C Prevention of Skeletal Muscle Myopathy

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Giorgio Vescovo and Luciano Dalla Libera⁽¹⁾

Internal Medicine II, S. Bortolo Hospital, Vicenza and (1) CNR Institute of Neuro- science, Unit for Neuromuscular Biology and Pathophysiology, Department of Bio- medical Sciences, University of Padova, Padova, Italy

Abstract

Heart failure is a syndrome leading to a skeletal myopathy with muscle atrophy and shift toward fast contracting fibres. It has been shown that the major cause of atrophy is muscle waste due to skeletal muscle myonuclei apoptosis. Apoptosis is triggered by circulating cytokines and their second messengers, in particular sphingolipids. We report here several attempts to block apoptosis. Thalidomide, an inhibitor of TNFα biosynthesis, was used without success. More successful was the attempt with blocking the Angiotensin II receptors in that apoptosis and atrophy could be prevented. Another useful approach was the block of the apoptotic cascade involving sphyngomyelinase and caspases. This was done both in vivo and in vitro with a natural occurring substance: carnitine, that has been shown to be able to block both sphyngomyelinase and caspases. Aim of our previous studies was also to shed some light on the pathophysiology of the heart failure myopathy, but at the same time to see whether apoptosis could be inhibited with subsequent prevention of the myopathy (atrophy and MHCs shift) and improvement of exercise capacity and symptoms. Since these data suggest that it is possible to block the apoptotic cascade and prevent muscle waste and atrophy, the next step is to push research into heart failure in humans and maybe to other myopathies in which it has been shown that apoptosis plays a determinant role in producing myocyte loss.

Key words: angiotensin II, apoptosis, carnitine, chronic heart failure, myosin heavy chains, skeletal muscle, sphingosine, TNFα.

Basic Appl Myol 13 (3): 121-129, 2003

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hronic heart failure (CHF) is characterised as a clinical disorder by exercise intolerance. The symptoms making a subject stop exercising are usually shortness of breath or fatigue. During exercise, there is an early and prolonged release of lactate from the exercising muscle in patients with CHF, even during light exercise [49]. At first sight, fatigue may be thought simply due to failure of perfusion of the exercising musculature and to the consequent early onset of intramuscular acidosis; however, evidence increasingly points to the existence of intrinsic abnormalities of muscle metabolism and structure in patients with CHF. In fact there is a close correlation between exercise capacity and measurements of metabolic gas exchange, particularly peak VO2. However peak VO2

poorly correlates with indices of central haemodynamic function [15, 56] and with measurements of peripheral blood flow as suggested by Wilson et al. [70]. These lat- ter showed in fact that exertional fatigue is due to skeletal muscle dysfunction rather than to reduced skeletal muscle blood flow. For these reasons investigations have been carried out looking at skeletal muscle abnormalities:

changes in histology [33], mitochondria [15], oxidative enzyme activity [57] and high energy phosphate handling [37] have been reported. Minotti et al. [43] showed that there is no reduction in mean force per unit area, imply- ing that myofibril force production was normal, but that loss of skeletal muscle mass is an important determinant of muscle strength.

Despite data showing major benefits of medical therapy in terms of reduced mortality in CHF, little progress has been made in improving the quality of life in these patients. Quality of life for the majority of patients is limited by two symptoms: fatigue and shortness of breath. We have shown that in the gastrocnemius of patients with CHF there is a shift from the slow to the fast myosin heavy chain (MHC) isoforms [63]. The magnitude of this shift correlates with the severity of CHF.

Similar observations were made by Sullivan et al. [58].

It is possible that a high percentage of glycolytic fibres reduces exercise capacity because of the early appear- ance of anaerobic metabolism, due to the prevalence of fast fatigueable fibres with low anaerobic threshold.

