Resistance exercise acutely increases MHC and mixed muscle protein synthesis rates in 78-84 and 23-32 yr olds

We determined whether short-term weight-lifting exercise increases the synthesis rate of the major contractile proteins, myosin heavy chain (MHC), actin, and mixed muscle proteins in nonfrail elders and younger women and men. Fractional synthesis rates of mixed, MHC, and actin proteins were determined in seven healthy sedentary 23- to 32-yr-old and seven healthy 78- to 84-yr-old participants in paired studies done before and at the end of a 2-wk weight-lifting program. The in vivo rate of incorporation of 1-[(13)C]leucine into vastus lateralis MHC, actin, and mixed proteins was determined using a 14-h constant intravenous infusion of 1-[(13)C]leucine. Before exercise, the mixed and MHC fractional synthetic rates were lower in the older than in the younger participants (P < or = 0.04). Baseline actin protein synthesis rates were similar in the two groups (P = not significant). Over a 2-wk period, participants completed ten 1- to 1. 5-h weight-lifting exercise sessions: 2-3 sets per day of 9 exercises, 8-12 repetitions per set, at 60-90% of maximum voluntary muscle strength. At the end of exercise, MHC and mixed protein synthetic rates increased in the younger (88 and 121%) and older participants (105 and 182%; P < 0.001 vs. baseline). These findings indicate that MHC and mixed protein synthesis rates are reduced more than actin in advanced age. Similar to that of 23-32 yr olds, the vastus lateralis muscle in 78-84 yr olds retains the capacity to increase MHC and mixed protein synthesis rates in response to short-term resistance exercise.     

Turnover of myosin heavy and light chains in cultured embryonic chick cardiac and skeletal muscle

The relative rates of synthesis and breakdown of myosin heavy and light chains were studied in primary cell cultures of embryonic chick cardiac and skeletal muscle. Measurements were made after 4 days in culture, at which time both skeletal and cardiac cultures were differentiated and contracted spontaneously. Following a 4-hr pulse of radioactive leucine, myosin and its heavy and light chains were extracted to 90% or greater purity and the specific activities of the proteins were determined. In cardiac muscle, myosin heavy chains were synthesized approximately 1.6 times the rate of myosin light chains, and in skeletal muscle, heavy chains were synthesized at approximately 1.4 times the rate of light chains. Relative rates of degradation of muscle proteins were determined using a dual-isotope technique. In general, the soluble and myofibrillar proteins of both types of muscle had decay rates proportional to their molecular weights (larger proteins generally had higher decay rates) based on analyses utilizing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A notable exception to this general rule was myosin heavy chains, which had decay rates only slightly higher than the myosin light chains. Direct measurements on purified proteins indicated that the heavy chains of myosin were turning over at a slightly greater rate (approximately 20%) than the myosin light chains in both cardiac and skeletal muscle. The reasons for the apparent discrepancy between these measurements of myosin heavy and light chain synthesis and degradation are discussed.     

Coordinate synthesis of two myosins in wild-type and mutant nematode muscle during larval development

In this paper we examine the role of two myosins in body-wall muscle cells of the nematode Caenorhabditis elegans. Large populations of nematodes are synchronized, and the synthesis and accumulation of myosin heavy chains and total protein are followed through postmitotic larval development. Growth is exponential with time for both the wild-type N2 and the body-wall muscledefective mutant E675, with a longer doubling time for the mutant. Utilizing the electrophoretic polymorphism of the E675 myosin heavy chains, we show that distinguishable classes of heavy chains accumulate differentially throughout development. Immunochemical measurements confirm a similar result in N2. Total myosin heavy chain accumulation is also quantitatively similar for the two strains. Myosin heavy chain relative synthetic rates as determined by pulse-labeling are constant throughout development and are equivalent for the two strains. The final fraction of accumulated unc-54 to total heavy chains of approximately 0.63 equals the constant synthetic fraction of approximately 0.62.Since myosin heavy chain accumulation and relative synthesis are equivalent, we conclude that the turnover of heavy chains is also similar in N2 and E675 despite the extensive structural and functional disruption within body-wall muscle cells of the latter strain. Since the accumulated fraction of unc-54 myosin heavy chains reaches a plateau at the constant synthetic fraction, myosin accumulation In the body-wall muscle cells may be attributed to a constant ratio of synthetic rates of the two body-wall myosin species. The coordinate synthesis of two myosins in the same body-wall muscle cells is discussed.     

