Oxidative capacities of cardiac and skeletal muscles of heart transplant recipients: mitochondrial effects of cyclosporin-A and its vehicle Cremophor-EL.

Chronic immunosuppressive treatment was suspected to alter maximal muscle oxidative capacity (Vmax ) of heart transplant recipients, leading to a limitation of their exercise tolerance. It remains undefined whether the mitochondrial respiratory chain (MRC) of right ventricle (RV) and vastus lateralis (VL) muscles were altered by immunosuppressants and/or their vehicles. Vmax was measured polarographically in saponin-skinned fibres of RV and VL biopsies of patients and compared with Vmax of healthy VL and myocardium. Effects of increasing concentrations (1-10-100 µM) of Sandimmune(®) , its vehicle, Cyclosporine (CsA) in ethanol (EtOH), or EtOH alone were tested. The vehicle's effects on MRC complexes were investigated using specific substrates and inhibitors. Ten months after grafting, Vmax of RV and VL of immunosuppressed patients were similar to their Vmax at time of transplantation and to that of control tissues. In Vitro, Sandimmune(®) significantly decreased Vmax while CsA in EtOH or EtOH exerted small and similar effects. Effects of the vehicle were higher than (RV) or identical to (VL) those of Sandimmune(®) . The sites of action of the vehicle on MRC were located on complexes I and IV. While unchanged under chronic immunosuppressive therapy, Vmax of RV and VL muscles was depressed by acute exposure to intravenous Sandimmune(®) in vitro, an effect attributed to its vehicle by inhibition of complexes I and IV of the MRC. This work provides an in vitro proof of a toxic effect on the mitochondria respiratory chain of the vehicle used in the intravenous formulation of Sandimmune(®) but with no clinical consequences in chronically immunosuppressed patients.

Impairment of maximal aerobic power with moderate hypoxia in endurance athletes: do skeletal muscle mitochondria play a role?

This study investigates the role of central vs. peripheral factors in the limitation of maximal oxygen uptake (Vo(2max)) with moderate hypoxia [inspired fraction (Fi(O(2))) =14.5%]. Fifteen endurance-trained athletes performed maximal cycle incremental tests to assess Vo(2max), maximal cardiac output (Q(max)), and maximal arteriovenous oxygen (a-vO(2)) difference in normoxia and hypoxia. Muscle biopsies of vastus lateralis were taken 1 wk before the cycling tests to evaluate maximal muscle oxidative capacity (V(max)) and sensitivity of mitochondrial respiration to ADP (K(m)) on permeabilized muscle fibers in situ. Those athletes exhibiting the largest reduction of Vo(2max) in moderate hypoxia (Severe Loss group: -18 +/- 2%) suffered from significant reductions in Q(max) (-4 +/- 1%) and maximal a-vO(2) difference (-14 +/- 2%). Athletes who well tolerated hypoxia, as attested by a significantly smaller drop of Vo(2max) with hypoxia (Moderate Loss group: -7 +/- 1%), also display a blunted Q(max) (-9 +/- 2%) but, conversely, were able to maintain maximal a-vO(2) difference (+1 +/- 2%). Though V(max) was similar in the two experimental groups, the smallest reduction of Vo(2max) with moderate hypoxia was observed in those athletes presenting the lowest apparent K(m) for ADP in the presence of creatine (K(m+Cr)). In already-trained athletes with high muscular oxidative capacities, the qualitative, rather than quantitative, aspects of the mitochondrial function may constitute a limiting factor to aerobic ATP turnover when exercising at low Fi(O(2)), presumably through the functional coupling between the mitochondrial creatine kinase and ATP production. This study suggests a potential role for peripheral factors, including the alteration of cellular homeostasis in active muscles, in determining the tolerance to hypoxia in maximally exercising endurance-trained athletes.

Deciphering the metabolic and mechanical contributions to the exercise-induced circulatory response: insights from eccentric cycling.

