Author + information
- Received December 14, 1995
- Revision received May 21, 1996
- Accepted June 3, 1996
- Published online October 1, 1996.
- ELIANE LAMPERT and
- BERTRAND METTAUER
- HANS HOPPELERa
- JEAN LONSDORFER*
- ↵*Address for correspondence Dr. Jean Lonsdorfer, Service des Explorations Fonctionnelles Respiratoires et de l'Exercice, Hôpitaux Universitaires de Strasbourg, 67091 Strasbourg Cedex, France.
Objectives. This study sought to define the ultrastructural characteristics of skeletal muscle in heart transplant recipients (HTRs) in relation to exercise capacity compared with that in age-matched control subjects.
Background. Muscle structural features seem to play an important role in the limitation of exercise capacity of HTRs long after transplantation.
Methods. The structure of the vastus lateralis muscle was analyzed by ultrastructural morphometry in 16 HTRs and 20 healthy control subjects. Maximal oxygen consumption (peakV̇O2) was determined by an incremental exercise test.
Results. Peak V̇O2 was significantly lower (by 35%) in HTRs. Fiber size, volume density of mitochondria and intramyocellular lipid deposits were not significantly different between HTRs and control subjects. In contrast, the capillary density and the capillary/fiber ratio were both significantly reduced in HTRs (by 24% and 27%, respectively).
Conclusions. A normal volume density of mitochondria and a reduced capillary network are the main characteristics of muscle ultrastructure in HTRs by 10 months after transplantation. The muscle structural abnormalities and reduced exercise capacity might be related to immunosuppressive therapy with cyclosporine and corticosteroids as well as deconditioning.
Heart transplantation represents an effective therapeutic procedure for patients with end-stage heart disease, and survival has been improved by the introduction of cyclosporine therapy. Recent studies report a 5-year survival rate of ∼70% . Despite normalization of oxygenation and most hemodynamic variables, heart transplant recipients (HTRs) still complain of abnormal leg fatigue and exhibit a limited exercise capacity [2, 3]. Peripheral muscle factors seem to play a major role in this limitation [4–7]. Abnormalities of skeletal muscle structure and abnormal skeletal muscle metabolic responses to submaximal exercise [9, 10]have been well documented in patients with chronic heart failure. The depressed oxidative capacity of skeletal muscle in these patients appears to be independent of total limb perfusion as well as muscle atrophy . Because HTRs have all experienced chronic heart failure before undergoing grafting, we hypothesized that their reduced functional exercise capacity could be related to a lowered oxidative capacity of the locomotor muscles. We also considered that lifelong immunosuppressive treatment with cyclosporine and prednisone might alter skeletal muscle structure and function because negative effects on muscle performance have been described for prednisone in humans and cyclosporine in animal models [14, 15].
Patients and normal subjects. We studied 16 clinically orthotopic stable HTRs (15 men, 1 woman) who had undergone transplantation 10 ± 3 months before the present investigation. The etiology of heart failure was ischemic heart disease in 5 patients, idiopathic dilated cardiomyopathy in 10 and valvular disease in 1. The patients were free of clinical and biological signs of rejection as confirmed by recent myocardial biopsy. They also had normal rest left ventricular systolic function as shown by echocardiography. All 16 patients were treated according to a standard immunosuppressive protocol with cyclosporine, azathioprine and prednisone, and 15 were treated for moderate hypertension by calcium receptor antagonists. All patients had resumed their professional or private activities, but none was or had been involved in a formal long-term rehabilitation program beyond the standard postoperative rehabilitation.
For comparison, 20 normal sedentary control subjects (18 men, 2 women) were studied. Patients and normal control subjects were matched for age, body weight and body mass index (Table 1). All subjects gave written informed consent and were fully informed about the risks incurred with this study. The experimental protocol was approved by the local institutional ethics committee.