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Muscle atrophy by itself is a determinant of exercise capacity and the debate on its possible causes in CHF is still open. We have found in the leg muscles of rats with experimental CHF [67] an increased number of apoptotic nuclei and a decreased muscle fibres cross sectional area [9]. The pro-apoptotic factor caspase-3 was significantly increased, while Bcl-2, that is antiapoptotic, was significantly dropped [68]. Apoptosis in the skeletal muscle is paralleled by increased levels of circulating TNFα and sphingosine (SPH). They are known to produce muscle waste by triggering apoptosis [25]. We think that muscle atrophy is apoptosis- dependent and a similar picture was found in biopsies of the vastus lateralis of patients with severe heart failure where muscle atrophy and apoptosis [1, 68] were present. There is a strong inverse correlation between the number of apoptotic myonuclei, the degree of muscle atrophy [68] and peak VO2 [66]. The degree of fibres atrophy correlates with muscle endurance, and exercise capacity, suggesting that muscle strength depends on mass and is in turn a determinant of exercise capacity.

Aim of our studies was to shed some light on the pathophysiology of the heart failure myopathy, but at the same time to see whether apoptosis could be inhibited with subsequent prevention of the myopathy (atrophy and MHCs shift) and improvement of exercise capacity and symptoms. In case it was possible to block the apoptotic cascade and prevent muscle waste and atrophy, this research could be extended to heart failure in humans and to other myopathies in which it has been shown that apoptosis plays a determinant role in producing myocyte loss.

Materials and Methods

We here described three different therapeutical treatment for preventing skeletal muscle myopathy in an experimental model of heart failure: the monocrotaline treated rat. In the rat, monocrotaline induces right ventricle hypertrophy followed by right ventricle dilatation and failure. In these animals we found skeletal muscle atrophy secondary to myocyte nuclei apoptosis and shift of MHCs toward the fast glycolytic isoforms, confirming previously published data [9, 65, 67]. The presence of CHF is supported, beyond the post-mortem findings of pleural, pericardial and peritoneal effusions, by the right ventricle dilatation, as indicated by the decreased ratio right ventricle mass/right ventricle volume (RVM/RVV) index [9, 65, 67] and by the elevated plasma levels of Ang II [11, 12].

The occurrence of apoptosis in our study was con- firmed by different techniques, including TUNEL stain- ing, immunoblotting, immunohistochemistry, confocal microscopy, ELISA of DNA ladder, TNFα and SPH determination. Activated caspases 3 and 9 were also measured, this to ensure that the caspase cascade, which inevitably leads to programmed cell death, was initiated (see Figure 1). All these methods are described in full details in Dalla Libera et al [13].

Results and Discussion

Effect of thalidomide on the skeletal muscle in heart failure

In CHF, both the negative inotropism and the cardiomyocyte cell loss by apoptosis have been attributed to the heart’s production of TNFα [41, 42]. Recently, it has been argued that both the cardiomyocyte apoptosis [25] and the negative inotropism [26, 47] are a conse- quence of TNFα-triggered sphingolipid production.

Several lines of evidence suggest that sphingolipids are important mediators of apoptosis acting as intracellular signals [20]. Sphingolipid signaling molecules are derived from sphingomyelin and include ceramide, sphingosine (SPH), sphingosine-1-phosphate (S1P) and shin- gosylphosphoryl choline. Ceramide and SPH are intracellular second messengers activated by the sphingomyelin signal transduction cascade in response to inflammatory cytokines, like TNFα, IFNγ, and IL-1β, to hormones first messengers, such as vitamin D and dex- amethasone, to anti-neoplastic agents, and to ischaemia (see for a review reference [52]). Sphingolipids mediate a variety of cellular responses including calcium- dependent stimulus-secretion coupling [75], neutrophil activation [72], cell proliferation [76], platelet activation [74] and apoptosis [22]. The interaction of TNFα with its receptor TNFR1 leads to the rapid degradation of sphingomyelin with the consequent generation of SPH [70]. Cardiomyocytes express the TNFR1 [26] as well as essential elements of the sphingolipid signaling sys- tem [19, 47, 51], and it has been hypothesized that it may act as the second messenger for TNFα, in triggering cardiac cell apoptosis [25].

