3 Electrophoretic separation of MHCs

The method is an improvement of that published by Carraro and Catani [20]and described in detail by Vescovo et al. [10]. Muscles were homogenised and solubilised in 2.3% sodium dodecyl sulphate (SDS), 10% glycerol, 0.5% 2-mercaptoethanol, and 6.25 mM Tris–HCl, pH 6.8. The running buffer was added to the sample in a ratio of 1:1 (v/v). Analytical SDS-polyacrylamide gel electrophoresis of MHC was performed on 7.5% polyacrylamide slabs with 37.5% (v/v) glycerol. The stacking gel was composed of 37.5% glycerol, 4% T–acrylamide–Bis (36.5:1), 12.5 mM Tris–HCl (pH 6.8), and 0.1% SDS. Running buffer was made of 50 mM Tris, 384 mM glycine, pH 8.3, and 0.2% SDS. Separation of MHC was achieved using constant current at 4 mA. After 12 h the running buffer was changed and electrophoresis restarted with the same parameters for another 24 h. Gel containing approximately 0.2 μg of protein per band was stained with 0.1% Coomassie brilliant blue in 5% acetic acid–40% methanol and destained with 40% methanol–7% acetic acid. Identification of individual MHCs was performed in a separate series of experiments by immunoblotting the gel bands with a panel of monoclonal antibodies [10].

2.4 Assessment of MHCs content

The percent distribution of MHC1, MHC2a and MHC2b was determined by densitometric scan of the stained slab gels. A GS300 transmittance reflectance scanning densitometer (Hoefer Scientific Instruments) connected to a McIntosh SE Apple computer was used. Data were processed with GS370 densitometry software. A linear response is obtained on densitometry when 0.1–2 μg of individual MHC is analysed. Quantitative densitometry was performed using internal MHC standards electrophoresed in the same gel as described by Sandri et al. [21].

2.5 Skeletal muscle blood flow determination

Skeletal muscle blood flow was determined according to the Glenny et al. method [22]in another group of animals: six CHF, ten Con, eight Hyp and six ConHyp rats were studied. The rats were anaesthetised with pentobarbital (45 mg/kg body weight) and a cannula was introduced into the carotid artery for the injection of fluorescent microspheres. Red microspheres [FluoSpheres polystyrene, 15μm, red fluorescent (580/605)] for blood flow determination were supplied as suspension in 10 ml of 0.15 M NaCl with 0.02% Tween 20 and 0.02% thimerosal. The suspension containing 1.0·106 microspheres/ml (Molecular Probes Europe) was vigorously vortexed for 3–5 min and ultrasonicated. Via the carotid cannula it was injected into the left ventricle over 10 s in a volume of 400 μl (4·105 microspheres). The cannula was then flushed with 0.5 ml of heparinised saline. Starting 10 s before the injection of the fluorospheres, a reference blood sample was collected for 90 s from the femoral artery cannula at a constant rate of 0.51 ml/min. The skeletal muscles (Sol and EDL) were excised, weighed and digested overnight in 4 ml of 4 M KOH. The digested tissue pieces were then individually filtered through 10 μm pore polycarbonate filters (Poretics, Livermore, CA, USA) by use of negative pressure. The tubes and filtering burettes (Millipore) were washed twice with 2.0% Tween, and the collected fluid was filtered to pick up residual microspheres. Filters were placed in polypropylene test tubes and the fluorescent dye eluted from the retained microspheres by 1 ml of Cellosolve acetate. Fluorescence was determined by a spectrofluorophotometer. Tissue blood flow could then be calculated with respect to the reference blood sample, using the following equation: Embedded Image In our series of experiments the relationship between number of microspheres and fluorescence intensity was linear (r>0.99) and the estimated number of microspheres per sample was between 100 and 200. The detection limit of the fluorescent microspheres method is two- to fourfold higher than that of other previously employed methods in which radioactive and coloured microspheres were used.

