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.     

Effect of heart failure on the regulation of skeletal muscle protein synthesis,breakdown, and apoptosis

Heart failure is often characterized by skeletal muscle atrophy. The mechanisms underlying muscle wasting, however, are not fully understood. We studied 30 Dahl salt-sensitive rats (10 male, 20 female) fed either a high-salt (HS; n = 15) or a low-salt (LS; n = 15) diet. This strain develops cardiac hypertrophy and failure when fed a HS diet. LS controls were matched to HS rats for gender and duration of diet. Body mass, food intake, and muscle mass and composition were measured. Skeletal muscle protein synthesis was measured by isotope dilution. An additional group of 27 rats (HS, n = 16; LS; n = 11) were assessed for expression of genes regulating protein breakdown and apoptosis. Gastrocnemius and plantaris muscles weighed less (16 and 22%, respectively) in HS than in LS rats (P < 0.01). No differences in soleus or tibialis anterior weights were found. Differences in muscle mass were abolished after data were expressed relative to body size, because HS rats tended (P = 0.094) to weigh less. Lower body mass in HS rats was related to a 16% reduction (P < 0.01) in food intake. No differences in muscle protein or DNA content, the protein-to-DNA ratio, or muscle protein synthesis were found. Finally, no differences in skeletal muscle gene expression were found to suggest increased protein breakdown or apoptosis in HS rats. Our results suggest that muscle wasting in this model of heart failure is not associated with alterations in skeletal muscle metabolism. Instead, muscle atrophy was related to reduced body weight secondary to decreased food intake. These findings argue against the notion that heart failure is characterized by a skeletal muscle myopathy that predisposes to atrophy.     

β-Adrenergic receptor blockade blunts postexercise skeletal muscle mitochondrial protein synthesis rates in humans

β-Adrenergic receptor (AR) signaling is a regulator of skeletal muscle protein synthesis and mitochondrial biogenesis in mice. We hypothesized that β-AR blockade blunts postexercise skeletal muscle mitochondrial protein synthesis rates in adult humans. Six healthy men (mean ± SD: 26 ± 6 yr old, 39.9 ± 4.9 ml·kg(-1)·min(-1) peak O(2) uptake, 26.7 ± 2.0 kg/m(2) body mass index) performed 1 h of stationary cycle ergometer exercise (60% peak O(2) uptake) during 1) β-AR blockade (intravenous propranolol) and 2) administration of saline (control). Skeletal muscle mitochondrial, myofibrillar, and sarcoplasmic protein synthesis rates were assessed using [(2)H(5)]phenylalanine incorporation into skeletal muscle proteins after exercise. The mRNA content of signals for mitochondrial biogenesis was determined using real-time PCR. β-AR blockade decreased mitochondrial (from 0.217 ± 0.076 to 0.135 ± 0.031%/h, P < 0.05), but not myofibrillar or sarcoplasmic, protein synthesis rates. Peroxisome proliferator-activated receptor-γ coactivator-1α mRNA was increased ～2.5-fold (P < 0.05) at 5 h compared with 1 h postexercise but was not influenced by β-AR blockade. We conclude that decreased β-AR signaling during cycling can blunt the postexercise increase in mitochondrial protein synthesis rates without affecting mRNA content.     

Detection of a specific isoform of alpha-actinin with antisera directed against dystrophin

We have characterized a protein immunologically related to dystrophin, the protein product of the Duchenne muscular dystrophy gene. We identify this related protein as a fast-twitch glycolytic isoform (mouse extensor digitorum longus-specific) of myofibrillar alpha-actinin. This specific isoform of alpha-actinin exhibits a more restricted pattern of expression in skeletal muscle than fast-twitch-specific isoforms of both myosin and Ca2+-ATPase. Our results provide evidence that dystrophin and myofibrillar alpha-actinin are related proteins, reinforcing the previous data concerning the sequence homologies noted between nonmuscle cytoskeletal alpha-actinin and dystrophin. In addition, we describe the first antisera directed against a specific myofibrillar skeletal muscle isoform of alpha-actinin.     