Apoptosis is involved in myocyte loss both in CHF [45] and in cardiomyopathies [23]. In the heart apoptosis can be triggered by activation of cytokines, such as TNFα [25] or by hormones, such as AngII [29]. We have shown that apoptosis is accompanied by increased levels of circulating sphingosine, a phospholipid which is the second messenger of TNFα [23], and that is able to induce skeletal myocyte apoptosis both in vivo and in vitro [12]. Although clinical trials aimed to block TNFα Figure 1. Skeletal muscle apoptosis in CHF. A: double labelling for TUNEL and laminin. Arrows indicate apoptotic nuclei (magnification x400). A’: double exposure for laminin and Hoechst. B: Im- munofluorescence for activated Caspase 3 and dystrophin from a CHF animal. Thin, granulated pattern indicates diffuse cytosolic distribution of Caspase 3. Positive fibers (with red dots) are sur- rounded by negative fibers (black). Sarcolemma is stained in green (magnification x400).

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with specific antibodies have failed [34], there is a com- pelling evidence that apoptosis can be inhibited with favourable consequences on muscle atrophy.

We tested the hypothesis that thalidomide, used as a blocker of TNFα synthesis, may have effects on the skeletal muscle in CHF. Although thalidomide and its analogues have a broad spectrum of properties ranging from anti-inflammatory [7, 8] to anti-angiogenetic [8] and anti-tumoral activities [55], which may induce several dichotomous variation in Interleukins and Interferons secretion, its major action is to inhibit TNFα synthesis [7, 24, 46, 60]. The rationale for blocking TNFα with thalidomide, was to prevent the well known detrimental car- diovascular effects of TNFα itself. It has in fact been demonstrated that TNFα is elevated in CHF together with other pro-inflammatory cytokines [17, 40] and in patients with cardiac cachexia and muscle waste [5].

We have also demonstrated a link between the degree of muscle atrophy and the magnitude of skeletal muscle apoptosis [68] suggesting that apoptosis could be one of the major causes of skeletal muscle waste. Preventing muscle atrophy could be one of the targets for improving exercise capacity in patients with CHF, since muscle trophism is an independent predictor and determinant of it [36, 64, 68, 69]. At the same time we have previously shown that TNFα plasma levels and skeletal muscle apoptosis can be lowered, preventing the development of muscle atrophy, even by intervening on other mechanisms such as angiotensin II receptors [54].

The effects of thalidomide, an inhibitor of TNFα production [7, 24, 46, 60], were studied in a well established model of CHF, the monocrotaline treated rat [9, 67].

Monocrotaline is able to produce pulmonary hypertension followed by right ventricle (RV) hypertrophy and failure.

TNFα was significantly elevated in CHF rats, confirming that this cytokine is increased in CHF [11, 12, 54, 67].

While in the heart a cause-effect relationship between TNFα and apoptosis is well established [25], the same is not demonstrated for skeletal muscle. The result of the cytotoxic effect of TNFα on skeletal muscle could be due to its second messengers SPH, that is released by cardiac myocytes when TNFα binds to their surfaces [12, 25]. In fact SPH by itself is able to induce skeletal muscle apop- tosis both in vivo and in vitro [12].

The working hypothesis for blocking TNFα, is therefore to prevent skeletal muscle apoptosis and muscle atrophy, for further improving EC and symptoms. Re- cently, Embrel, a specific p75 TNFα receptor fusion protein, by reducing the biological activity of TNFα of 50%, was able to improve exercise capacity (EC) [14], indicating that TNFα is an important therapeutic target in CHF. In our study, where we used thalidomide to block the synthesis of TNFα, the plasma levels of this cytokine as well as SPH were substantially unchanged.

At the same time apoptosis was still ongoing as demonstrated by the persistence of elevated TUNEL positivity, by the presence of DNA ladder and activated Caspases.

