HIF-1-driven skeletal muscle adaptations to chronic hypoxia: molecular insights into muscle physiology.

Skeletal muscle is a metabolically active tissue and the major body protein reservoir. Drop in ambient oxygen pressure likely results in a decrease in muscle cells oxygenation, reactive oxygen species (ROS) overproduction and stabilization of the oxygen-sensitive hypoxia-inducible factor (HIF)-1α. However, skeletal muscle seems to be quite resistant to hypoxia compared to other organs, probably because it is accustomed to hypoxic episodes during physical exercise. Few studies have observed HIF-1α accumulation in skeletal muscle during ambient hypoxia probably because of its transient stabilization. Nevertheless, skeletal muscle presents adaptations to hypoxia that fit with HIF-1 activation, although the exact contribution of HIF-2, I kappa B kinase and activating transcription factors, all potentially activated by hypoxia, needs to be determined. Metabolic alterations result in the inhibition of fatty acid oxidation, while activation of anaerobic glycolysis is less evident. Hypoxia causes mitochondrial remodeling and enhanced mitophagy that ultimately lead to a decrease in ROS production, and this acclimatization in turn contributes to HIF-1α destabilization. Likewise, hypoxia has structural consequences with muscle fiber atrophy due to mTOR-dependent inhibition of protein synthesis and transient activation of proteolysis. The decrease in muscle fiber area improves oxygen diffusion into muscle cells, while inhibition of protein synthesis, an ATP-consuming process, and reduction in muscle mass decreases energy demand. Amino acids released from muscle cells may also have protective and metabolic effects. Collectively, these results demonstrate that skeletal muscle copes with the energetic challenge imposed by O2 rarefaction via metabolic optimization.

Mechanotransduction in striated muscle via focal adhesion kinase.

Contractile tissues demonstrate a pronounced capacity to remodel their composition in response to mechanical challenges. Descriptive evidence suggests the upstream involvement of the phosphotransfer enzyme FAK (focal adhesion kinase) in the molecular control of load-dependent muscle plasticity. Thereby FAK evolves as a myocellular transducer of mechanical signals towards downstream transcript expression in myofibres. Recent advances in somatic gene therapy now allow the exploration of the functional involvement of this enzyme in mechanotransduction in intact muscle.

Regulation of ubiquitin-proteasome system, caspase enzyme activities, and extracellular proteinases in rat soleus muscle in response to unloading.

In the present study, we determined the impact of 5 and 10 days of muscle deconditioning induced by hindlimb suspension (HS) on the ubiquitin-proteasome system of protein degradation and caspase enzyme activities in rat soleus muscles. A second goal was to determine whether activities of matrix metalloproteinase-2/9 (MMP-2/9) and urokinase-type/tissue-type plasminogen activator (PAs) were responsive to HS. As expected, HS led to a pronounced atrophy of soleus muscle. Level of ubiquitinated proteins, chymotrypsin-like activity of 20S proteasome, and Bcl-2-associated gene product-1 protein level were all transitory increased in response to 5 days of HS. These changes may thus potentially account for the decrease in muscle mass observed in response to 5 days of HS. Caspase-3 activity was significantly increased throughout the experimental period, whereas activities of caspase-6, another effector caspase, and caspase-9, the mitochondrial-dependent activator of both caspase-3 and -6, were only increased in response to 10 days of HS. This suggests that caspase-3 may be regulated through mitochondrial-independent and mitochondrial-dependent mechanisms in response to HS. Finally, MMP-2/9 activities remained unchanged, whereas PAs activities were increased after 5 days of HS. Overall, these data suggest that time-dependent regulation of intracellular and extracellular proteinases are important in setting the new phenotype of rat soleus muscle in response to HS.

Validation of a new calibration method for human muscle microdialysis at rest and during exercise.