Myofibrillar and collagen protein synthesis in human skeletal muscle in young men after maximal shortening and lengthening contractions

We aimed to determine whether there were differences in the extent and time course of skeletal muscle myofibrillar protein synthesis (MPS) and muscle collagen protein synthesis (CPS) in human skeletal muscle in an 8.5-h period after bouts of maximal muscle shortening (SC; average peak torque = 225 +/- 7 N.m, means +/- SE) or lengthening contractions (LC; average peak torque = 299 +/- 18 N.m) with equivalent work performed in each mode. Eight healthy young men (21.9 +/- 0.6 yr, body mass index 24.9 +/- 1.3 kg/m2) performed 6 sets of 10 maximal unilateral LC of the knee extensors on an isokinetic dynamometer. With the contralateral leg, they then performed 6 sets of maximal unilateral SC with work matched to the total work performed during LC (10.9 +/- 0.7 vs. 10.9 +/- 0.8 kJ, P = 0.83). After exercise, the participants consumed small intermittent meals to provide 0.1 g.kg(-1).h(-1) of protein and carbohydrate. Prior exercise elevated MPS above rest in both conditions, but there was a more rapid rise after LC (P < 0.01). The increases (P < 0.001) in CPS above rest were identical for both SC and LC and likely represent a remodeling of the myofibrillar basement membrane. Therefore, a more rapid rise in MPS after maximal LC could translate into greater protein accretion and muscle hypertrophy during chronic resistance training utilizing maximal LC.     

Oxidative stress of myosin contributes to skeletal muscle dysfunction in rats with chronic heart failure

Intrinsic muscle abnormalities affecting skeletal muscle are often reported during chronic heart failure (CHF). Because myosin is the molecular motor of force generation, we sought to determine whether its dysfunction contributes to skeletal muscle weakness in CHF and, if so, to identify the underlying causative factors. Severe CHF was induced in rats by aortic stenosis. In diaphragm and soleus muscles, we investigated in vitro mechanical performance, myosin-based actin filament motility, myosin heavy (MHC) and light (MLC) chain isoform compositions, MLC integrity, caspase-3 activation, and oxidative damage. Diaphragm and soleus muscles from CHF exhibited depressed mechanical performance. Myosin sliding velocities were 16 and 20% slower in CHF than in sham in diaphragm (1.9 +/- 0.1 vs. 1.6 +/- 0.1 microm/s) and soleus (0.6 +/- 0.1 vs. 0.5 +/- 0.1 microm/s), respectively (each P < 0.05). The ratio of slow-to-fast myosin isoform did not differ between sham and CHF. Immunoblots with anti-MLC antibodies did not detect the presence of protein fragments, and no activation of caspase-3 was evidenced. Immunolabeling revealed oxidative damage in CHF muscles, and MHC was the main oxidized protein. Lipid peroxidation and expression of oxidized MHC were significantly higher in CHF than in shams. In vitro myosin exposure to increasing ONOO(-) concentrations was associated with an increasing amount of oxidized MHC and a reduced myosin velocity. These data provide experimental evidence that intrinsic myosin dysfunction occurs in CHF and may be related to oxidative damage to myosin.     

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.     

Aging, muscle fiber type, and contractile function in sprint-trained athletes.

Biopsy samples were taken from the vastus lateralis of 18- to 84-yr-old male sprinters (n = 91). Fiber-type distribution, cross-sectional area, and myosin heavy chain (MHC) isoform content were identified using ATPase histochemistry and SDS-PAGE. Specific tension and maximum shortening velocity (V(o)) were determined in 144 single skinned fibers from younger (18-33 yr, n = 8) and older (53-77 yr, n = 9) runners. Force-time characteristics of the knee extensors were determined by using isometric contraction. The cross-sectional area of type I fibers was unchanged with age, whereas that of type II fibers was reduced (P < 0.001). With age there was an increased MHC I (P < 0.01) and reduced MHC IIx isoform content (P < 0.05) but no differences in MHC IIa. Specific tension of type I and IIa MHC fibers did not differ between younger and older subjects. V(o) of fibers expressing type I MHC was lower (P < 0.05) in older than in younger subjects, but there was no difference in V(o) of type IIa MHC fibers. An aging-related decline of maximal isometric force (P < 0.001) and normalized rate of force development (P < 0.05) of knee extensors was observed. Normalized rate of force development was positively associated with MHC II (P < 0.05). The sprint-trained athletes experienced the typical aging-related reduction in the size of fast fibers, a shift toward a slower MHC isoform profile, and a lower V(o) of type I MHC fibers, which played a role in the decline in explosive force production. However, the muscle characteristics were preserved at a high level in the oldest runners, underlining the favorable impact of sprint exercise on aging muscle.     

