Effect of short-term training on mitochondrial ATP production rate in human skeletal muscle

Seven untrainedvolunteers [3 men, 4 women, 20.1 ± 2.0 (SD) yr, 66.0 ± 11.0 kg, 171 ± 13 cm] participated in a 10-day cycle exercisetraining program. Resting muscle samples were obtained from vastuslateralis before and after 5 and 10 days of training. Mitochondrial ATPproduction rate (MAPR) was assayed in isolated mitochondria by using abioluminescence technique and referenced to the activity of glutamatedehydrogenase in the muscle sample. MAPR increased 136 and 161% after10 days of training for the mitochondrial substrate combinationspyruvate + palmitoyl-L-carnitine + -ketoglutarate + malate andpalmitoyl-L-carnitine + malate, respectively. Total muscle glutamate dehydrogenase and citrate synthaseactivity increased 53 and 16%, respectively, after 5 days but did notsignificantly increase further after 10 days. The results from thepresent study indicate that MAPR, measured by using the substratecombinations pyruvate + palmitoyl-L-carnitine + -ketoglutarate + malate andpalmitoyl-L-carnitine + malate, can rapidly increase in response to endurance training.     

Effect of trifluoperazine on skeletal muscle mitochondrial respiration

The effect of trifluoperazine on the respiration of porcine liver and skeletal muscle mitochondria was investigated by polarographic and spectroscopic techniques. Low concentrations of trifluoperazine (88 nmol/mg protein) inhibited both the ADP- and Ca²⁺-stimulated oxidation of succinate, and reduced the values of the respiratory control index and the $\text{ADP}\text{O}$ and $\text{Ca}{}^{\mathrm{2+}}\text{O}$ ratio. High concentrations inhibited both succinate and ascorbate plus tetramethyl-p-phenylenediame (TMPD) oxidations, and uncoupler (carbonyl cyanide p-trifluromethoxyphenylhydrazone) and Ca²⁺-stimulated respiration. Porcine liver mitochondria were more sensitive to trifluoperazine than skeletal muscle mitochondria. Trifluoperazine inhibited the electron transport of succinate oxidation of skeletal muscle mitochondria within the cytochrome b-c₁ and cytochrome c₁-aa₃ segments of the respiratory chain system. 233 nmol trifluoperazine/mg protein inhibited the aerobic steady-state reduction of cytochrome c₁ by 92% with succinate as substrate, and of cytochrome c and cytochrome aa₃ by 50–60% with ascorbate plus TMPD as electron donors. Trifluoperazine can thus inhibit calmodulin-independent reactions particularly when used at high concentrations.     

Evidence for mitochondrial thioesterase 1 as a peroxisome proliferator-activated receptor-alpha-regulated gene in cardiac and skeletal muscle

The physiological role of mitochondrial thioesterase 1 (MTE1) is unknown. It was proposed that MTE1 promotes fatty acid (FA) oxidation (FAO) by acting in concert with uncoupling protein (UCP)3. We previously showed that ucp3 is a peroxisome proliferator-activated receptor-alpha (PPAR alpha)-regulated gene, allowing induction when FA availability increases. On the assumption that UCP3 and MTE1 act in partnership to increase FAO, we hypothesized that mte1 is also a PPAR alpha-regulated gene in cardiac and skeletal muscle. Using real-time RT-PCR, we characterized mte1 gene expression in rat heart and soleus muscles. Messenger RNA encoding for mte1 was 3.2-fold higher in heart than in soleus muscle. Cardiac mte1 mRNA exhibited modest diurnal variation, with 1.4-fold higher levels during dark phase. In contrast, skeletal muscle mte1 mRNA remained relatively constant over the course of the day. High-fat feeding, fasting, and streptozotocin-induced diabetes, interventions that increase FA availability, muscle PPAR alpha activity, and muscle FAO rates, increased mte1 mRNA in heart and soleus muscle. Conversely, pressure overload and hypoxia, interventions that decrease cardiac PPAR alpha activity and FAO rates, repressed cardiac mte1 expression. Specific activation of PPAR alpha in vivo through WY-14643 administration rapidly induced mte1 mRNA in cardiac and skeletal muscle. WY-14643 also induced mte1 mRNA in isolated adult rat cardiomyocytes dose dependently. Expression of mte1 was markedly lower in hearts and soleus muscles isolated from PPAR alpha-null mice. Alterations in cardiac and skeletal muscle ucp3 expression mirrored that of mte1 in all models investigated. In conclusion, mte1, like ucp3, is a PPAR alpha-regulated gene in cardiac and skeletal muscle.     