Microdialysis presents the unique possibility to measure metabolite concentrations in human interstitial fluid. During exercise, the recovery of these metabolites should be precisely monitored since it is known to increase greatly with muscle blood flow. The loss of ethanol, perfused at low concentration, can be accurately measured and reflects the changes in dialysis conditions. We evaluated whether using the relationship determined in resting metabolic conditions between the loss of ethanol, as reference substance, and the recovery for lactate or glucose would allow us to calculate precisely the concentration of these substances and their variations during exercise. Using the new catheter calibration method (slope method), the error of estimation of lactate and glucose in vitro was limited to -0.6 (5.8)% and -0.7 (6.2)%, respectively. In resting human muscle, the slope method proved to be as accurate as an established calibration technique ("no net flux method") to evaluate interstitial lactate concentration [1.82 (0.58) vs 1.83 (0.47) mM, respectively]. During dynamic knee-extension exercise or light neuromuscular electrical stimulation, the estimated interstitial lactate and glucose concentrations varied differently, but their time course changes remained consistent with their respective plasma values. We conclude that, after an initial calibration step, the slope method allows accurate measurement of interstitial muscle metabolites and it could be used to monitor rapid metabolic changes during exercise.

Molecular adaptations of neuromuscular disease-associated proteins in response to eccentric exercise in human skeletal muscle.

The molecular events by which eccentric muscle contractions induce muscle damage and remodelling remain largely unknown. We assessed whether eccentric exercise modulates the expression of proteinases (calpains 1, 2 and 3, proteasome, cathepsin B+L), muscle structural proteins (alpha-sarcoglycan and desmin), and the expression of the heat shock proteins Hsp27 and alphaB-crystallin. Vastus lateralis muscle biopsies from twelve healthy male volunteers were obtained before, immediately after, and 1 and 14 days after a 30 min downhill treadmill running exercise. Eccentric exercise induced muscle damage as evidenced by the analysis of muscle pain and weakness, creatine kinase serum activity, myoglobinaemia and ultrastructural analysis of muscle biopsies. The calpain 3 mRNA level was decreased immediately after exercise whereas calpain 2 mRNA level was increased at day 1. Both mRNA levels returned to control values by day 14. By contrast, cathepsin B+L and proteasome enzyme activities were increased at day 14. The alpha-sarcoglycan protein level was decreased immediately after exercise and at day 1, whereas the desmin level peaked at day 14. alphaB-crystallin and Hsp27 protein levels were increased at days 1 and 14. Our results suggest that the differential expression of calpain 2 and 3 mRNA levels may be important in the process of exercise-induced muscle damage, whereas expression of alpha-sarcoglycan, desmin, alphaB-crystallin and Hsp27 may be essentially involved in the subsequent remodelling of myofibrillar structure. This remodelling response may limit the extent of muscle damage upon a subsequent mechanical stress.

Assembly of the cellular powerhouse: current issues in muscle mitochondrial biogenesis.

Mitochondrial biogenesis occurs in muscle in response to chronic exercise, resulting in fatigue resistance. The assembly of the organelle is initiated by contraction-induced signals, which lead to the transcriptional activation of nuclear genes. This is accompanied by alterations in mRNA stability, as well as increases in protein import and mitochondrial DNA copy number, leading to a greater muscle mitochondrial content.

Expression and activity of caspases 1 and 3 in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are characterized by abnormal growth of committed progenitors in clonogenic assay, with reduced number of colonies and decreased colony/cluster ratio. It has been suggested that excessive apoptosis is the cause of marrow failure in MDS. We studied the expression of caspase-1 (interleukin-1beta-converting enzyme, ICE) and caspase-3 (CPP32/apopain) in marrow mononuclear cells, and the growth pattern of committed progenitors in a series of 83 MDS cases. The percentage of apoptotic cells as detected by TUNEL technique, and the percentage of caspase-3-positive cells were significantly higher in refractory anemia (RA) and RA with ringed sideroblasts (RAS) than in chronic myelomonocytic leukemia (CMML), refractory anemia with excess of blasts (RAEB) and RAEB in transformation (RAEB-T). Spontaneous growth of CFU-GM was associated with a higher percentage of blasts, and with a lower expression of caspase-3 and caspase-1. The yield of CFU-E, BFU-E, and CFU-GM (in the presence of growth factors) was decreased by comparison to normal marrow, but large individual differences were observed in all cytological categories. Inhibition of caspase-1 and caspase-3 activities by specific inhibitors resulted in a significant increase of the production of all types of colonies (up to 50-fold of control). In the presence of caspase-3 inhibitor, the number of BFU-E and CFU-E was in the range of normal values in most cases of RA and RAS. In addition, caspase-1 and -3 protease activities were detectable by fluorogenic assay in all cases studied. Western blot analysis confirmed the expression of caspase-3, including the cleaved (activated)-p17 form in most cases of RA/RAS analyzed. It is concluded that caspase-3 is implicated in the increased apoptosis observed in MDS and that inhibition of its activity can restore at least partially the growth of committed progenitors.