Thalidomide, in our study, was used at the same doses employed, and shown to be efficacious, for the treatment of TB, AIDS and leprosy in humans [60]. There are no studies at present indicating that higher doses may be effective in experimental models of CHF and we cannot therefore exclude that higher doses or a longer period of treatment may be required in CHF, although the second hypothesis seems unlikely since in our experimental model two weeks treatment with other drugs were able to produce improvements in skeletal muscle changes [11]. The results our study, with all the limitations of animal investigations, should be taken into account pathophysiologically and clinically, now that the cytokine hypothesis is under investigaton in large randomized clinical trials, and lively discussed because of contradictory results with TNFα blockers such as Embrel (RENAISSANCE), and because some other molecules are going to be tested.

Effects on skeletal muscle of the Angiotensin II type 1 (AT1) receptor blocker Irbesartan

We have demonstrated that Irbesartan, a drug which se- lectively blocks the AT1 receptor, was not able to prevent RV hypertrophy, as shown by the ratio left ventricle mass/right ventricle mass (LVM/RVM) index that did not differ in Irbesartan from the CHF group. The occurrence of RV dilatation was however partially prevented.

RVM/RVV was in fact significantly lower than control and higher than CHF. This confirms that a certain degree of compensated hypertrophy with a lower degree of failure was present in the monocrotaline rats that had two weeks Irbesartan. We can therefore assume that Irbesar- tan produced only a partial improvement in the hemodynamic pattern. A similar finding was observed in the Nifedipine treated animals, where RV dilatation was prevented to an even higher degree and a greater deal of compensated RV hypertrophy was found. Nifedipine was used in order to obtain favorable hemodynamic changes in a model of CHF due to pulmonary hypertension, without directly interfering with AngII receptors.

Despite similar hemodynamic effects Irbesartan greatly differed from Nifedipine in term of biological effects.

This is in fact the first demonstration in vivo that an AT1

blocker can produce beneficial effects on the skeletal muscle of rats with experimental CHF. These effect could not be obtained with the calcium blocker. In Irbesartan MHCs pattern was in fact similar to that of control with a partial improvement in the degree of tibialis anterior muscle (TA) atrophy (higher TAW/BW and myocyte cross sectional area (CSA)). In the Nifedipine group however, neither MHC composition, nor indices of muscle atrophy were different from CHF animals.

We can reasonably speculate that this effect of Irbesar- tan on muscle atrophy can be secondary to the lower levels of apoptosis. The absolute number of TUNEL positive cells was in fact lower in Irbesartan when compared to CHF rats. In the Nifedipine rats, where apoptosis was detected at even higher level than CHF, muscle atrophy was

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worse. Pro-Caspase-3, and activated Caspases 3 and 9, that are compulsory steps in the death receptor signaling pathway, were equally decreased in Irbesartan, while remained high in Nifedipine rats. Bcl-2, that has a protec- tive effect, was significantly higher in Irbesartan than in CHF and similar to control.

The role of TNFα in CHF is not entirely understood.

We know that in CHF the circulating TNFα is increased both in man [17] and in animal models [12]. TNFα blockade with specific antagonists (Enbrel) can improve the clinical status of CHF patients [13]. TNFα levels par- allel the severity of CHF as well as the number of Tunel positive skeletal myocytes [12]. Moreover TNFα is known to worsen CHF by depressing cardiac contractility [47]. Though still debated, TNFα has been shown to trigger apoptosis [25]. We know that in the heart it induces sphingolipids production, such as sphingosin and ceramide, that are in turn mediators of apoptosis [52].

In this study TNFα was significantly increased in CHF animals when compared to control, and remained high in Nifedipine rats. In Irbesartan rats it was slightly decreased, reaching borderline significance. The TNFα figures therefore resemble those of TUNEL, Pro- Caspase-3 and Bcl-2, maybe suggesting the existence of a link between TNFα and apoptosis. We can only speculate why TNFα is decreased with Irbesartan. We may hypothesize that the less compromised haemody- namics may have interfered with TNFα synthesis which in CHF is linked to the clinical status [14, 47]. If that was the case, even the Nifedipine rats, which had a similar haemodynamic improvement should have shown a TNFα reduction. We therefore think that the interplay between TNFα, AngII receptors and apoptosis is much more complicated.