Skeletal muscle myofibrillar protein metabolism in heart failure: relationship to immune activation and functional capacity

Chronic heart failure is characterized by changes in skeletal muscle that contribute to physical disability. Most studies to date have investigated defects in skeletal muscle oxidative capacity. In contrast, less is known about how heart failure affects myofibrillar protein metabolism. Thus we examined the effect of heart failure on skeletal muscle myofibrillar protein metabolism, with a specific emphasis on changes in myosin heavy chain (MHC) protein content, synthesis, and isoform distribution in 10 patients with heart failure (63 +/- 3 yr) and 11 controls (70 +/- 3 yr). In addition, we examined the relationship of MHC protein metabolism to inflammatory markers and physical function. Although MHC and actin protein content did not differ between groups, MHC protein content decreased with increasing disease severity in heart failure patients (r = -0.748, P < 0.02), whereas actin protein content was not related to disease severity. No difference in MHC protein synthesis was found between groups, and MHC protein synthesis rates were not related to disease severity. There were, however, relationships between C-reactive protein and both MHC protein synthesis (r = -0.442, P = 0.05) and the ratio of MHC to mixed muscle protein synthesis (r = -0.493, P < 0.03). Heart failure patients showed reduced relative amounts of MHC I (P < 0.05) and a trend toward increased MHC IIx (P = 0.06). In regression analyses, decreased MHC protein content was related to decreased exercise capacity and muscle strength in heart failure patients. Our results demonstrate that heart failure affects both the quantity and isoform distribution of skeletal muscle MHC protein. The fact that MHC protein content was related to both exercise capacity and muscle strength further suggests that quantitative alterations in MHC protein may have functional significance.     

Phosphorylation of smooth muscle myosin heavy and light chains. Effects of phorbol dibutyrate and agonists

A number of different protein kinases phosphorylate purified heavy chains or the 20-kDa light chain of smooth muscle myosin. The physiological significance of these phosphorylation reactions has been examined in intact smooth muscle. Myosin heavy chain was slightly phosphorylated (0.08 mol of phosphate/mol) under control conditions in bovine tracheal tissue. Treatment with carbachol, isoproterenol, or phorbol 12,13-dibutyrate resulted in no significant change. In contrast, heavy chain was phosphorylated to 0.30 mol of phosphate/mol of heavy chain in tracheal smooth muscle cells in culture. This value increased significantly with ionomycin treatment. In control tissues, 9% of the light chain was monophosphorylated with 32P in the serine site phosphorylated by myosin light chain kinase. Carbachol (0.1 microM) alone resulted in contraction and 42% monophosphorylated light chain with 32P only in the serine site phosphorylated by myosin light chain kinase. Similarly, stimulation with histamine, 5-hydroxytryptamine, or KCl resulted in 32P incorporation into only the myosin light chain kinase serine site. Phorbol 12,13-dibutyrate (1 microM) alone resulted in 22% monophosphorylated light chain. However, only 25% of the 32P was in the myosin light chain kinase serine site, whereas 75% was in a serine site phosphorylated by protein kinase C. Phorbol 12,13-dibutyrate plus carbachol resulted in 27% monophosphorylated light chain; 75% of the 32P was in the myosin light chain kinase serine site, with the remainder in the protein kinase C serine site. These results indicate that phorbol esters act to increase phosphorylation of myosin light chain by protein kinase C. However, receptor-mediated stimulation or depolarization leading to tracheal smooth muscle contraction results in phosphorylation of myosin light chain by myosin light chain kinase alone.     

Role of contraction duration in inducing fast‐to‐slow contractile and metabolic protein and functional changes in engineered muscle

The role of factors such as frequency, contraction duration and active time in the adaptation to chronic low‐frequency electrical stimulation (CLFS) is widely disputed. In this study we explore the ability of contraction duration (0.6, 6, 60, and 600 sec) to induce a fast‐to‐slow shift in engineered muscle while using a stimulation frequency of 10 Hz and keeping active time constant at 60%. We found that all contraction durations induced similar slowing of time‐to‐peak tension. Despite similar increases in total myosin heavy (MHC) levels with stimulation, increasing contraction duration resulted in progressive decreases in total fast myosin. With contraction durations of 60 and 600 sec, MHC IIx levels decreased and MHC IIa levels increased. All contraction durations resulted in fast‐to‐slow shifts in TnT and TnC but increased both fast and slow TnI levels. Half‐relaxation slowed to a greater extent with contraction durations of 60 and 600 sec despite similar changes in the calcium sequestering proteins calsequestrin and parvalbumin and the calcium uptake protein SERCA. All CLFS groups resulted in greater fatigue resistance than control. Similar increases in GLUT4, mitochondrial enzymes (SDH and ATPsynthase), the fatty acid transporter CPT‐1, and the metabolic regulators PGC‐1α and MEF2 were found with all contraction durations. However, the mitochondrial enzymes cytochrome C and citrate synthase were increased to greater levels with contraction durations of 60 and 600 sec. These results demonstrate that contraction duration plays a pivotal role in dictating the level of CLFS‐induced contractile and metabolic adaptations in tissue‐engineered skeletal muscle. J. Cell. Physiol. 230: 2489–2497, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Actin and myosin contribute to mammalian mitochondrial DNA maintenance