2.6 Histology and morphometric analysis

On each muscle a cross-section was taken for histological examination. Sections were stained with Azan Mallory. The cross-sectional area of fibres and the volume fraction of extracellular space, which is composed by interstitium plus fibrosis, were calculated by means of a computerised interactive method using a Zeiss Ibas 2000 instrument, as described by Angelini et al. [23].

2.7 Statistical analysis

Student's t-test for unpaired data and linear regression was used when appropriate. Mean±S.D. are reported. A 5% difference was considered statistically significant.

3 Results

3.1 Postmortem examination (Table 1)

A postmortem examination of the rats was carried out in order to assess the presence or absence of CHF and of compensated cardiac hypertrophy in the different groups of animals according to the criteria listed in Section 2. The presence of CHF was confirmed in 16 rats (CHF group). Concentric compensated right ventricle hypertrophy was detected in eight rats (Hyp group). No alterations were found in the control rats. The occurrence of CHF was also confirmed by the RVW/BW, by the lung weight/BW, by the presence of ascites and pleural effusions and by the β-ANP assay which showed significantly higher levels of this peptide in the decompensated animals (see Table 1).

View this table:
Table 1

Right ventricle weight/body weight (RVW/BW), lung weight/body weight, ascites, pleural effusions and β-ANP in the Con, CHF, ConHyp and Hyp rats

Lung weight/BW (mg/g)6.26±0.27b12.59±0.72b5.72±0.136.20±0.35
Ascites (ml)01.14±0.8800
Pleural effusions (ml)01.52±0.4500
Plasma β-ANP (fmol/ml)35.89±5.07c357.87±25.85c39.57±2.7643.2±0.94
  • a p<0.01, bp<0.01, cp<0.001.

3.2 Body weight in CHF, Hyp and control rats

The Hyp rats and their relative controls showed similar BW (214.3±18.6 g vs. 232.1±22.9 respectively; p=NS), which were both significantly (p=0.001) higher than those of the CHF animals and their relative controls. Between these latter groups BW also differed significantly (116.2±28.3 vs. 159.2±21.2; p=0.005).

3.3 Sol and EDL weight (Table 2)

Sol weight was significantly higher in Hyp rats than in CHF (p<0.001). No statistically significant differences were found between Hyp and ConHyp (p=NS), while CHF rats showed a lower Sol weight when compared to Con (p<0.002). Similarly EDL weight was identical in the Hyp and ConHyp groups (p=NS), though a significant difference could be noted between CHF and Con rats (p<0.002). As expected CHF rats showed a much lower EDL weight than Hyp rats (p<0.0001).

View this table:
Table 2

EDL and Sol weight in CHF and Con rats, Hyp and ConHyp rats

Muscle weight (mg)49.7±12.279.8±14.2101.3±9.0105.9±10.4
Muscle weight (mg)45.5±9.870.15±11.878.1±11.790.1±13.4
Relative muscle weight0.43±0.060.51±0.100.47±0.040.45±0.02
Relative muscle weight0.40±0.060.44±0.060.41±0.040.33±0.02
  • Relative muscle weight (muscle weight/body weight) in the same four groups of animals.

    ap<0.002, bp<0.0001, cp<0.001, dp<0.002, ep<0.009.

3.4 Relative muscle weights (Table 2)

The Sol weight/BW was similar in CHF and Con rats (p=NS). In the Hyp the Sol relative muscle weight was significantly higher than in the ConHyp (p<0.009). EDL weight/BW of CHF rats was slightly though not significantly lower than that of Con (p=0.09). In the Hyp and ConHyp groups the ratio was almost identical (p=NS).

3.5 Histology and morphometric analysis

There were no differences in fibres cross-sectional area between CHF and Hyp animals and their relative controls (1292±375 μ2 in CHF vs. 1427±491 in Con, p=0.2; 1391±343 in Hyp vs. 1479±448 in Con Hyp, p=NS). The fibrosis and interstitial volume fractions, measured altogether, ranged from 3.8 to 4.2%, and were therefore similar in the four groups of animals for both EDL and Sol.

3.6 MHCs electrophoretic pattern

MHC isoforms were separated as described in Section 2and are presented in Fig. 1. In rat skeletal muscle three MHC isoforms (MHC1, MHC2b and MHC2a) were separated on the basis of their relative mobility.