Training at high exercise intensity promotes qualitative adaptations of mitochondrial function in human skeletal muscle

This study explored mitochondrial capacities to oxidize carbohydrate and fatty acids and functional optimization of mitochondrial respiratory chain complexes in athletes who regularly train at high exercise intensity (ATH, n = 7) compared with sedentary (SED, n = 7). Peak O(2) uptake (Vo(2max)) was measured, and muscle biopsies of vastus lateralis were collected. Maximal O(2) uptake of saponin-skinned myofibers was evaluated with several metabolic substrates [glutamate-malate (V(GM)), pyruvate (V(Pyr)), palmitoyl carnitine (V(PC))], and the activity of the mitochondrial respiratory complexes II and IV were assessed using succinate (V(s)) and N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (V(TMPD)), respectively. Vo(2max) was higher in ATH than in SED (57.8 +/- 2.2 vs. 31.4 +/- 1.3 ml.min(-1).kg(-1), P < 0.001). V(GM) was higher in ATH than in SED (8.6 +/- 0.5 vs. 3.3 +/- 0.3 micromol O(2).min(-1).g dry wt(-1), P < 0.001). V(Pyr) was higher in ATH than in SED (8.7 +/- 1.0 vs. 5.5 +/- 0.2 micromol O(2).min(-1).g dry wt(-1), P < 0.05), whereas V(PC) was not significantly different (5.3 +/- 0.9 vs. 4.4 +/- 0.5 micromol O(2).min(-1).g dry wt(-1)). V(S) was higher in ATH than in SED (11.0 +/- 0.6 vs. 6.0 +/- 0.3 micromol O(2).min(-1).g dry wt(-1), P < 0.001), as well as V(TMPD) (20.1 +/- 1.0 vs. 16.2 +/- 3.4 micromol O(2).min(-1).g dry wt(-1), P < 0.05). The ratios V(S)/V(GM) (1.3 +/- 0.1 vs. 2.0 +/- 0.1, P < 0.001) and V(TMPD)/V(GM) (2.4 +/- 1.0 vs. 5.2 +/- 1.8, P < 0.01) were lower in ATH than in SED. In conclusion, comparison of ATH vs. SED subjects suggests that regular endurance training at high intensity promotes the enhancement of maximal mitochondrial capacities to oxidize carbohydrate rather than fatty acid and induce specific adaptations of the mitochondrial respiratory chain at the level of complex I.     

Effect of postexercise carbohydrate supplementation on glucose uptake-associated gene expression in the human skeletal muscle

We previously found that the exercise-induced elevation in GLUT4 mRNA of rat muscle can be rapidly down-regulated when glucose is given immediately following exercise. The purpose of this study was to determine the effect of postexercise carbohydrate diet on GLUT4 and hexokinase (HK) II mRNA levels in the human skeletal muscle. Eight untrained male subjects (age, 20.7+/-3.1 years) exercised for 60 min on a cycle ergometer at a 70-75% maximal oxygen consumption. The postexercise dietary treatment was performed in a crossover design. Immediately after the exercise, a diet with 70% carbohydrate content (1 g per kilogram of body weight; 356+/-19.8 kcal) was given to half of the subjects (eaten in 10 min) followed by a 3-h recovery, while the control subjects remained unfed for 3 h. Biopsies were performed on the deep portion of the vastus lateralis muscle of all subjects immediately after the exercise and 3 h after the carbohydrate ingestion. Blood glucose and serum insulin concentrations were measured every 30 min for 3 h. At the end of the 3-h recovery, blood glucose and serum insulin levels were not different from control levels, indicating that the oral carbohydrate was mostly disposed in the body within 3 h. In addition, GLUT4 and HK II mRNA levels were significantly lowered in the exercised human skeletal muscle in subjects receiving the carbohydrate diet. In conclusion, the present study demonstrates that GLUT4 mRNA and HK II mRNA in the exercised human skeletal muscle were significantly lowered by a high-carbohydrate diet.     