Mitochondrial DNA maintenance and segregation are dependent on the actin cytoskeleton in budding yeast. We found two cytoskeletal proteins among six proteins tightly associated with rat liver mitochondrial DNA: non-muscle myosin heavy chain IIA and β-actin. In human cells, transient gene silencing of MYH9 (encoding non-muscle myosin heavy chain IIA), or the closely related MYH10 gene (encoding non-muscle myosin heavy chain IIB), altered the topology and increased the copy number of mitochondrial DNA; and the latter effect was enhanced when both genes were targeted simultaneously. In contrast, genetic ablation of non-muscle myosin IIB was associated with a 60% decrease in mitochondrial DNA copy number in mouse embryonic fibroblasts, compared to control cells. Gene silencing of β-actin also affected mitochondrial DNA copy number and organization. Protease-protection experiments and iodixanol gradient analysis suggest some β-actin and non-muscle myosin heavy chain IIA reside within human mitochondria and confirm that they are associated with mitochondrial DNA. Collectively, these results strongly implicate the actomyosin cytoskeleton in mammalian mitochondrial DNA maintenance.     

Two different forms of beta myosin heavy chain are expressed in human striated muscle

Summary We have found evidence for two beta-like myosin heavy chains in humans, one cardiac and one skeletal. The cDNA sequences of the cardiac beta myosin heavy chain cDNA clone pHMC3 and the skeletal beta-like myosin heavy chain cDNA clone pSMHCZ, were compared to each other. It was found that the 3 untranslated regions as well as 482 nucleotides specifying the carboxyl coding region, were 100% homologous. Further examination revealed that the skeletal clone pSMHCZ diverges from the human cardiac beta myosin heavy chain cDNA clone pHMC3 at the 5 end. We present evidence in this report which indicates that the cardiac beta myosin heavy chain mRNA is expressed in skeletal muscle tissues. The human cardiac beta myosin heavy chain cDNA clone, pHMC3, which codes for a portion of the light meromyosin section of the myosin heavy chain, was used as a probe for S1 nuclease mapping studies with RNA derived from cardiac tissue, smooth muscle and skeletal muscle tissues consisting of fast-twitch, slow-twitch and mixed fast- and slow-twitch muscle fibres. Two probes were used to examine the expression of the mRNA. One probe (406 nucleotides) constitutes the 3 untranslated region and a portion of the coding region of the beta cardiac myosin heavy chain cDNA clone, which is 100% homologous to pSMHCZ, the skeletal cDNA clone. The other constitutes the majority of the coding region (1017 nucleotides) of the cardiac clone pHMC3 in which the first 216 nucleotides from the labelled end are 100% homologous to the skeletal clone pSMHCZ. In the soleus muscle, which is rich in slow-twitch type I muscle fibres, the expression of the cardiac beta myosin heavy chain mRNA was very prominent. In gastrocnemius muscle, a mixed fibre muscle, the expression of this mRNA was detected to a lesser degree than that for the soleus muscle. In vastus lateralis and vastus medialis, which consist of predominantly type II, fast-twitch fibres, there were trace amounts of the cardiac beta myosin heavy chain mRNA. When expression of this mRNA was tested in smooth muscle tissue none could be detected.     

Oxidation of myosin heavy chain and reduction in force production in hyperthyroid rat soleus.