Zidovudine (AZT) induced alterations in mitochondrial biogenesis in rat striated muscles.

Zidovudine (AZT) and didanosine (ddI), two drugs used in the treatment of AIDS, are also known to cause mitochondrial abnormalities. We investigated the physiological relevance of the mitochondrial defects by measuring in situ skeletal muscle performance and cytochrome c oxidase (CYTOX) enzyme activity in heart muscle, red highoxidative (RG) and white low-oxidative (WG) portions of the gastrocnemius muscle of control (n = 17), AZT-(n = 14), or ddI-treated (n = 11) rats for 28 days. We also evaluated the hypothesis that AZT treatment could alter the expression of the mitochondrial transcription factor A (mtTFA), a key molecule involved in mitochondrial DNA (mtDNA) replication and transcription. AZT had a pronounced effect on blood pressure and skeletal muscle performance, which were significantly decreased during contractile activity at 2 and 5 Hz, compared with control. A significant decrease in CYTOX activity in heart and RG, but not WG muscles, was also evident. In the heart, this was accompanied by an apparent compensatory increase in mtTFA mRNA level that could not be attributed to enhanced transcriptional activation mediated by nuclear respiratory factor 1 (NRF-1). In contrast with AZT, no effect of ddI was found on the extent of fatigue or muscle enzyme activity. These results indicate that AZT induces mitochondrial defects primarily in muscles with the highest oxidative capacities (heart and RG). The long-term effects of AZT on mitochondrial biogenesis have the potential to reduce muscle performance, but the effects on performance in this short-term study were likely due to an inability of the AZT-treated animals to maintain blood pressure during contractile activity.

Cytochrome c transcriptional activation and mRNA stability during contractile activity in skeletal muscle.

We evaluated contractile activity-induced alterations in cytochrome c transcriptional activation and mRNA stability with unilateral chronic stimulation (10 Hz, 3 h/day) of the rat tibialis anterior (TA) muscle for 1, 2, 3, 4, 5, and 7 days (n = 3-11/group). Transcriptional activation was assessed by direct plasmid DNA injection into the TA with a chloramphenicol acetyltransferase (CAT) reporter gene linked to 326 bp of the cytochrome c promoter. Cytochrome c mRNA in stimulated muscles increased by 1.3- to 1. 7-fold above control between 1 and 7 days. Cytochrome c protein was increased after 5 days of stimulation to reach levels that were 1. 9-fold higher than control by 7 days. Cytochrome c mRNA stability, determined with an in vitro decay assay, was greater in stimulated TA than in control between 2 and 4 days, likely mediated by the induction of a cytosolic factor. In contrast, cytochrome c transcriptional activation was elevated only after 5 days of stimulation when mRNA stability had returned to control levels. Thus the contractile activity-induced increase in cytochrome c mRNA was due to an early increase in mRNA stability, followed by an elevation in transcriptional activation, leading to an eventual increase in cytochrome c protein levels.

Calcium-dependent regulation of cytochrome c gene expression in skeletal muscle cells. Identification of a protein kinase c-dependent pathway.