抗阻训练对增龄大鼠骨骼肌线粒体功能的影响*

目的: 从线粒体动力学的角度,探讨抗阻运动对增龄大鼠骨骼肌线粒体功能的影响。方法: 40只雄性SD大鼠随机分为4组:2月龄安静对照组(C1组)、2月龄抗阻运动训练组(R1组)、6月龄安静对照组(C2组)、6月龄抗阻运动训练组(R2组),每组10只。C1、C2组正常喂养,R1、R2组大鼠进行跑台坡度为35°,速度为15 m/min的抗阻运动,一次跑动15 s,间歇30 s,4次为一组,组间间歇3 min,3组为一次循环,一天为2个循环,循环间歇10 min,每周6 d,共8周。采用Western blot法测定各组大鼠股四头肌线粒体融合蛋白2(Mfn2)、GTP酶1(DRP1) 蛋白含量,使用流式细胞仪测定各组大鼠股四头肌线粒体膜电位(ΔΨm)、活性氧(ROS)和游离钙(Ca²⁺)水平。结果: ① 与C1组相比,R1组大鼠DRP1蛋白升高(P＜0.01)、Mfn2蛋白无显著变化,C2组大鼠DRP1、Mfn2蛋白均降低(P均＜0.01);与C2组相比,R2组大鼠DRP1、Mfn2蛋白均升高(P＜0.01,P＜0.05);与R1组相比,R2组DRP1、Mfn2蛋白均降低(P＜0.01,P＜0.05)。② 与C1组相比,R1组Ca²⁺含量降低(P＜0.01)、C2组Ca²⁺含量升高(P＜0.01);与C2组相比,R2组Ca²⁺含量降低(P＜0.01);与R1组相比,R2组Ca²⁺含量升高(P＜0.01)。③ 与C1组相比,R1组ROS含量有所上升,但无显著性差异,C2组ROS含量升高(P＜0.01);与C2组相比,R2组ROS含量降低(P＜0.01);与R1组相比,R2组ROS含量升高(P＜0.01)。④ 与C1组相比,C2组ΔΨm降低(P＜0.01);与C2组相比,R2组ΔΨm升高(P＜0.01);与R1组相比,R2组ΔΨm有所降低,但无统计学差异。结论: 大鼠增龄过程中股四头肌线粒体出现Ca²⁺堆积、活性氧增多、线粒体膜电位下降、融合蛋白减少等现象,抗阻训练可有效改善这些变化。     

Opening of potassium channels modulates mitochondrial function in rat skeletal muscle

We have investigated the presence of diazoxide- and nicorandil-activated K+ channels in rat skeletal muscle. Activation of potassium transport in the rat skeletal muscle myoblast cell line L6 caused a stimulation of cellular oxygen consumption, implying a mitochondrial effect. Working with isolated rat skeletal muscle mitochondria, both potassium channel openers (KCOs) stimulate respiration, depolarize the mitochondrial inner membrane and lead to oxidation of the mitochondrial NAD-system in a strict potassium-dependent manner. This is a strong indication for KCO-mediated stimulation of potassium transport at the mitochondrial inner membrane. Moreover, the potassium-specific effects of both diazoxide and nicorandil on oxidative phosphorylation in skeletal muscle mitochondria were completely abolished by the antidiabetic sulfonylurea derivative glibenclamide, a well-known inhibitor of ATP-regulated potassium channels (K(ATP) channels). Since both diazoxide and nicorandil facilitated swelling of de-energised mitochondria in KSCN buffer at the same concentrations, our results implicate the presence of a mitochondrial ATP-regulated potassium channel (mitoK(ATP) channel) in rat skeletal muscle which can modulate mitochondrial oxidative phosphorylation.     