From the present results we can hypothesize that the favorable effects of Irbesartan on apoptosis are likely to be secondary to a direct AT1-mediated antiapoptotic effect, rather than to hemodynamic improvements. In fact Nifedipine-treated rats, that show a similar degree of compensated RV hypertrophy, with an even lower dilatation of the RV, do not exhibit any skeletal muscle change.

The role of AngII receptors on apoptosis is far from being elucidated. There are in fact several observations suggesting that the AT2 stimulation mediates apoptosis, through ERK inhibition [28], ceramide accumulation [73], activation of MKP1 with subsequent inhibition of MAPKinase and Bcl2 dephosphorylation [73]. Apop- tosis can be blocked by PD-123319 and by PD 123177 [30, 31] that are specific AT2 blockers. In contrast some Authors [27] have shown that AT1 blockade with Losar- tan can equally protect from apoptosis. AT1 stimulation can in fact lead to an increase in FAS, together with a fall in cNOS and Blc-2 levels. Li et al. [31] and Leri et al. [27] have also demonstrated that myocyte stretch- induced apoptosis can be blocked by Losartan, that is able to increase levels of Bcl-2 and decrease Bax and p53. Similar observations have been made in the heart

of spontaneously hypertensive rats (SHR) [19]. The ACE-inhibitor Captopril has been shown to reduce apoptosis in SHR with CHF [32].

AngII-induced apoptosis can be blocked in postinfarc- ted hypertrophied ventricular myocytes by AT1 blockers, while AT2 antagonists had no impact on these cellular events [27]. The conflict whether AT1 or AT2 receptor mediate apoptosis can be mediated by the observation that tissues that express primarily AT2 exhibit AT2- mediated apoptosis, while tissues that express primarily AT1 exhibit AT1-mediated apoptosis [31]. Moreover it has been demonstrated that in many tissues, skeletal muscle included, the majority of physiological responses to AngII occurs through its accumulation that is AT1- mediated [62]. These observations allow us to speculate on the mechanism by which Irbesartan, a highly specific AT1 blocker, may have prevented apoptosis by acting through a receptorial mechanism involving the AT1 receptor. This specific action can be brought about even in the presence of very high levels of AngII. In fact in Irbe- sartan rats we found a three/four fold increase in AngII, which was similar to those of CHF and Nifedipine. It may be also speculated that excessive AT2 stimulation, due to AT1 blockade with Irbesartan, may reduce the number of apoptotic cells in the skeletal muscle of CHF rats. A recent observation in man [35] shows however that AT2 gene is not expressed in the skeletal muscle of men with CHF. AngII levels are in keeping with those of the literature both for normal [59] and AngII blockers- treated rats, that show a four to five fold increase in AngII. CHF by itself and the reflex sympathetic tone are likely to be the cause of AngII elevation in Nifedipine.

That AngII receptor blockade and ACE-inhibition can play a favorable effect on skeletal muscle fibers type, MHCs and exercise capacity has been previously demonstrated by us [64]. These findings are a further insight in the pathophysiology of the CHF myopathy, allowing speculations on the pathogenesis of this syndrome. In fact muscle atrophy is the main “muscular” determinants of the reduced exercise capacity in CHF. This is due to the reduced muscle force and endurance [36] and to the shift toward the fast and more fatigable fibers [33]. Irbesartan has shown the potential for reducing skeletal muscle apoptosis and atrophy, at the same time protecting from the shift toward the fast MHCs, further contributing to a possible improvement in exercise tolerance and symptoms [6]. The reason for MHCs shift remains to be established, together with the interplay between AT1 and AT2

receptors in the genesis and prevention of apoptosis.

L-Carnitine as a tool for blocking apoptosis and skeletal muscle myopathy in heart failure

L-carnitine is a quaternary amine that is fundamental in skeletal muscle metabolism, in that promotes fatty acids oxydation [48], and that has been shown effective in producing a selective trophic effect on Type I and IIa skeletal muscle fibres [21]. In our study L-carnitine muscle levels are decreased in CHF rats. Plasma levels of carnitine in

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the CHF animals are even higher that those of controls, indicating that liver biosynthesis is intact (the absence of liver damage is also proved by the normal transaminases).