Mitochondrial biogenesis can occur rapidly in mammalian skeletal muscle subjected to a variety of physiological conditions. However, the intracellular signal(s) involved in regulating this process remain unknown. Using nuclearly encoded cytochrome c, we show that its expression in muscle cells is increased by changes in cytosolic Ca2+ using the ionophore A23187. Treatment of myotubes with A23187 increased cytochrome c mRNA expression up to 1.7-fold. Transfection experiments using promoter-chloramphenicol acetyltransferase constructs revealed that this increase could be transcriptionally mediated since A23187 increased chloramphenicol acetyltransferase activity by 2.5-fold. This increase was not changed by KN62, an inhibitor of Ca2+/calmodulin-dependent kinases II and IV, and it was not modified by overexpression of protein kinase A and cAMP response element-binding protein, demonstrating that the A23187 effect was not mediated through Ca2+/calmodulin-dependent kinase- or protein kinase A-dependent pathways. However, treatment of myotubes with staurosporine or 12-O-tetradecanoylphorbol-13-acetate reduced the effect of A23187 on cytochrome c transactivation by 40-50%. Coexpression of the Ca2+-sensitive protein kinase C isoforms alpha and betaII, but not the Ca2+-insensitive delta isoform, exaggerated the A23187-mediated response. The short-term effect of A23187 was mediated in part by mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2) since its activation peaked 2 h after A23187 treatment, and cytochrome c transactivation was reduced by PD98089, a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor. These results demonstrate the existence of a Ca2+-sensitive, protein kinase C-dependent pathway involved in cytochrome c expression and implicate Ca2+ as a signal in the up-regulation of nuclear genes encoding mitochondrial proteins.

Contractile activity-induced adaptations in the mitochondrial protein import system.

We previously demonstrated that subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial subfractions import proteins at different rates. This study was undertaken to investigate 1) whether protein import is altered by chronic contractile activity, which induces mitochondrial biogenesis, and 2) whether these two subfractions adapt similarly. Using electrical stimulation (10 Hz, 3 h/day for 7 and 14 days) to induce contractile activity, we observed that malate dehydrogenase import into the matrix of the SS and IMF mitochondia isolated from stimulated muscle was significantly increased by 1.4-to 1.7-fold, although the pattern of increase differed for each subfraction. This acceleration of import may be mitochondrial compartment specific, since the import of Bcl-2 into the outer membrane was not affected. Contractile activity also modified the mitochondrial content of proteins comprising the import machinery, as evident from increases in the levels of the intramitochondrial chaperone mtHSP70 as well as the outer membrane import receptor Tom20 in SS and IMF mitochondria. Addition of cytosol isolated from stimulated or control muscles to the import reaction resulted in similar twofold increases in the ability of mitochondria to import malate dehydrogenase, despite elevations in the concentration of mitochondrial import-stimulating factor within the cytosol of chronically stimulated muscle. These results suggest that chronic contractile activity modifies the extra- and intramitochondrial environments in a fashion that favors the acceleration of precursor protein import into the matrix of the organelle. This increase in protein import is likely an important adaptation in the overall process of mitochondrial biogenesis.

L-carnitine stimulation of mitochondrial oxidative phosphorylation rate in isolated rat skeletal muscle mitochondria.

L-carnitine effects on mitochondrial adenosine triphosphate (ATP) production rate were investigated in mitochondria isolated from rat extensor digitorum longus (EDL) muscle. Kinetic parameters, apparent Michaelis constant for free adenosine diphosphate (ADP) (K(m)) and apparent maximal ATP production rate (Vmax), were determined in absence and presence of L-carnitine. Although the K(m) remained unchanged in response to L-carnitine addition when pyruvate + malate was oxidized, the affinity of isolated mitochondria towards ADP was decreased when pyruvate + palmitoylcarnitine + malate was used as substrate. As a result of L-carnitine addition, a slight non-significant (P < 0.07) increase in Vmax was observed with pyruvate + malate while the increase reached the level of significance with pyruvate + palmitoylcarnitine + malate. Moreover, a positive correlation was obtained when mitochondrial ATP production rate was plotted against the difference between values measured with and without L-carnitine. Our data suggest that addition of L-carnitine to isolated rat skeletal muscle mitochondria can stimulate mitochondrial oxidative phosphorylation rate under conditions where the inhibitory effect of acetyl-CoA accumulation on pyruvate dehydrogenase complex activity is optimized. A change in the control of mitochondrial ATP flux by pyruvate dehydrogenase (PDH) complex might also occur with increasing mitochondrial ATP production rate, reinforcing the L-carnitine effects.