Fig. 1

Electrophoretic separation on polyacrylamide SDS slab-gel of myosin heavy chains (MHC) (arrows indicate the three isoforms: MHC1 “slow type”, MHC2a “fast oxidative” and MHC2b “fast glycolytic”) separated on the basis of their relative mobility in order from fastest to slowest. (a) Extensor digitorum longus (EDL) from Con rat. (b) EDL from CHF rat. (c) Sol from Con rat. (d) Sol from CHF rat.

3.6.1 EDL [Fig. 2(a and b)]

MHC2a in Con rats was 35.8±8.6% vs. 30.5±6.1 of the CHF (p<0.05). MHC2b was significantly lower in the Con than in the CHF rats (61.0±9.6 vs. 68.5±6.6, p=0.017). A similar shift toward the fast type isoforms was not present in the Hyp rats that showed a percentage of MHC2a and MHC2b similar to that of the ConHyp (33.6±6.7 vs. 28.3±3.1, p=NS and 71.6±3.1 vs. 66.3±6.7 p=NS respectively).

Fig. 2

(a) Percent distribution on myosin heavy chain (MHC) 2a and 2b in the extensor digitorum longus (EDL) in the CHF and Con rats, showing an increased expression of the fast glycolytic isoform MHC2b in CHF. *p<0.05, +p=0.017. (b) Percent distribution of myosin heavy chain (MHC) 2a and 2b in the extensor digitorum longus (EDL) in the Hyp and ConHyp rats, showing no changes in MHCs pattern with compensated hypertrophy. *p=NS, +p=NS.

3.6.2 Sol [Fig. 3(a and b)]

The Sol showed changes in the isozyme pattern similar to those seen in EDL with a shift toward the fast MHCs. CHF animals showed in fact a significantly lower percentage of MHC1 when compared to Con (87.6±3.4 vs. 91.9±5.2, p=0.02), while MHC2a was significantly higher (12.04±3.5 vs. 7.9±5.2, p=0.028). In the Hyp group no changes were detected when compared to ConHyp either for MHC1 (89.03±5.4 vs. 93.9±4.04, p=NS) or MHC2a (10.9±5.4 vs. 6.06±4.04, p=NS).

Fig. 3

(a) Percent distribution of myosin heavy chain (MHC) 1 and 2a in the Sol of CHF and Con rats, showing an increased expression of the fast oxidative MHC2a in CHF. *p=0.028, +p=0.02. (b) Percent distribution of myosin heavy chain 1 and 2a in the Sol of Hyp and ConHyp rats, showing no changes in MHCs pattern with compensated hypertrophy. *p=NS, +p=NS.

There was a significant correlation between MHC1 and β-ANP in the CHF group (r=0.64, p<0.01) and a borderline significance for RVW/BW (r=0.5, p=0.05). There was no correlation in the Hyp group between these parameters.

3.7 Effect of 2 weeks monocrotaline-treatment on skeletal muscle

Two weeks of treatment with monocrotaline did not affect either Sol (MHC2a 8.8±1.4 vs. 7.8±1.3, p=NS), or EDL MHC composition (MHC2a 32.3±1.2 vs. 30.4±4.6, p=NS). The degree of atrophy was similar in the monocrotaline and controls both in EDL (0.43±0.01 in the monocrotaline vs. 0.46±0.02 in controls, p=NS) and Sol (0.45±0.03 in the monocrotaline vs. 0.44±0.02 in controls, p=NS). Right ventricle hypertrophy was not present in the monocrotaline-treated rats as shown by the RVW/BW which was identical to that of the controls (0.078±0.02 vs. 0.084±0.01, p=NS).

3.8 Correlation between degree of muscle atrophy and MHC composition

The degree of muscle atrophy was correlated with the percent distribution of MHCs within the CHF and Hyp groups analysed separately.