Exercise testing. Patients and subjects underwent a maximal graded exercise tolerance test using an upright bicycle ergometer (Medifit), starting at a workload of 20 W, with increases of 20 W every 2 min. The tests were discontinued at the onset of generalized or thigh fatigue. Maximal oxygen consumption (peak V̇O2 [ml/min per kg]) was measured using a commercially available, breath by breath metabolic measurement chart (Medisoft). Cardiac frequency was measured by an electrocardiographic (ECG) recorder (Schiller).
Muscle biopsy, electron microscopy and morphometry. Biopsy specimens were taken from the vastus lateralis muscle at midthigh level, under local anesthesia, using the technique of Bergström . All subjects were instructed not to perform any physical exercise for 24 h before biopsy. The muscle tissue samples were processed for electron microscopy by fixation in a 6.25% solution of glutaraldehyde, as previously described . Four randomly chosen blocks from each biopsy were used for stereologic analysis. The orientation of the sections was essentially transverse with regard to the muscle fiber axis. Capillary number, fiber number and fiber cross-sectional area were estimated at a final magnification of 1,500. Five micrographs/block (20 micrographs/biopsy) were taken in consecutive frames of slotted grids (type R. 100 A, Veco Co, Amsterdam, The Netherlands) yielding > 100 muscle fiber profiles for analysis in each biopsy. A final magnification of 24,000 was used for estimation of the volumes of mitochondria and intracellular lipid droplets/unit volume of muscle fiber (Fig. 1). Ten micrographs/block (40 micrographs/biopsy) were taken with a routine sampling procedure in consecutive frames of 200-square mesh grids. Pictures of the 35-mm films were projected on a screen fitted with quadratic line grids. Point counting was performed with an A 100 grid (100 test points) for the lower magnification and a B 36 grid (144 test points) for the higher magnification . Estimates of variables were obtained according to standard stereologic procedures.
Statistical analysis. Results are reported as mean value ± SD. Comparisons of the results between the two groups were performed using analysis of variance and the Student t test for unpaired observations. Linear regression analysis was used to correlate the volume density of mitochondria with peak V̇O2 and capillary density with volume density of mitochondria. Comparison of regression lines was performed using covariance analysis. Statistical significance was set at p < 0.05.
Patients and control subjects. Anthropometric characteristics of HTRs and control subjects are reported in Table 1. Peak V̇O2 in HTRs was significantly decreased by 35% compared with that in control subjects. The results of the morphometric analysis of the lateral vastus muscle in HTRs and normal subjects are reported in Table 2. On qualitative inspection, muscle tissue appeared normal in all biopsy specimens, and we could not detect any differences between biopsy samples from HTRs patients and control subjects. Likewise, neither ultrastructural composition of the muscle fiber nor fiber size was significantly different between HTRs and control subjects. However, both capillary density and capillary/fiber ratio showed significant decreases of 24% and 27%, respectively, compared with that in control subjects.
A significant linear relation was found between capillary/fiber ratio and peak V̇O2 (r = 0.55, p < 0.01) and between volume density of mitochondria and peak V̇O2 (r = 0.61, p < 0.01) in normal subjects but not in HTRs (Fig. 2).
A significant linear relation between volume density of mitochondria and capillary density was found in both groups (Fig. 3). The slope of the regression lines was similar in the two groups and showed a nonsignificantly lower y-intercept for patients.
Heart transplantation improves functional class IV heart failure outcome, survival and quality of life and substantially ameliorates the central hemodynamic variables in HTRs. Surprisingly, their functional exercise capacity remains very much reduced. We therefore hypothesized that a decreased oxidative capacity of the periphery (i.e., of leg muscles) might be a major contributing factor for the lack of functional improvement after heart transplantation. An ultrastructural study in patients with heart failure had shown abnormalities of skeletal muscle tissue closely related to their exercise capacity. Patients with severely impaired peak V̇O2 (12.6 ± 2.5 ml/min per kg) showed a significant decrease in volume density of total mitochondria and capillary length . These observations agree with results of other studies indicating major alterations in skeletal muscle histologic appearance and biochemistry, with reduced mitochondrial enzymes [18, 19]and muscle atrophy in patients with heart failure.