Oxygen uptake during submaximal incremental and constant work load exercises in hypoxia.

The effect of acute hypoxia on oxygen uptake (VO2) was studied during incremental (IE) and constant work load exercises. Twenty-two healthy subjects performed two incremental exercises on a bicycle ergometer under normoxic (21% O2) and hypoxic (10.4% O2) conditions. Fifteen subjects performed a constant work load exercise at the same absolute power (CAP) (116 +/- 33 W), while seven other subjects performed three constant work load exercises at the same relative power (CRP) (50, 60 and 70% of VO2max) in both conditions. VO2 was defined as extraventilatory when the estimation of respiratory muscles O2 consumption was subtracted from the total VO2. During IE, the slope of the linear regression relating VO2 to work rate was higher in normoxia than in hypoxia (11.6 +/- 1.2 ml.l-1.W-1 vs 10.1 +/- 1.1 ml.l-1.W-1, p < 0.01). During CAP, VO2 was lower in normoxia than in hypoxia (1.88 +/- 0.45).min-1 vs 1.96 +/- 0.42 l.min-1, p < 0.01) whereas extraventilatory VO2 was not significantly different (1.80 +/- 0.441.min-1 vs 1.77 +/- 0.36) l.min-1). During CRP, the slope relating VO2 to power output computed from the three work loads was not statistically different between normoxia and hypoxia (delta VO2/delta w = 11.9 +/- 3.1 ml.min-1.W-1 vs 12.3 +/- 1.2 ml.min-1.W-1). These findings showed that during CRP, the metabolic efficiency (delta VO2/delta W) was the same in normoxia and in hypoxia. During CAP, the respiratory muscles O2 consumption might have accounted for the difference in VO2 consumption between hypoxia and normoxia.

Mitochondrial biogenesis in skeletal muscle in response to endurance exercises.

Repeated bouts of endurance exercise stimulates mitochondrial biogenesis in skeletal muscle. The synthesis of mitochondrial proteins involves a coordinated expression of both nuclear and mitochondrial genes. During this process, multiples sites of regulation have been identified at the transcriptional and translational levels. After their synthesis, mitochondrial proteins originating from the nuclear genome are imported into newly synthesized preexisting membranes and directed to one of the four mitochondrial subcompartments. The detailed mechanisms of the endurance training-induced mitochondrial biogenesis are still poorly understood. In particular, much work is needed to identify the molecular signals able to stimulate and coordinate the expression of mitochondrial proteins in response to endurance training. This will be a great help in the future to understand clearly the intimate mechanisms of mitochondrial biogenesis in skeletal muscle and the factors involved in endurance exercise performance.

Effect of a 6-week endurance training programme and branched-chain amino acid supplementation on histomorphometric characteristics of aged human muscle.

The effects of a 6-week endurance training programme and a branched-chain amino acid (BCAA) supplementation were investigated on skeletal muscle histomorphometric characteristics of elderly men. Seventeen elderly men, age (63 +/- 5 years), height (173 +/- 5 cm) and weight (75 +/- 8 kg) were included in the study. One group (n = 9) received an oral BCAA supplementation for 6 weeks (16, 2 and 2 g per day of leucine, isoleucine and valine, respectively), while another group (n = 8) received a placebo. During these 6 weeks, subjects trained on a Monark cycle ergometer at 75 +/- 9% of their maximal heart rate for 1 h/day, 4 days/week. Muscle biopsy samples taken at rest before and after endurance training were analyzed for capillarization, fibre type distribution and fibre area. As a result of endurance training, maximal oxygen uptake was significantly increased by about 5% in control and BCAA supplemented groups (P < 0.01). The number of capillaries per fibre and in contact with type I fibres was significantly increased (P < 0.05), this effect being similar in control and BCAA supplemented groups. The percentage distribution and area of type I, type IIa and type IIb fibres did not differ between the two groups and remained unchanged with endurance training. It is concluded that skeletal muscle of elderly men can adapt to a 6-week endurance training programme and that a BCAA supplementation does not further enhance the induced histomorphometric changes.