The low levels of muscle carnitine detected in the CHF rats are therefore probably due to low muscle uptake.

There are recent observations that L-carnitine, beyond the well known metabolic effect, possesses some more complex activities in regulating gene expression and activity of caspases [44]. In our study L-carnitine was unable to prevent the development of heart failure, but, despite that, we could detect favourable changes in the tibialis anterior muscle. These consisted in a decreased degree of muscle atrophy, as demonstrated by the CSA of tibialis anterior fibres. This was in fact significantly higher than that of CHF rats, although it wasn’t brought to the levels of control. It may be suggested that the reduction in muscle atrophy may be due to correction of metabolic impairement and to the lower levels of apoptosis observed in the CHF carnitine-treated animals.

Metabolic impairment

In the CHF rats we have shown a profoundly altered glucose metabolism: in fact, after monocrotaline treatment, skeletal muscle GLUT-4 expression dra- matically increases. The contemporary administration of L-carnitine does not counterbalance the monocrotaline-induced GLUT-4 over-expression, therefore, suggesting that L-carnitine does not play a role in modulating insulin responsiveness of skeletal muscles in monocrotaline-treated rats.

In a mouse model of skeletal muscle GLUT-4 overex- pression it was demonstrated that the increased glycolysis in muscle was associated both with increased serum lactate levels and with increased flux of this metabolite through the Cori-cycle [61].

The glycolysis rates were increased in these animals while their glucose oxidation rates seemed to be consid- erably lower than those of glycolysis (probably secondary to inhibition of pyruvate dehydrogenase complex by acetyl-CoA derived from increased substrate oxidation). By a parallelism between our model and the transgenic model, it is possible to postulate that L-carnitine could have an important role in regulating glucose metabolism in skeletal muscle of monocrotaline treated rats. This has been also shown in the rat perfused heart, where physiologic concentrations of carnitine mimics insulin-like metabolic effects by increasing glucose oxidation [50].

By increasing muscle L-carnitine levels in the CHF rats may stimulate glucose oxidation by the action of L- carnitine on inner mitochondrial L-carnitine acetyltrans- ferase, which enhances conversion of mitochondrial acetyl-CoA to cytoplasmic acetylcarnitine, resulting in a decrease in the inner mitochondrial acetyl-CoA ratio. A decrease in this ratio will stimulate the pyruvate dehydrogenase complex, the enzyme that converts pyruvate to acetyl-CoA, and is the rate limiting step of glucose oxidation. As a result of an increase of L-carnitine in muscle overexpressing GLUT-4, a greater proportion of

the pyruvate derived from glycolysis as well as pyruvate derived from lactate can be oxidized.

The reported changes in MHCs composition after L- carnitine treatment can be as well due to the well known trophic metabolic effect of L-carnitine on type 2a skeletal muscle fibres [21].

Apoptosis

In the L-carnitine treated CHF rats we found a substantially lower degree of TUNEL positive nuclei and DNA break strands (ELISA ladder), which were accompanied by a lower expression of caspases and by an increased expression of Bcl-2. We can speculate on the mechanisms by which L-carnitine may have prevented apoptosis:

a) by blocking TNFα and sphingolipids activation cascade as previously shown in the heart [3];

Figure 2. Percent of total of MHC2a in CHR rats and after GH treatment. CHF: heart failure rats;

CHF+GH: heart failure rats treated with growth hormone. Data are expressed as mean ± SD. * P

< 0.0001; # P < 0.001.

Figure 3. Single fibers cross-sectional area in CHF rats and after treatment with GH. CHF: heart failure rats; CHF+GH: heart failure rats treated with growth hormone. Data are expressed as mean ± SD. * P < 0.0001; # P < 0.01.

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b) by inhibiting the cleavage of caspases substrates at mitochondrial level, making it a general caspases inhibitor [44].