Only very few studies have focused on skeletal muscle function after heart transplantation. In HTRs up to 18 months after transplantation, Braith et al. found a deficit of leg strength that could partially explain the decrease in peak V̇O2 of these patients. To our knowledge, the present study is the first complete ultrastructural analysis of skeletal muscle tissue in HTRs to show that after heart transplantation, patients essentially have a normal mitochondrial volume density. Both interfibrillar and subsarcolemmal populations appear qualitatively and quantitatively unaltered. There is no muscle atrophy, as evidenced by the normal fiber cross-sectional area. In contrast, muscle capillarity is reduced significantly compared with that in age-matched control subjects. These unexpected findings led us to reconsider our initial hypothesis and ask whether the mitochondria might be functionally impaired or whether the limit to exercise capacity might be related to the lower capillary counts, or both. Because of the descriptive nature of the present study, we can only hypothesize about the etiology of our observations.
Normal mitochondria volumes combined with low peakV̇O2. Drexler et al. found significantly lower values of mitochondrial volume densities closely related to lower peakV̇O2 values (12.6 ml/min per kg) in patients with severe heart failure. Our mitochondrial density values differ considerably from those of Drexler et al. in absolute terms. Drexler et al. report unusually high mitochondrial volume densities for patients (5.67% vs. 4.37% present study) as well as for control subjects (7.75% vs. 4.82% present study). In fact, their values for control subjects are similar to those reported in a number of studies for endurance-trained athletes . In part, this discrepancy could be due to the larger magnification used for the morphometric analysis by Drexler et al. (×60,000 vs. approximately ×24,000 in most other studies). It has been shown and discussed previously that larger mitochondrial volume densities can be obtained when higher magnifications are used for morphometric evaluation of mitochondria. More pertinent than the absolute numerical values for mitochondrial volume densities, however, is the fact that when identical techniques were used, we showed similar mitochondrial volumes in the muscles of HTRs and control subjects, whereas Drexler et al. found significant differences.
In the present study, we tried to understand how a significantly reduced peak exercise capacity could be combined with a normal muscle volume density of mitochondria. Several lines of evidence indicate that the immunosuppressive therapy, in particular, cyclosporine, could be responsible, at least in part. A recent report demonstrates the persistence of abnormalities of oxidative intermediates in exercising skeletal muscle by means of phosphorus-31 magnetic resonance spectroscopy >1 year after heart transplantation. That study showed a systematically reduced submaximal exercise phosphocreatine (PCr) inorganic phosphate (Pi) ratio [(PCr)/(PCr + Pi)], a trend toward a lower submaximal intracellular pH and a lower apparent effective maximal rate of mitochondrial adenosine triphosphate synthesis (Vmax) with cyclosporine treatment. Likewise, a recent animal study reported a significant decrease of maximal coupled and uncoupled skeletal muscle mitochondrial respiration in vitro during cyclosporine administration. Moreover, in their in vivo study, Mercier et al. showed that a 14-day administration of high doses of cyclosporine to rats decreased their submaximal endurance exercise time and their skeletal muscle mitochondrial respiration. The cyclosporine-induced deterioration of mitochondrial function could thus explain the rapid increase of plasma lactate levels during incremental exercise in HTRs [2, 5, 7, 22, 23]. Taken together, these findings suggest that mitochondrial function could be impaired by cyclosporine treatment and that, as a consequence, a normal mitochondrial volume would produce less adenosine triphosphate. This contention needs to be supported by direct evidence from appropriate experiments in HTRs. Because renal transplant recipients treated by glucocorticoids alone have no impairment of total oxidative phosphorylation capacity , and there are no current data on azathioprine muscle toxicity, these drugs are less likely to be responsible for the discrepancy between muscular function and muscle mitochondrial content.