Metabolic demand and muscle mechanical tension are closely coupled during exercise, making their respective drives to the circulatory response difficult to establish. This coupling being altered in eccentric cycling, we implemented an experimental design featuring eccentric vs. concentric constant-load cycling bouts to gain insights into the control of the exercise-induced circulatory response in humans. Heart rate (HR), stroke volume (SV), cardiac output (Q), oxygen uptake (V(.-)(O(2))), and electromyographic (EMG) activity of quadriceps muscles were measured in 11 subjects during heavy concentric (heavy CON: 270 +/- 13 W; V(.-)(O(2)) = 3.59 +/- 0.20 l/min), heavy eccentric (heavy ECC: 270 +/- 13 W, V(.-)(O(2)) = 1.17 +/- 0.15 l/min), and light concentric (light CON: 70 +/- 9 W, V(.-)(O(2)) = 1.14 +/- 0.12 l/min) cycle bouts. Using a reductionist approach, the circulatory responses observed between heavy CON vs. light CON (difference in V(.-)(O(2)) and power output) was ascribed either to metabolic demand, as estimated from heavy CON vs. heavy ECC (similar power output, different V(.-)(O(2))), or to muscle mechanical tension, as estimated from heavy ECC vs. light CON (similar V(.-)(O(2)), different power output). 74% of the Q response was determined by the metabolic demand, also accounting for 65% and 84% of HR and SV responses, respectively. Consequently, muscle mechanical tension determined 26%, 35%, and 16% of the Q, HR, and SV responses, respectively. Q was significantly related to V(.-)(O(2)) (r(2) = 0.83) and EMG activity (r(2) = 0.82; both P < 0.001). These results suggest that the exercise-induced circulatory response is mainly under metabolic control and support the idea that the level of muscle activation plays a role in the cardiovascular regulation during cycle exercise in humans.

Exercise training in normobaric hypoxia in endurance runners. I. Improvement in aerobic performance capacity.

This study investigates whether a 6-wk intermittent hypoxia training (IHT), designed to avoid reductions in training loads and intensities, improves the endurance performance capacity of competitive distance runners. Eighteen athletes were randomly assigned to train in normoxia [Nor group; n = 9; maximal oxygen uptake (VO2 max) = 61.5 +/- 1.1 ml x kg(-1) x min(-1)] or intermittently in hypoxia (Hyp group; n = 9; VO2 max = 64.2 +/- 1.2 ml x kg(-1) x min(-1)). Into their usual normoxic training schedule, athletes included two weekly high-intensity (second ventilatory threshold) and moderate-duration (24-40 min) training sessions, performed either in normoxia [inspired O2 fraction (FiO2) = 20.9%] or in normobaric hypoxia (FiO2) = 14.5%). Before and after training, all athletes realized 1) a normoxic and hypoxic incremental test to determine VO2 max and ventilatory thresholds (first and second ventilatory threshold), and 2) an all-out test at the pretraining minimal velocity eliciting VO2 max to determine their time to exhaustion (T(lim)) and the parameters of O2 uptake (VO2) kinetics. Only the Hyp group significantly improved VO2 max (+5% at both FiO2, P < 0.05), without changes in blood O2-carrying capacity. Moreover, T(lim) lengthened in the Hyp group only (+35%, P < 0.001), without significant modifications of VO2 kinetics. Despite similar training load, the Nor group displayed no such improvements, with unchanged VO2 max (+1%, nonsignificant), T(lim) (+10%, nonsignificant), and VO2 kinetics. In addition, T(lim) improvements in the Hyp group were not correlated with concomitant modifications of other parameters, including VO2 max or VO2 kinetics. The present IHT model, involving specific high-intensity and moderate-duration hypoxic sessions, may potentialize the metabolic stimuli of training in already trained athletes and elicit peripheral muscle adaptations, resulting in increased endurance performance capacity.

Muscular mitochondrial function in amyotrophic lateral sclerosis is progressively altered as the disease develops: a temporal study in man.

We performed repeated analysis of mitochondrial respiratory function in skeletal muscle (SM) of patients with early-stage sporadic amyotrophic lateral sclerosis (SALS) to determine whether mitochondrial function was altered as the disease advanced. SM biopsies were obtained from 7 patients with newly diagnosed SALS, the same 7 patients 3 months later, and 7 sedentary controls. Muscle fibers were permeabilized with saponin, then skinned and placed in an oxygraphic chamber to measure basal and maximal adenosine diphosphate (ADP)-stimulated respiration rates and to assess mitochondrial regulation by ADP. We found that the maximal oxidative phosphorylation capacity of muscular mitochondria significantly increased, and muscular mitochondrial respiratory complex IV activity significantly decreased as the disease advanced. This temporal study demonstrates for the first time that mitochondrial function in SM in human SALS is progressively altered as the disease develops.

Exercise training in normobaric hypoxia in endurance runners. II. Improvement of mitochondrial properties in skeletal muscle.