The partially reduced levels of TNFα and SPH found in this paper may have blunted the phospholipid- induced apoptosis.

In our study the mitochondrial pathway is certainly involved, in that activated caspases 3 and 9 are inhibited and Bcl-2 is increased. In vitro inhibition of stauro- sporine-induced apoptosis, that in C2C12 cells acts via the mitochondrial cascade [39, 53], strengthen this hypothesis. It is not surprising that L-carnitine may be active at mitochondrial level in that it accumulates at this site. It has been recently suggested by Mercadier and coworkers [3] that ceramide generation is linked to mitochondrial metabolism. If that was the case we would not be dealing with two so independent pathways. It is hard to split the beneficial effects of L-carnitine among its action on myocyte metabolism and its role in modulating apoptosis. We cannot also exclude indirect effects on TNFα, SPH and AngII downregulation. The com- plexity of the interplay between these molecules has been previously shown by our group in the same animal model: Ang II receptor blockade was in fact able to decrease apoptosis, but also TNFα [11] and SPH [10].

We know that skeletal muscle bulk is one of the major determinants of exercise capacity, in that muscle strength is related to bulk [36, 69]. Muscle waste is also linked to prognosis: patients with cardiac cachexia, in whom muscle waste is extreme, have a very poor prognosis [4]. It is therefore clear how blocking apoptosis and improving oxidative metabolism may prevent skeletal muscle bulk loss, improve exercise capacity and maybe prognosis in the CHF patients.

It may be speculated on the clinical relevance of these findings. In agreement with previous studies carried out in patients with CHF, muscle levels of L-Carnitine are decreased [38] and plasma levels are increased [16], confirming a muscle deficiency and a reduced uptake of L-carnitine both in man and in our animal model of CHF. It is clear that interventions trying to restore normal levels of carnitine at muscle level have the potential for improving skeletal muscle metabolism and also throphism. Although a large randomized trial with L- carnitine on exercise capacity [18] has been published, it has shown neutral results on the long term outcome.

Only in a subgroup of patients with preserved ejection fraction, and in another small study there were partially positive effects of the active treatment on peak oxygen consumption [2]. We think that a conclusive interpreta- tion cannot be drawn yet. Our data show only a partial improvement in preventing apoptosis, atrophy, cytokine release and SPH formation. In overt heart failure, if production of cytokines and sphingolipids is over- whelming, as it is in sickest or cachectic patients, the consequent apoptosis may be only partially modulated.

It is therefore possible that intervention may be effective

only in well defined subgroups of patients. However it is of importance the understanding of the mechanisms by which L-carnitine is acting in that they may contrib- ute to shed some light on the pathophysiology of the CHF myopathy, leading to prevention of atrophy, pres- ervation of exercise capacity and to design clinical trials targeted on particular populations looking not only at clinical but even at biological end-points.

Perspectives

There are several observations that growth hormone (GH) can improve haemodynamic and exercise capacity in CHF. It is now well known that exercise capacity in CHF is linked to skeletal muscle myopathy, and muscle atrophy is one of its major and independent determinants.

We are currently testing the hypothesis that GH treatment can prevent skeletal muscle atrophy and MHC shift.

Two weeks after monocrotaline injection the rats were treated daily with recombinant human GH (1.0 mg/kg/die) sc in the neck. After two additional weeks, when in the monocrotaline treated animals overt heart failure has developed, rats were killed and tibialis anterior muscle excised. Myosin heavy chain composition and single fibers cross sectional area (CSA) were assessed.

As shown in Figures 2 and 3 GH treatment is able to reverse, in a significant manner, both the MHC shift and muscle atrophy as determined by CSA. These are findings very promising that deserve further investigations.

Address correspondence to:

Giorgio Vescovo MD PhD FESC, Internal Medicine II, Ospedale S. Bortolo, Viale Rodolfi, 36100 Vicenza (Italy), tel. 0039 0444992462, fax 0039 0444993655, Email ldl@bio.unipd.it.

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