Human vastus lateralis and soleus muscles display divergent cellular contractile properties

The purpose of this study was to investigate potential differences in single-fiber contractile physiology of fibers with the same myosin heavy chain isoform (MHC I and MHC IIa) originating from different muscles. Vastus lateralis (VL) and soleus biopsies were obtained from 27 recreationally active females (31 +/- 1 yr, 59 +/- 1 kg). A total of 943 single fibers (MHC I = 562; MHC IIa = 301) were isolated and examined for diameter, peak tension (Po), shortening velocity (Vo), and power. The soleus had larger (P < 0.05) fibers (MHC I +18%; MHC IIa +19%), higher MHC I Vo (+13%), and higher MHC I Po (+18%) compared with fibers from the VL. In contrast, fibers from the VL had higher (P < 0.05) specific tension (MHC I +18%; MHC IIa +20%), and MHC I normalized power (+25%) compared with the soleus. There was a trend for MHC IIa soleus fibers to have higher Vo [MHC IIa +13% (P = 0.058)], whereas VL MHC IIa fibers showed a trend for higher normalized power compared with soleus fibers [MHC IIa +33% (P = 0.079)]. No differences in absolute power were detected between muscles. These data highlight muscle-specific differences in single-fiber contractile function that should serve as a scientific basis for consideration when extending observations of skeletal muscle tissue from one muscle of interest to other muscles of origin. This is important when examining skeletal muscle adaptation to physical states such as aging, unloading, and training.     

Troponin C isoform composition determines differences in Sr(2+)-activation characteristics between rat diaphragm fibers

Single fibers of rat diaphragm containing different naturally occurring combinations of myofibrillar protein isoforms were used to evaluate the contribution of troponin C (TnC) isoforms to fiber type-related differences with respect to sensitivity to Sr²⁺ of the contractile system. Mechanically skinned fibers were studied for their isometric force vs. Sr²⁺ concentration ([Sr²⁺]) relationships and then analyzed electrophoretically for myofibrillar protein isoform composition. Our data demonstrate that fiber-type differences in Sr²⁺ dependence of contractile activation processes are primarily determined by the TnC isoform composition, with the slow isoform conferring on average a sevenfold greater sensitivity to Sr²⁺ than the fast isoform. Moreover, the ratio of TnC isoforms determined functionally from the force-pSr (–log₁₀ [Sr²⁺]) curves is tightly (r² = 0.97) positively correlated with that estimated electrophoretically. Together, these results validate the use of Sr²⁺ activation characteristics to distinguish fibers containing different proportions of fast and slow TnC isoforms and to study the mechanisms by which divalent cations activate the contractile apparatus. We also found that the functionally and electrophoretically determined ratios of TnC isoforms present in a fiber display similar sigmoidal relationships with the ratio of myosin heavy chain (MHC) isoform types expressed. These relationships 1) offer further insight in the functional and molecular expression of TnC in relation to the molecular expression of MHC isoform types and 2) may provide the basis for predicting sensitivity to Sr²⁺, TnC, and MHC isoforms in pure and hybrid skeletal muscle fibers. muscle contraction; skeletal muscle; myofibrillar proteins; single fiber; sensitivity to strontium; sensitivity to calcium     

Chronic heart failure reduces Akt phosphorylation in human skeletal muscle: relationship to muscle size and function