3.8.1 CHF group

In the Sol of the 15 CHF rats mentioned in Section 2.1, no correlation was found either for MHC1 or MHC2a (r=0.15 p=NS, r=0.10 p=NS respectively) (Fig. 4(a and b)). The same was true for EDL degree of atrophy and both MHC2a (r=0.14, p=NS) and MHC2b (r=0.25, p=NS) (Fig. 5a and 5b).

Fig. 5

(a) Linear regression between EDL degree of atrophy (EDL weight/body weight) and percentage of myosin heavy chain 2a (MHC2a), showing no correlation between these two parameters in the CHF group (n=15). (b) Linear regression between EDL degree of atrophy (EDL weight/body weight) and percentage of myosin heavy chain 2b (MHC2b), showing no correlation between these two parameters in the CHF group (n=15).

Fig. 4

(a) Linear regression between degree of Sol atrophy (Sol weight/body weight) and percentage of myosin heavy chain 1 (%MHC1), showing no correlation between these two parameters in the CHF group (n=15). (b) Linear regression between Sol degree of atrophy (Sol weight/body weight) and percentage of myosin heavy chain 2a (MHC2a), showing no correlation between these two parameters in the CHF group (n=15).

3.8.2 Hyp group [Fig. 6(a and b)]

In the animals mentioned in Section 2.1, the degree of atrophy of Sol was significantly correlated with both MHC2a (r=0.6, p=0.028) and MHC1 (r=0.6, p=0.029). Similar results were obtained for EDL, being r=0.64 (p=0.015) for MHC2a and r=0.65 (p=0.015) for MHC2b (Fig. 7(a and b).

Fig. 7

(a) Linear regression between EDL degree of muscle atrophy (EDL weight/body weight) and percentage of myosin heavy chain 2a (MHC2a), showing in the Hyp and ConHyp groups a significant correlation between these two parameters (n=13). (b) Linear regression between EDL degree of atrophy (EDL weight/body weight) and percentage of myosin heavy chain 2b (MHC2b), showing in the Hyp and ConHyp groups a significant correlation between these two parameters (n=13).

Fig. 6

(a) Linear regression between Sol degree of atrophy (Sol weight/body weight) and percentage of myosin heavy chain 1 (MHC1), showing in the Hyp and ConHyp groups a significant correlation between these two parameters (n=13). (b) Linear regression between Sol degree of atrophy (Sol weight/body weight) and percentage of myosin heavy chain 2a (MHC2a), showing in the Hyp and ConHyp groups a significant correlation between these two parameters (n=13).

3.9 Skeletal muscle blood flow (Table 3)

3.9.1 CHF group

The skeletal muscle blood flow of the Sol was 0.11±0.03 ml/min/g of tissue. This was significantly lower (p<0.05) than that of Con rats (0.22±0.03). The blood flow of EDL was not significantly different from that of Con rats (0.08±0.02 vs. 0.06±0.02, p=NS).

View this table:
Table 3

Skeletal muscle blood flow in CHF, Con, Hyp and ConHyp rats

Sol CHFSol ConEDL CHFEDL ConSol HypSol ConHypEDL HypEDL ConHyp
Blood flow0.11±0.03*0.22±0.03*0.06±0.020.08±0.020.11±0.040.18±0.050.10±0.020.12±0.03

3.9.2 Hyp group

The Sol blood flow in Hyp rats was 0.11±0.04. This was not statistically different from that measured in the ConHyp rats (0.18±0.05, p=NS). In the EDL there was a slight, but not significant, reduction in blood flow when compared to the ConHyp rats (0.10±0.02 vs. 0.12±0.03, p=NS).