Decreased capillary density and capillary/fiber ratio. Because there is no difference in muscle fiber cross-sectional area between HTRs and control subjects, the decrease in both capillary density and capillary/fiber ratio represents a true decrease in the extent of the muscle capillary network in HTRs. Interestingly, the magnitude of this decrease is similar to that observed by Drexler et al. in patients with congestive heart failure. If the hypothesis of a cyclosporine-induced mitochondrial dysfunction is correct, then the observation of a reduced capillarity would be compatible with a reduced mitochondrial function combined with a normal mitochondrial volume density. This would offset the mitochondria/capillary relation, as demonstrated in Fig. 3. In contrast, it cannot be excluded that mitochondrial oxidative phosphorylation is throttled by a decreased oxygen supply as a consequence of the reduction of the capillary network.
Current knowledge of the pathophysiologic effects of exercise after heart transplantation offers alternative explanations for the reduced capillarity observed in HTRs. Continued impairment of lower limb blood flow during exercise has been observed in patients with heart failure [24, 25]. Because all HTRs were former patients with heart failure, the reduced muscle capillarity might have persisted in our patients after grafting. This view could be contested on the grounds that recent studies [11, 26]have demonstrated that many patients with chronic cardiac failure and severe exertional fatigue are not primarily limited by an impaired muscle blood flow.
It cannot be ruled out that the immunosuppressive drugs taken by our patients affect muscle capillarity. Cyclosporine is known to cause acute renal vasoconstriction without systemic vasoconstriction at rest . A significant vasopressor effect of cyclosporine has been suggested to occur during exercise . Corticosteroids induce a well known myopathy with pronounced skeletal muscle atrophy. It is noteworthy that a decrease in capillary density similar to that seen in our patients has been described in renal transplant recipients treated by corticosteroids . The decreased muscle capillary density in HTRs is all the more surprising because patients were, without exception, receiving vasodilator therapy to treat moderate hypertension. Because vasodilators have been shown to increase muscle capillary density , one would have expected these drugs to positively affect muscle capillarity in our patients.
A further pathogenic mechanism that needs to be taken into consideration is deconditioning. Muscle deconditioning, including a significant reduction in capillary density, has been shown after prolonged immobilization [31, 32]. Because all our patients had end-stage heart failure (many of them were bedridden for several months), some deconditioning might have persisted after transplantation. However, the absence of a decrease in fiber size argues somewhat against deconditioning as the leading reason for our observations.
The current descriptive study does not allow us to factor out the contributions of the different pathogenic mechanisms that lead to the reduction in capillaries and the discrepancy in skeletal muscle mitochondrial content and aerobic exercise performance.
Conclusions. Morphometric analysis of skeletal muscle structure revealed that HTRs have qualitatively and quantitatively normal mitochondria but a significant reduction in the extent of the capillary network. The functional exercise capacity of these patients is severely impaired, as indicated by a low symptom-limited maximal oxygen uptake. Adverse drug effects on mitochondria and capillaries and, less likely, deconditioning may be of importance in limiting exercise capacity. In view of these results, it is suggested that the effects of antirejection therapy on muscle function as well as possible countermeasures, such as exercise training, need to be evaluated to improve quality of life in HTRs.
We express sincere gratitude to Helgard Claassen and Sylvie Beyhurst, for excellent and skillful technical assistance, as well as to Mireille Trautmann, for secretarial assistance.
A.1 Abbreviations and Acronyms
HTRs = heart transplant recipients
peak V̇O2 = maximal oxygen consumption
↵1 This work was supported in part by Grant 31-30946.91 from the Swiss National Science Foundation, Bern and by the Institute of Sports Sciences, Magglingen, Switzerland.
- Received December 14, 1995.
- Revision received May 21, 1996.
- Accepted June 3, 1996.
- THE AMERICAN COLLEGE OF CARDIOLOGY
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