Patients with chronic heart failure (HF) frequently lose muscle mass and function during the course of the disease. A reduction in anabolic stimuli to the muscle has been put forth as a potential mechanism underlying these alterations. The present study examined the hypothesis that skeletal muscle tissue from HF patients would show reduced IGF-1 expression and phosphorylation of signaling molecules downstream of receptor activation. To isolate the unique effect of HF on these variables, we limited the confounding effects of muscle disuse and/or acute disease exacerbation by recruiting controls (n = 11) with similar physical activity levels as HF patients (n = 11) and by testing patients at least 6 mo following any bouts of disease exacerbation/hospitalization. IGF-1 expression in skeletal muscle was similar between patients and controls. Despite this, HF patients were characterized by reduced levels of phospho-Akt/Akt (S473; -43%; P < 0.05), whereas no differences were found in total Akt protein content or phospho- or total protein content of mammalian target of rapamycin (mTOR; S2448), glycogen synthase kinase-3β (GSK-3β; S9), eukaryotic translation initiation factor 4E binding protein-1 (eIF4E-BP; T37/46), p70 ribosomal S6 kinase (p70 S6K; T389), or eIF2Bε (S540). Reduced phospho-Akt/Akt levels and phospho-mTOR/mTOR were related to decreased skeletal muscle myosin protein content (r = 0.602; P < 0.02) and knee extensor isometric torque (r = 0.550; P < 0.05), respectively. Because patients and controls were similar for age, muscle mass, and physical activity, we ascribe the observed alterations in Akt phosphorylation and its relationship to myosin protein content to the unique effects of the HF syndrome.     

Neuromuscular electrical stimulation increases muscle protein synthesis in elderly type 2 diabetic men

Physical activity is required to attenuate the loss of skeletal muscle mass with aging. Short periods of muscle disuse, due to sickness or hospitalization, reduce muscle protein synthesis rates, resulting in rapid muscle loss. The present study investigates the capacity of neuromuscular electrical stimulation (NMES) to increase in vivo skeletal muscle protein synthesis rates in older type 2 diabetes patients. Six elderly type 2 diabetic men (70 ± 2 yr) were subjected to 60 min of one-legged NMES. Continuous infusions with l-[ring-(13)C(6)]phenylalanine were applied, with blood and muscle samples being collected regularly to assess muscle protein synthesis rates in both the stimulated (STIM) and nonstimulated control (CON) leg during 4 h of recovery after NMES. Furthermore, mRNA expression of key genes implicated in the regulation of muscle mass were measured over time in the STIM and CON leg. Muscle protein synthesis rates were greater in the STIM compared with the CON leg during recovery from NMES (0.057 ± 0.008 vs. 0.045 ± 0.008%/h, respectively, P < 0.01). Skeletal muscle myostatin mRNA expression in the STIM leg tended to increase immediately following NMES compared with the CON leg (1.63- vs. 1.00-fold, respectively, P = 0.07) but strongly declined after 2 and 4 h of recovery in the STIM leg only. In conclusion, this is the first study to show that NMES directly stimulates skeletal muscle protein synthesis rates in vivo in humans. NMES likely represents an effective interventional strategy to attenuate muscle loss in elderly individuals during bed rest and/or in other disuse states.     

Stimulation of myofibrillar synthesis by exercise is mediated by more efficient translation of mRNA.

Resistance exercises stimulate protein synthesis in human muscle, but the roles of changes in mRNA concentrations and changes in the efficiency of mRNA translation have not been defined. The present study was done to determine whether resistance exercise affects concentrations of total RNA, total mRNA, actin mRNA, or myosin heavy-chain mRNA (total and isoform specific). Eight subjects, 62-75 yr old, performed unilateral knee extensions at 80% of their one-repetition-maximum capacity on days 1, 3, and 6 of the study. On day 7, biopsies of exercised and nonexercised vastus lateralis muscles were obtained. Myofibrillar synthesis was determined by stable- isotope incorporation, and mRNA concentrations were determined by membrane hybridization and PCR-based methods. The exercise stimulated myofibrillar synthesis [30 +/- 6 (SE)%] without affecting RNA or mRNA concentrations. The effect of exercise on protein synthesis in individual subjects did not correlate with the effect on total RNA and mRNA concentrations. These data suggest that the stimulation of myofibrillar synthesis by resistance exercise is mediated by more efficient translation of mRNA.     

Near-maximal fractional oxygen extraction by active skeletal muscle in patients with chronic heart failure.