Mitochondrial ATP production rate in 55 to 73-year-old men: effect of endurance training.

The effect of 6-week endurance training on mitochondrial ATP production rate was investigated in 14 elderly men. Mean age, body weight and height were 63 +/- 6 yr, 75.6 +/- 9.2 kg and 174 +/- 4 cm, respectively. Subjects trained on a Monark cycle ergometer at 79 +/- 8% of their maximal heart rate for 1 h day-1, 4 days week-1. Muscle samples were obtained at rest, before and after endurance training, by a needle biopsy technique and used for determination of mitochondrial ATP production rate in isolated mitochondria and enzyme assays. Endurance training resulted in a significant increase in maximal oxygen uptake (L min-1) (P < 0.01). Citrate synthase activity, a mitochondrial marker enzyme, and hexokinase activity increased significantly (both P < 0.01) in response to training while 3-hydroxyacyl-CoA dehydrogenase and carnitine palmitoyltransferase I activities remained statistically unchanged. A higher mitochondrial ATP production rate was observed after endurance training with the substrate combinations pyruvate+palmitoyl-L-carnitine+L-glutamate+malate (P < 0.01), L-glutamate (P < 0.001), pyruvate+malate (P < 0.05) and palmitoyl-L-carnitine+malate (P < 0.01). The largest increase was obtained with L-glutamate (170%). Significant correlations were observed between the percent increase in citrate synthase activity and those of mitochondrial ATP production rates. It was concluded that the increased mitochondrial ATP production rate of aged human skeletal muscle with training seems mainly to occur through an increased mitochondrial content, and in a way similar to those observed in young men.

Energetic status and mitochondrial oxidative capacity of rat skeletal muscle in response to creatine analogue ingestion.

A creatine analogue, beta-guanidinopropionic acid (beta-GPA), was administered in the food (1% w/w) of 8 male rats for 6 weeks, while 8 control rats received a standard diet. Mitochondrial oxidative capacity and cytosolic modulators of mitochondrial oxidative phosphorylation (free ADP, ATP-to-free ADP ratio) were evaluated in the soleus and extensor digitorum longus (EDL) muscles. Mitochondrial adaptation to the diet was significantly different between muscles. Citrate synthase activity and mitochondrial ATP synthesis rate were 35 and 45% higher in EDL muscle, respectively, whereas they were virtually unchanged in the soleus muscle. In both muscles, 3-hydroxyacyl-CoA dehydrogenase activity remained unaffected. Regardless of muscle type, creatine, phosphocreatine and ATP concentrations, as well as the total adenine nucleotide content (ATP + ADP + AMP), were significantly lower in beta-GPA fed rats. Whereas free ADP concentration remained unchanged, a significantly greater decrease in ATP-to-free ADP ratio was observed in EDL than in the soleus muscle. It is suggested that regulation of mitochondrial oxidative phosphorylation, through changes in metabolite concentrations, could be an important factor to consider for mitochondrial adaptation induced by beta-GPA feeding.

ATP synthesis kinetic properties of mitochondria isolated from the rat extensor digitorum longus muscle depleted of creatine with beta-guanidinopropionic acid.

A creatine analogue, beta-guanidinopropionic acid (beta-GPA), was administered in the food (1% w/w) of 8 male rats while 8 control rats received a standard diet. Mitochondrial oxidative capacity and kinetic parameters of mitochondrial ATP synthesis, apparent maximal ATP synthesis rate (Vmax) and apparent Michaelis constant for free ADP (Km), were investigated in the extensor digitorum longus (EDL) muscle. Mitochondrial ATP synthesis rate was measured by a bioluminescent method over a large range of ADP concentration (2-30 microM). As a result of the diet, Vmax was significantly increased (P < 0.05) while Km remained unchanged at around 20 microM. Citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase activities were significantly increased (both P < 0.05). Vmax was tightly correlated with CS activity (P < 0.001; r = 0.84). It was concluded that the increase in maximal mitochondrial ATP synthesis rate after beta-GPA feeding in EDL muscle was essentially due to a general increase in mitochondrial enzyme concentrations.