Effect of weight reduction, obesity predisposition, and aerobic fitness on skeletal muscle mitochondrial function

We used (31)P magnetic resonance spectroscopy to measure maximal mitochondrial function in 12 obesity-prone women before and after diet-induced weight reduction and in 12 matched, never-obese, and 7 endurance-trained controls. Mitochondrial function was modeled after maximum-effort plantar flexion from the phosphocreatine recovery time constant (TC(PCr)), the ADP recovery time constant (TC(ADP)), and the rate of change in PCr during the first 14 s of recovery (OxPhos). Weight reduction was not associated with a significant change in mitochondrial function by TC(PCr), TC(ADP), or OxPhos. Mitochondrial function was not different between postobese and never-obese controls by TC(PCr) [35.1 +/- 2.5 (SE) vs. 34.6 +/- 2.5 s], TC(ADP) (22.9 +/- 1.8 vs. 21.2 +/- 1.8 s), or OxPhos (0.26 +/- 0. 03 vs. 0.25 +/- 0.03 mM ATP/s), postobese vs. never-obese, respectively. However, TC(ADP) was significantly faster (14.5 +/- 2. 3 s), and OxPhos was significantly higher (0.38 +/- 0.04 mM ATP/s) in the endurance-trained group. These results suggest that maximal mitochondrial function is not impaired in normal-weight obesity-prone women relative to their never-obese counterparts but is increased in endurance-trained women.     

Estimation of the mitochondrial redox state in human skeletal muscle during exercise

The mitochondrial redox (NAD+/NADH) state can be used as a reflection of oxygen availability within the mitochondrion. Previous studies using isolated muscle preparations suggest that active muscle is not hypoxic during lactate production, whereas experiments with humans come to the opposite conclusion. Six men exercised for 5 min at 75% maximal O2 consumption (VO2max) and then at 100% VO2max to exhaustion. Ammonia, oxoglutarate (alpha-ketoglutarate), and glutamate, as well as lactate, were measured in biopsies (vastus lateralis) taken at the end of each exercise. The three former metabolites were used to determine the mass action ratio of glutamate dehydrogenase and thus were used as an estimate of the mitochondrial redox state. Muscle lactate increased (P less than 0.05) to 14.5 and 24.5 mmol/kg wet wt after 75 and 100% VO2max, respectively. At both exercise intensities, muscle ammonia rose (P less than 0.05), glutamate fell (P less than 0.05) to only 30-35% of rest levels, and oxoglutarate declined (P less than 0.05). Despite the high levels of muscle lactate accumulation, the estimated mitochondrial redox rate rose 300% (P less than 0.05) in both exercise bouts. This response should increase the activity of key oxidative enzymes and promote increased VO2. Furthermore the data do not support the concept that muscle lactate is formed because of tissue hypoxia.     

Age-related mitochondrial genotypic and phenotypic alterations in human skeletal muscle

To have a clearer picture of how mitochondrial damages are associated to aging, a comprehensive study of phenotypic and genotypic alterations was carried out, analyzing with histochemical and molecular biology techniques the same skeletal muscle specimens of a large number of healthy subjects from 13 to 92 years old. Histochemical data showed that ragged red fibers (RRF) appear at about 40 years of age and are mostly cytochrome c oxidase (COX)-positive, whereas they are almost all COX-negative thereafter. Molecular analyses showed that the 4977 bp deletion of mitochondrial DNA (mtDNA(4977)) and the 7436 bp deletion of mtDNA (mtDNA(7436)) are already present in individuals younger than 40 years of age, but their occurrence does not change with age. After 40 years of age the number of mtDNA deleted species, as revealed by Long Extension PCR (LX-PCR), increases, the 10422 bp deletion of mtDNA (mtDNA(10422)) appears, although with a very low frequency of occurrence, and mtDNA content is more than doubled. Furthermore, mtDNA(4977) level directly correlates with that of COX-negative fibers in the same analyzed subjects. These data clearly show that, after 40 years of age, the phenotypic and genotypic mitochondrial alterations here studied appear in human skeletal muscle and that they are closely related.     

A new single nucleotide polymorphism in the ryanodine gene of chicken skeletal muscle

Some genes affect meat quality in chickens. We looked for polymorphisms in the Gallus gallus α-RyR gene (homologous to RyR 1) that could be associated with PSE (pale, soft and exudative) meat. Because RyR genes are over 100,000 bp long and code for proteins with about 5000 amino acids, primers were designed to amplify a fragment of hotspot region 2, a region with a high density of mutations in other species. Total blood DNA was extracted from 50 birds, 25 that had PSE meat and 25 normal chickens. The DNA samples were amplified by PCR, cloned, sequenced, and used to identify single nucleotide polymorphisms (SNPs). The amplified fragment of α-RyR was 604 nucleotides in length; 181 nucleotides were similar to two exons from a hypothetical turkey cDNA sequence for α-RyR. A non-synonymous nucleotide substitution (G/A) was identified in at least one of the three sequenced clones obtained from nine animals, six PSE (HAL+) birds and three normal (HAL-) birds; they were heterozygous for this mutation. This SNP causes a change from Val to Met in the α-RYR protein. Since the frequencies of this SNP were not significantly different in the PSE versus normal chickens, it appears that this mutation (in heterozygosity) does not alter the structure or function of the muscle protein, making it an inappropriate candidate as a genetic marker for PSE meat.     