We tested the hypothesis that a force reduction in hyperthyroid rat soleus muscle would be associated with oxidative modification in myosin heavy chain (MHC). Daily injection of thyroid hormone [3,5,3'-triiodo-L-thyronine (T3)] for 21 days depressed isometric forces of whole soleus muscle across a range of stimulus frequencies (P < 0.01). In fiber bundles, hyperthyroidism also led to pronounced reductions (P < 0.01) in both K+ - and 4-chloro-m-cresol-induced contracture forces. The degrees of the reductions were similar between these two contractures that were induced by distinct reagents. Treatment with T3 elicited a significant decrease ( approximately 14%; P < 0.05) in the relative content of MHC contained in myofibrillar proteins. The content of carbonyl groups in myofibrillar protein extracts was elevated (P < 0.05) by approximately 50% in T3-treated muscles. Immunoblot analyses on T3-treated muscles showed a greater increase (106%; P < 0.05) of the carbonyl content in MHC than in myofibrillar protein extracts. These data suggest that in hyperthyroidism the decrease in force production of skeletal muscles may stem primarily from failure in myofibrillar protein function resulting from oxidative modification of MHC.     

Myofibrillar protein turnover. Synthesis of protein-bound 3-methylhistidine, actin, myosin heavy chain and aldolase in rat skeletal muscle in the fed and starved states.

The turnover of 3-methylhistidine (N tau-methylhistidine) and in some cases actin, myosin heavy chain and aldolase in skeletal muscle was measured in a number of experiments in growing and adult rats in the fed and overnight-starved states. In growing fed rats in three separate experiments, measurements of the methylation rate of protein-bound 3-methylhistidine by either [14C]- or [3H]-methyl-labelled S-adenosylmethionine show that 3-methylhistidine synthesis is slower than the overall rate of protein synthesis indicated by [14C]tyrosine incorporation. Values ranged from 36 to 51%. However, in one experiment with rapidly growing young fed rats, acute measurements over 1 h showed that 3-methylhistidine synthesis could be increased to the same rate as the overall rate. After overnight starvation in these rats, the steady-state synthesis rate of 3-methylhistidine was 38.8% of the overall rate. This was a similar value to that in adult non-growing rats, in which measurements of the relative labelling of 3-methylhistidine and histidine after a single injection of [14C]histidine indicated that 3-methylhistidine synthesis was 37% of the overall rate in the fed or overnight-starved state. According to measurements of actin, myosin heavy-chain and aldolase synthesis in the over-night-starved state with young rats, with a variety of precursors, slow turnover of 3-methylhistidine results from the specific slow turnover of actin, since turnover rates of myosin heavy chain, mixed protein and aldolase were 2.5, 3 and 3.4 times faster respectively. However, in the fed state synthesis rates of actin were increased disproportionately to give similar rates for all proteins. These results show that (a) 3-methylhistidine turnover in muscle is less than half the overall rate in both young and adult rats, (b) slow 3-methylhistidine turnover reflects the specifically slow turnover of actin compared with myosin heavy chain and other muscle proteins, and (c) during growth the synthesis rate of actin is particularly sensitive to the nutritional state and can be increased to a similar rate to that of other proteins.     

Age-dependent regulation of skeletal muscle mitochondria by the thrombospondin-1 receptor CD47

CD47, a receptor for thrombospondin-1, limits two important regulatory axes: nitric oxide-cGMP signaling and cAMP signaling, both of which can promote mitochondrial biogenesis. Electron microscopy revealed increased mitochondrial densities in skeletal muscle from both CD47 null and thrombospondin-1 null mice. We further assessed the mitochondria status of CD47-null vs WT mice. Quantitative RT-PCR of RNA extracted from tissues of 3 month old mice revealed dramatically elevated expression of mRNAs encoding mitochondrial proteins and PGC-1α in both fast and slow-twitch skeletal muscle from CD47-null mice, but modest to no elevation in other tissues. These observations were confirmed by Western blotting of mitochondrial proteins. Relative amounts of electron transport enzymes and ATP/O₂ ratios of isolated mitochondria were not different between mitochondria from CD47-null and WT cells. Young CD47-null mice displayed enhanced treadmill endurance relative to WTs and CD47-null gastrocnemius had undergone fiber type switching to a slow-twitch pattern of myoglobin and myosin heavy chain expression. In 12 month old mice, both skeletal muscle mitochondrial volume density and endurance had decreased to wild type levels. Expression of myosin heavy chain isoforms and myoglobin also reverted to a fast twitch pattern in gastrocnemius. Both CD47 and TSP1 null mice are leaner than WTs, use less oxygen and produce less heat than WT mice. CD47-null cells produce substantially less reactive oxygen species than WT cells. These data indicate that loss of signaling from the TSP1-CD47 system promotes accumulation of normally functioning mitochondria in a tissue-specific and age-dependent fashion leading to enhanced physical performance, lower reactive oxygen species production and more efficient metabolism.     