Systemic oxygen uptake and deep femoral vein oxygen content were determined at peak exercise in 53 patients with chronic heart failure with impaired systolic function (mean left ventricular ejection fraction 0.18; n = 41) or preserved systolic function (mean left ventricular ejection fraction 0.70; n = 12) and in 6 age-matched sedentary normal subjects. At peak exercise, deep femoral vein oxygen content in heart failure patients with impaired systolic function and preserved systolic function were similar, both significantly lower than that of normal subjects (2.5 +/- 0.1, 2.9 +/- 0.2, and 5.0 +/- 0.1 ml/100 ml, respectively; P < 0.05). Deep femoral venous oxygen content was lower in patients with the greater impairment of aerobic capacity, regardless of the underlying systolic function (r = 0.72, P < 0.01). Fractional oxygen extraction in the skeletal muscle at peak exercise is enhanced in patients with chronic heart failure when compared with normal subjects, in proportion to the degree of aerobic impairment.     

The relationship between human skeletal muscle pyruvate dehydrogenase phosphatase activity and muscle aerobic capacity

Pyruvate dehydrogenase (PDH) is a mitochondrial enzyme responsible for regulating the conversion of pyruvate to acetyl-CoA for use in the tricarboxylic acid cycle. PDH is regulated through phosphorylation and inactivation by PDH kinase (PDK) and dephosphorylation and activation by PDH phosphatase (PDP). The effect of endurance training on PDK in humans has been investigated; however, to date no study has examined the effect of endurance training on PDP in humans. Therefore, the purpose of this study was to examine differences in PDP activity and PDP1 protein content in human skeletal muscle across a range of muscle aerobic capacities. This association is important as higher PDP activity and protein content will allow for increased activation of PDH, and carbohydrate oxidation. The main findings of this study were that 1) PDP activity (r(2) = 0.399, P = 0.001) and PDP1 protein expression (r(2) = 0.153, P = 0.039) were positively correlated with citrate synthase (CS) activity as a marker for muscle aerobic capacity; 2) E1α (r(2) = 0.310, P = 0.002) and PDK2 protein (r(2) = 0.229, P =0.012) are positively correlated with muscle CS activity; and 3) although it is the most abundant isoform, PDP1 protein content only explained ～ 18% of the variance in PDP activity (r(2) = 0.184, P = 0.033). In addition, PDP1 in combination with E1α explained ～ 38% of the variance in PDP activity (r(2) = 0.383, P = 0.005), suggesting that there may be alternative regulatory mechanisms of this enzyme other than protein content. These data suggest that with higher muscle aerobic capacity (CS activity) there is a greater capacity for carbohydrate oxidation (E1α), in concert with higher potential for PDH activation (PDP activity).     

Biochemical adaptation of cardiac and skeletal muscle to physical activity.

1. Female Wistar rats were randomly assigned to control (C) or exercising (T) groups and subsequently portioned into 1, 3, 5 and 10 day T and C groups. The T groups completed a progressive endurance running program. Biochemical indices of adaptation were measured in cardiac muscle and in plantaris and soleus muscles of C and T animals after their last exercise bout. 2. In cardiac muscle, myofibrillar ATPase activity was significantly elevated in the 3T (0.241 +/- 0.031) and 5T (0.242 +/- 0.013) groups (P less than or equal to 0.05) compared to their respective controls (3C = 0.187 +/- 0.015 and 5C = 0.190 +/- 0.007). 3. After 10 days of training cardiac myofibrillar ATPase activity was elevated by 17% but this was not significant (P greater than or equal to 0.05). 4. No changes in myofibrillar ATPase activity were seen in skeletal muscle (P greater than or equal to 0.05), however, hexokinase activity progressively increased and was significantly elevated in the 3T, 5T and 10T soleus and plantaris muscles of rats over controls (P less than or equal to 0.05). 5. Minimal nonsignificant changes were noted in the hexokinase activity of the hearts of all T groups (P greater than or equal to 0.05). 6. These results indicate that metabolic adaptation of the heart and skeletal muscles takes place after as little as three training sessions. 7. Although the adaptation of the skeletal muscles continually progresses, the adaptation of the heart appears to be transitory.(ABSTRACT TRUNCATED AT 250 WORDS)