This study investigates whether adaptations of mitochondrial function accompany the improvement of endurance performance capacity observed in well-trained athletes after an intermittent hypoxic training program. Fifteen endurance-trained athletes performed two weekly training sessions on treadmill at the velocity associated with the second ventilatory threshold (VT2) with inspired O2 fraction = 14.5% [hypoxic group (Hyp), n = 8] or with inspired O2 fraction = 21% [normoxic group (Nor), n = 7], integrated into their usual training, for 6 wk. Before and after training, oxygen uptake (VO2) and speed at VT2, maximal VO2 (VO2 max), and time to exhaustion at velocity of VO2 max (minimal speed associated with VO2 max) were measured, and muscle biopsies of vastus lateralis were harvested. Muscle oxidative capacities and sensitivity of mitochondrial respiration to ADP (Km) were evaluated on permeabilized muscle fibers. Time to exhaustion, VO2 at VT2, and VO2 max were significantly improved in Hyp (+42, +8, and +5%, respectively) but not in Nor. No increase in muscle oxidative capacity was obtained with either training protocol. However, mitochondrial regulation shifted to a more oxidative profile in Hyp only as shown by the increased Km for ADP (Nor: before 476 +/- 63, after 524 +/- 62 microM, not significant; Hyp: before 441 +/- 59, after 694 +/- 51 microM, P < 0.05). Thus including hypoxia sessions into the usual training of athletes qualitatively ameliorates mitochondrial function by increasing the respiratory control by creatine, providing a tighter integration between ATP demand and supply.

Coordinated changes in mitochondrial function and biogenesis in healthy and diseased human skeletal muscle.

We examined the transcriptional signaling cascade involved in the changes of mitochondrial biogenesis and mitochondrial function of skeletal muscle and of the exercise capacity of humans in response to long-term physical activity and chronic heart failure (CHF). Biopsy samples of vastus lateralis muscle were obtained from 18 healthy subjects with different fitness levels (assessed by maximal oxygen uptake, VO2 peak). We compared 9 sedentary subjects with 10 CHF patients undergoing transplantation. Muscle oxidative capacity was measured in permeabilized fibers (Vmax). Transcript levels of target genes were quantified by RT-PCR. In healthy subjects, VO2 peak was linearly related to Vmax (P<0.01) and to the gene expression of mitochondrial proteins and of the coactivator PGC-1alpha and its downstream transcription factors. A coordinate increase in PGC-1alpha and mRNA levels of proteins involved in degradation, fusion, and fission of mitochondria was observed associated with calcineurin activation. Despite decreased VO2 peak, in CHF patients skeletal muscles showed preserved Vmax in accordance with preserved markers and transcription factors of mitochondrial biogenesis and dynamics, with no calcineurin activation. The results provide strong support for a central role for PGC-1alpha and calcineurin activation in mitochondrial biogenesis in healthy and diseased human skeletal muscles.

Exercising with a denervated heart after cardiac transplantation.

Heart transplantation (HTR) is now an accepted life-extending procedure for those dying of intractable heart failure (CHF). HTR patients expect a high quality of life which implies a reasonable exercise capacity. Nevertheless HTR present unique exercise challenges with both central and peripheral factors of limitation that result in peak oxygen uptakes of 60-70% of age-matched normal subjects. Among central factors persistent chronotropic incompetence questions the occurrence and role of the graft reinnervation. Among peripheral factors the energetic impairement of the skeletal muscle seem to result more from microvascular abnormalities than from an actual deficit in oxidative capacity, questioning the mechanism of recovery from the CHF peripheral myopathy and the role of immunosuppressive drugs. Endurance and resistance training programs may reverse at least in part most but not all of these abnormalities. Training permits patients to engage in sports and even to participate in competitive events that are rewarding to them but also to the community because it promotes organ donation and confidence in medical achievements. Mechanisms of exercise impairments and improvements resulting from training are discussed in the perspective of current literature. Areas of future research and recommendations for the practice of sports after HTR are suggested.

Improving exercise capacity, 6 wk training tends to reduce circulating endothelin after heart transplantation.