Effects of cisplatin on mitochondrial function and autophagy-related proteins in skeletal muscle of rats

Cisplatin is widely known as an anti-cancer drug. However, the effects of cisplatin on mitochondrial function and autophagy-related proteins levels in the skeletal muscle are unclear. The purpose of this study was to investigate the effect of different doses of cisplatin on mitochondrial function and autophagy-re-lated protein levels in the skeletal muscle of rats. Eight-week-old male Wistar rats (n = 24) were assigned to one of three groups; the first group was administered a saline placebo (CON, n = 10), and the second and third groups were given 0.1 mg/kg body weight (BW) (n = 6), and 0.5 mg/kg BW (n = 8) of cisplatin, respectively. The group that had been administered 0.5 mg cisplatin exhibited a reduced BW, skeletal muscle tissue weight, and mitochondrial function and upregulated levels of autophagy-related proteins, including LC3II, Beclin 1, and BNIP3. Moreover, this group had a high LC3 II/I ratio in the skeletal muscle; i.e., the administration of a high dose of cisplatin decreased the muscle mass and mitochondrial function and increased the levels of autophagy-related proteins. These results, thus, suggest that reducing mitochondrial dysfunction and autophagy pathways may be important for preventing skeletal muscle atrophy following cisplatin administration.     

Cryopreservation of mitochondria and mitochondrial function in cardiac and skeletal muscle fibers

Long-term preservation of muscle mitochondria for consequent functional analysis is an important and still unresolved challenge in the clinical study of metabolic diseases and in the basic research of mitochondrial physiology. We here present a method for cryopreservation of mitochondria in various muscle types including human biopsies. Mitochondrial function was analyzed after freeze-thawing permeabilized muscle fibers using glycerol and dimethyl sulfoxide as cryoprotectant. Using optimal freeze-thawing conditions, high rates of adenosine 5(')-diphosphate-stimulated respiration and high respiratory control were observed, showing intactness of mitochondrial respiratory function after cryopreservation. Measurement of adenosine 5(')-triphosphate (ATP) formation showed normal rates of ATP synthesis and ATP/O ratios. Intactness of the outer mitochondrial membrane and functional coupling between mitochondrial creatine kinase and oxidative phosphorylation were verified by respiratory cytochrome c and creatine tests. Simultaneous confocal imaging of mitochondrial flavoproteins and nicotinamide adenine dinucleotide revealed normal intracellular arrangement and metabolic responses of mitochondria after freeze-thawing. The method therefore permits, after freezing and long-term storage of muscle samples, mitochondrial function to be estimated and energy metabolism to be monitored in situ. This will significantly expand the scope for screening and exchange of human biopsy samples between research centers, thus providing a new basis for functional analysis of mitochondrial defects in various diseases.     

Relationship between insulin sensitivity and in vivo mitochondrial function in skeletal muscle

Recent data have shown that individuals with low insulin sensitivity (S(I)) also have reduced whole body maximal oxygen uptake. The objectives of this study were to determine 1) whether muscle mitochondrial function was independently related to S(I) after being adjusted for known determinants of S(I) and 2) whether lower S(I) among African-American (AA) vs. Caucasian-American (CA) women was due to lower muscle mitochondrial function among AA women. Subjects were 37 CA and 22 AA premenopausal women (age: 33.6 +/- 6.3 yr). Mitochondrial function [time constant of ADP (ADP(tc))] was assessed during a 90-s unilateral isometric contraction using (31)P magnetic resonance spectroscopy, S(I) with an intravenous glucose tolerance test, body composition by dual-energy X-ray absorptiometry, and visceral adipose tissue (VAT) with computed tomography. ANOVA was used to compare AA and CA groups, and multiple linear regression modeling was used to identify independent predictors of S(I). Between-race comparisons indicated that muscle oxidative capacity was lower among AAs vs. CAs (ADP(tc): 25.6 +/- 9.8 vs. 21.4 +/- 9.9 s). Multiple linear regression models for the dependent variable S(I) contained 1) VAT and race and 2) VAT, race, and ADP(tc). Significant independent effects for all predictor variables were observed in both the first (r(2) = 0.345) and second (r(2) = 0.410) models. The partial correlation for race was lower in the second model (-0.404 vs. -0.300), suggesting that muscle mitochondrial function contributed to the racial difference in S(I). Lower muscle mitochondrial function among AAs may in part explain lower S(I) among them.     