Hypoxia differentially regulates muscle oxidative fiber type and metabolism in a HIF-1α-dependent manner

Loss of skeletal muscle oxidative fiber types and mitochondrial capacity is a hallmark of chronic obstructive pulmonary disease and chronic heart failure. Based on in vivo human and animal studies, tissue hypoxia has been hypothesized as determinant, but the direct effect of hypoxia on muscle oxidative phenotype remains to be established. Hence, we determined the effect of hypoxia on in vitro cultured muscle cells, including gene and protein expression levels of mitochondrial components, myosin isoforms (reflecting slow-oxidative versus fast-glycolytic fibers), and the involvement of the regulatory PPAR/PGC-1α pathway. We found that hypoxia inhibits the PPAR/PGC-1α pathway and the expression of mitochondrial components through HIF-1α. However, in contrast to our hypothesis, hypoxia stimulated the expression of slow-oxidative type I myosin via HIF-1α. Collectively, this study shows that hypoxia differentially regulates contractile and metabolic components of muscle oxidative phenotype in a HIF-1α-dependent manner.     

Exercise training reverses downregulation of HSP70 and antioxidant enzymes in porcine skeletal muscle after chronic coronary artery occlusion

Oxidative stress is associated with muscle fatigue and weakness in skeletal muscle of ischemic heart disease patients. Recently, it was found that endurance training elevates protective heat shock proteins (HSPs) and antioxidant enzymes in skeletal muscle in healthy subjects and antioxidant enzymes in heart failure patients. However, it is unknown whether coronary ischemia and mild infarct without heart failure contributes to impairment of stress proteins and whether exercise training reverses those effects. We tested the hypothesis that exercise training would reverse alterations in muscle TNF-alpha, oxidative stress, HSP70, SOD (Mn-SOD, Cu,Zn-SOD), glutathione peroxidase (GPX), and catalase (CAT) due to chronic coronary occlusion of the left circumflex (CCO). Yucatan swine were divided into three groups (n = 6 each): sedentary with CCO (SCO); 12 wk of treadmill exercise training following CCO (ECO); and sham surgery controls (sham). Forelimb muscle mass-to-body mass ratio decreased by 27% with SCO but recovered with ECO. Exercise training reduced muscle TNF-alpha and oxidative stress (4-hydroxynonenal adducts) caused by CCO. HSP70 levels decreased with CCO (-45%), but were higher with exercise training (+348%). Mn-SOD activity, Mn-SOD protein expression, and Cu,Zn-SOD activity levels were higher in ECO than SCO by 72, 82, and 112%, respectively. GPX activity was 177% greater in ECO than in SCO. CAT trended higher (P = 0.059) in ECO compared with SCO. These data indicate that exercise training following onset of coronary artery occlusion results in recovery of critical stress proteins and reduces oxidative stress.     

Resistance exercise load does not determine training-mediated hypertrophic gains in young men

We have reported that the acute postexercise increases in muscle protein synthesis rates, with differing nutritional support, are predictive of longer-term training-induced muscle hypertrophy. Here, we aimed to test whether the same was true with acute exercise-mediated changes in muscle protein synthesis. Eighteen men (21 ± 1 yr, 22.6 ± 2.1 kg/m(2); means ± SE) had their legs randomly assigned to two of three training conditions that differed in contraction intensity [% of maximal strength (1 repetition maximum)] or contraction volume (1 or 3 sets of repetitions): 30%-3, 80%-1, and 80%-3. Subjects trained each leg with their assigned regime for a period of 10 wk, 3 times/wk. We made pre- and posttraining measures of strength, muscle volume by magnetic resonance (MR) scans, as well as pre- and posttraining biopsies of the vastus lateralis, and a single postexercise (1 h) biopsy following the first bout of exercise, to measure signaling proteins. Training-induced increases in MR-measured muscle volume were significant (P < 0.01), with no difference between groups: 30%-3 = 6.8 ± 1.8%, 80%-1 = 3.2 ± 0.8%, and 80%-3= 7.2 ± 1.9%, P = 0.18. Isotonic maximal strength gains were not different between 80%-1 and 80%-3, but were greater than 30%-3 (P = 0.04), whereas training-induced isometric strength gains were significant but not different between conditions (P = 0.92). Biopsies taken 1 h following the initial resistance exercise bout showed increased phosphorylation (P < 0.05) of p70S6K only in the 80%-1 and 80%-3 conditions. There was no correlation between phosphorylation of any signaling protein and hypertrophy. In accordance with our previous acute measurements of muscle protein synthetic rates a lower load lifted to failure resulted in similar hypertrophy as a heavy load lifted to failure.