Short-term survival is no longer the pivotal issue after heart transplantation but, most heart-transplant (Htx) patients still present with increased circulating endothelin-1 (ET) and reduced exercise capacity. ET-1 limits both exercise-induced vasodilation and blood flow redistribution toward acting muscles and might be accessible to training. This study was performed to investigate the effect of training on ET-1 and whether an eventual training-induced improvement in exercise capacity may be related to reduced baseline or exercise circulating ET-1 in Htx patients. Five Htx patients performed a maximal bicycle exercise test and an endurance exercise test before and after a training program of 18 exercises sessions during 6 wk. ET-1 was determined by radioimmunoassay at rest, end endurance exercise and 30 min recovery, before and after training. Training improved significantly Htx's maximal oxygen uptake (+13.1 +/- 4.8%; p < 0.05) and also reduced significantly the endurance exercise-induced heart rate increase. Resting ET-1 was increased in Htx (5.98 +/- 1.88 vs. 1.61 +/- 0.25 pmol/L in controls; p < 0.01) but although ET-1 modulation might participate in training-induced beneficial effects, training failed to modulate either resting or exercise ET-1 plasma level. Training-induced improvement in exercise capacity might not mainly due to decreased ET-1 after heart transplantation. Further supporting the usefulness of training, these preliminary data suggest that improved exercise capacity may not be mainly due to decreased ET-1 in Htx patients. Further, larger scale studies will be needed to investigate whether an impaired nitric oxide pathway stimulation might explain such results and whether a longer training program can reduce local ET-1, arising from working muscles after heart transplantation.

Eccentric cycle exercise: training application of specific circulatory adjustments.

PURPOSE: Despite identical oxygen uptake (VO2), enhanced heart rate (HR) and cardiac output (Q) responses have been reported in eccentric (ECC) versus concentric (CON) cycle exercise. The aim of this study was to describe the specific circulatory adjustments (HR and stroke volume (SV)) to incremental ECC cycle exercise in order to: 1) determine the HR values leading to identical VO2 in ECC and CON cycling; and 2) estimate the interindividual variability of this HR correspondence between the two exercise modes, with emphasis upon rehabilitation and training purposes. METHODS: Eight healthy male subjects (age, 28 +/- 2 yr) participated in this study. They performed CON and ECC cycle incremental exercises (power output increases of 50 W every 3 min). Breath-by-breath gas exchange analysis and beat-by-beat thoracic impedancemetry were used to determine VO2 and Q, respectively. RESULTS: At the same metabolic power (VO2 of 1.08 +/- 0.05 L x min(-1) in CON vs 1.04 +/- 0.06 L x min in ECC), SV was not different, but HR was 17% higher in ECC (P < 0.01), leading to a 27% enhanced Q (P < 0.01). Q and HR net adjustments (exercise minus resting values) in ECC versus CON muscle involvement demonstrated important interindividual variability with coefficients of variation amounting to 32% and 30%, respectively. CONCLUSION: In practice, if a given level of VO2 is to be reached, ECC HR has to be set above the CON one. Taking into account the interindividual variability of the circulatory adjustments in ECC versus CON muscle involvement, a precise HR correspondence can be established individually from the VO2/HR relationship obtained using ECC incremental testing, allowing prescription of accurate target HR for rehabilitation or training purposes.

Evaluation of quantitative and qualitative aspects of mitochondrial function in human skeletal and cardiac muscles.

Techniques and protocols of assessment of mitochondrial properties are of physiological and physiopathological important significance. A precise knowledge of the advantages and limitations of the different protocols used to investigate the mitochondrial function, is therefore necessary. This report presents examples of how the skinned (or permeabilized) fibers technique could be applied for the polarographic determination of the actual quantitative and qualitative aspects of mitochondrial function in human muscle samples. We described and compared the main available respiration protocols in order to sort out which protocol seems more appropriate for the characterization of mitochondrial properties according to the questions under consideration: quantitative determination of oxidative capacities of a given muscle, characterization of the pattern of control of mitochondrial respiration, or assessment of a mitochondrial defect at the level of the respiratory chain complexes. We showed that while protocol A, using only two levels of the phosphate acceptor adenosine diphosphate (ADP) concentration and the adjunction of creatine, could be used for the determination of quantitative changes in very small amount of muscle samples, the ADP sensitivity of mitochondrial respiration was underestimated by this protocol in muscles with high oxidative capacities. The actual apparent Km for ADP and the role of functional activation of miCK in ATP production and energy transfer in oxidative muscles, are well-assessed by protocol B (in the absence of creatine) together with protocol C (in the presence of creatine) that use increasing concentrations of ADP ranging from 2.5-2000 microM. Protocol D is well-adapted to investigate the potential changes at different levels of the respiratory chain, by the use of specific substrates and inhibitors. As can be seen from the present data and the current review of previous reports in the literature, a standardization of the respiration protocols is needed for useful comparisons between studies.

Preserved response of mitochondrial function to short-term endurance training in skeletal muscle of heart transplant recipients.