Effects of weight loss and physical activity on skeletal muscle mitochondrial function in obesity

The current study was undertaken to address responsiveness of skeletal muscle mitochondrial electron transport chain (ETC) activity to weight loss (WL) and exercise in overweight or obese, sedentary volunteers. Fourteen middle-aged participants (7 male/7 female) had assessments of mitochondrial ETC activity and mitochondrial (mt)DNA in vastus lateralis muscle, obtained by percutaneous biopsy, before and after a 16-wk intervention. Mean WL was 9.7 (1.5%) and the mean increase in Vo(2 max) was [means (SD)] 21.7 (3.7)%. Total ETC activity increased significantly, from 0.13 (0.02) to 0.19 (0.03) U/mU creatine kinase (CK; P < 0.001). ETC activity was also assessed in mitochondria isolated into subsarcolemmal (SSM) and intermyofibrillar (IMF-M) fractions. In response to intervention, there was a robust increase of ETC activity in SSM (0.028 (0.007) to 0.046 (0.011) U/mU CK, P < 0.001), and in IMF-M [0.101 (0.015) to 0.148 (0.018) U/mU CK, P < 0.005]. At baseline, the percentage of ETC activity contained in the SSM fraction was low and remained unchanged following intervention [19 (3) vs. 22 (2)%], despite the increase in ETC activity. Also, muscle mtDNA content did not change significantly [1665 (213) vs. 1874 (214) mtDNA/nuclear DNA], denoting functional improvement rather than proliferation of mitochondria as the principal mechanism of enhanced ETC activity. Increases in ETC activity were correlated with energy expenditure during exercise sessions, and ETC activity in SSM correlated with insulin sensitivity after adjustment for Vo(2 max). In summary, skeletal muscle ETC activity is increased by WL and exercise in previously sedentary obese men and women. We conclude that improved skeletal muscle ETC activity following moderate WL and improved aerobic capacity contributes to associated alleviation of insulin resistance.     

PGC-1alpha integrates insulin signaling, mitochondrial regulation, and bioenergetic function in skeletal muscle

The pathophysiology underlying mitochondrial dysfunction in insulin-resistant skeletal muscle is incompletely characterized. To further delineate this we investigated the interaction between insulin signaling, mitochondrial regulation, and function in C2C12 myotubes and in skeletal muscle. In myotubes elevated insulin and glucose disrupt insulin signaling, mitochondrial biogenesis, and mitochondrial bioenergetics. The insulin-sensitizing thiazolidinedione pioglitazone restores these perturbations in parallel with induction of the mitochondrial biogenesis regulator PGC-1alpha. Overexpression of PGC-1alpha rescues insulin signaling and mitochondrial bioenergetics, and its silencing concordantly disrupts insulin signaling and mitochondrial bioenergetics. In primary skeletal myoblasts pioglitazone also up-regulates PGC-1alpha expression and restores the insulin-resistant mitochondrial bioenergetic profile. In parallel, pioglitazone up-regulates PGC-1alpha in db/db mouse skeletal muscle. Interestingly, the small interfering RNA knockdown of the insulin receptor in C2C12 myotubes down-regulates PGC-1alpha and attenuates mitochondrial bioenergetics. Concordantly, mitochondrial bioenergetics are blunted in insulin receptor knock-out mouse-derived skeletal myoblasts. Taken together these data demonstrate that elevated glucose and insulin impairs and pioglitazone restores skeletal myotube insulin signaling, mitochondrial regulation, and bioenergetics. Pioglitazone functions in part via the induction of PGC-1alpha. Moreover, PGC-1alpha is identified as a bidirectional regulatory link integrating insulin-signaling and mitochondrial homeostasis in skeletal muscle.