4 Discussion

In this paper we describe, for the first time, biochemical and structural modifications occurring in the skeletal muscle of a well established model of CHF [14, 15], together with measurements of blood flow. We have in fact found an increased expression of more fatiguable MHCs isoforms in muscles with different function and bioenergetic characteristics. In the “fast twitch” EDL the fast glycolytic MHC2b was increased as was the fast oxidative MHC2a in the “slow twitch” Sol. We have also reported on changes in muscle trophism in CHF and Hyp animals. CHF rats have in fact a loss of EDL muscle bulk as indicated by EDL weight/BW, while Sol, which is principally involved in maintaining posture, showed no sign of atrophy. We could detect neither changes in fibres' cross-sectional area, nor replacement fibrosis. Moreover no correlation was found between muscle changes in terms of MHCs composition, that can be considered a specific biochemical marker of the myopathy, and the occurrence of atrophy. We have also shown that compensated hypertrophy is accompanied by none of the above mentioned biochemical, trophic, or morphologic changes. In the same muscles we have measured nutritive resting blood flow which was only slightly reduced in the Sol, but not in EDL. It is widely accepted that CHF is characterised by the occurrence of a specific myopathy with intrinsic abnormalities of muscle fibres [1, 24, 25]. This seems to be true especially in the leg muscles, but also occurs in other areas [3, 26, 27]. In our CHF rats the occurrence of a skeletal muscle myopathy is documented by the specific changes in MHCs pattern that reflect changes in fibre type. These are similar to those reported by Simonini et al. [28]in a rat model of CHF due to MI (myocardial infarction). However what triggers the myopathy is still debated. The present data do not support previously published hypotheses in which detraining and the consequent atrophy had been advocated as the possible cause of the CHF myopathy [29, 30]. This is in keeping with a previous report of our group [10]that showed in disuse atrophy in man the preferential synthesis of the slow MHC1 and with those of Simonini et al. [31]who showed no explanation in the reduced activity for the changes occurring in MHCs expression in CHF rats. Though we tried to limit physical activity in the Hyp and Con rats by restricting them in small cages, rats with severe CHF might have exercised less, but it should not have influenced skeletal muscle myosin gene expression. It has been in fact shown by Simonini et al. by monitoring locomotor activity of CHF rats, that different levels of physical activity do not influence MHCs gene expression [31]. It has been also debated for long time whether changes in nutritive blood flow could limit exercise tolerance and play an important role in the development of this myopathy [13, 32, 33]. As previously suggested by Massie et al. in man [9]our data do not support this hypothesis for two reasons: first of all because the resting blood flow in EDL was unchanged, secondly because the slight reduction found in the Sol of CHF rats do not explain changes in MHCs composition of the same order of magnitude of those occurring in the EDL. In this case blood flow reduction could simply represent a flow redistribution toward the more oxygen demanding, fast muscles, in order to preserve precise movements.

Measurement of blood flow with fluorescent microspheres is an extremely reliable method, certainly superior to radioactive or coloured microspheres [34]. The reproducibility of the method is fairly high both in terms of absolute number of microspheres per sample, and linearity between number of microspheres and fluorescence intensity.

We can exclude a direct effect of monocrotaline on the skeletal muscle beyond the toxic effect, that is known to be selective, on the two specific target organs, namely liver and lung [14, 15]. Hyp animals had in fact the same dose of alkaloid as CHF rats, without showing any histologic damage or, more importantly, any sign of MHCs redistribution. Moreover the specifically designed experiment with 2 weeks monocrotaline-treatment showed no changes in MHCs pattern, when the heart showed no sign of hypertrophy or failure, and the skeletal muscle no trophic changes. The monocrotaline-treated rat is an unique model for studying the heart failure syndrome [16, 35, 36]. The diagnosis of overt heart failure vs. compensated right ventricle hypertrophy is in fact well established and easily done allowing comparison between these two pathophysiological conditions [16], although the occurrence of CHF in our animals was strengthened by the measurement of β-ANP.

In conclusion our study suggests that muscle abnormalities of the CHF syndrome are neither due to detraining atrophy, nor to blood flow reduction. It may well be that cytokines (TNFα), insulin resistance or other neuro–hormonal mechanisms may contribute to its development [11, 37]. We can also speculate on the pathophysiology of this myopathy since it has been recently demonstrated in the rat with CHF that mRNA levels encoding β-MHC are depressed, whereas those encoding MHC2b are increased. It suggests that the redistribution in MHC composition is not due to a selective myosin or fibre atrophy, but rather to a specific shift in isomyosin synthesis [28]. At the same time muscle waste can occur because of nonselective fibre loss, possibly mediated by apoptosis [38].


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