OBJECTIVES: We sought to determine whether intrinsic mitochondrial function and regulation were altered in heart transplant recipients (HTRs) and to investigate the response of mitochondrial function to six-week endurance training in these patients. BACKGROUND: Despite the normalization of central oxygen transport during exercise, HTRs are still characterized by limited exercise capacity, which is thought to result from skeletal muscle metabolic abnormalities. METHODS: Twenty HTRS agreed to have vastus lateralis biopsies and exercise testing: before and after training for 12 of them and before and after the same control period for eight subjects unwilling to train. Mitochondrial respiration was evaluated on saponin-permeabilized muscle fibers in the absence or presence (maximum respiration rate [V(max)]) of saturating adenosine diphosphate. RESULTS: Mitochondrial function was preserved at the level of sedentary subjects in untrained HTRs, although they showed 28 +/- 5% functional aerobic impairment (FAI). After training, V(max), citrate synthase, cytochrome c oxidase, and mitochondrial creatine kinase (CK) activities were significantly increased by 48%, 40%, 67%, and 53%, respectively (p < 0.05), whereas FAI decreased to 12 +/- 5% (p < 0.01). The control of mitochondrial respiration by creatine and mitochondrial CK was also improved (p < 0.01), suggesting that phosphocreatine synthesis and transfer by the mitochondrial CK become coupled to oxidative phosphorylation, as shown in trained, healthy subjects. CONCLUSIONS: In HTRs, the mitochondrial properties of skeletal muscle were preserved and responded well to training, reaching values of physically active, healthy subjects. This suggests that, in HTRs, immunosuppressive drugs do not alter the intrinsic muscle oxidative capacities and that the patients' physical handicap results from nonmitochondrial mechanisms.

Mitochondrial electron transport chain function is enhanced in inspiratory muscles of patients with chronic obstructive pulmonary disease.

In chronic obstructive pulmonary disease, inspiratory muscles face increased resistive and elastic workloads and therefore increased energy requirements. The adaptive response of these muscles to this higher energy demand includes increased oxidative enzymes and changes in contractile protein expression but the consequences on mitochondrial function and energy metabolism have not been assessed so far. We investigated the in situ properties of the mitochondria of costal diaphragm and external intercostal muscles using the skinned fiber technique in 9 emphysematous and 11 age-matched control patients. Biopsies obtained during thoracic surgery were placed in an oxygraphic chamber to measure maximal oxygen uptake. We observed that the maximal oxidative capacity of diaphragm and external intercostal muscles increased significantly in the emphysematous group compared with the control group (+135 and +37%, respectively). Significant correlations were found between the maximal oxidative capacity and patients' pulmonary indexes of obstruction (diaphragm: r = -0.637, intercostal: r = -0.667, p < 0.005) and hyperinflation (diaphragm: r = 0.639, p < 0.003, intercostal: r = 0.634, p < 0.01). Slow myosin heavy chain isoform increased in the diaphragm of the emphysematous group, with significant relationships between indexes of obstruction and hyperinflation and activities of biochemical mitochondrial markers. Thus, severe emphysema was associated with increased mitochondrial capacity and efficiency in the inspiratory muscles, supporting an endurance training-like effect.

Mitochondrial respiratory chain function in skeletal muscle of ALS patients.

Evidence implicating mitochondrial dysfunction in the central nervous system of patients with sporadic amyotrophic lateral sclerosis (SALS) has recently been accumulating. In contrast, data on mitochondrial function in skeletal muscle in SALS are scarce and controversial. We investigated the in situ properties of muscle mitochondria in patients with early-stage SALS and sedentary (SED) controls using the skinned fiber technique to determine whether respiration of muscle tissue is altered in early-stage SALS in comparison with SED. Musculus vastus lateralis biopsies were obtained from 7 SED group members and 14 patients with early-stage SALS (mean disease duration, 9 months). Muscle fibers were permeabilized with saponine and then skinned and placed in an oxygraphic chamber to measure basal (V(0)) and maximal (V(max)) adenosine diphosphate-stimulated respiration rates and to assess mitochondrial regulation by adenosine diphosphate. Muscle oxidative capacity, evaluated with V(max), was identical in patients in the SALS and SED groups (V(0): SALS, 1.1 +/- 0.1; SED, 0.8 +/- 0.1, micromol 0(2). min(-1). gm(-1)dw and V(max): SALS, 3.1 +/- 0.3; SED, 2.5 +/- 0.3, micromol 0(2). min(-1). gm(-1)dw). This study shows an absence of large mitochondrial damage in skeletal muscle of patients with early-stage SALS, suggesting that mitochondrial dysfunction in the earlier stages of SALS is almost certainly not systemic.