Myosin Turnover Research Articles

Endurance Training: Volume-Dependent Adaptational Changes in Myosin

The purpose of this study was to find the effect of different endurance training volumes on the composition and turnover of myosin. Sixteen-week-old male rats of the Wistar strain were divided into three different volume-based training groups. Changes in myosin heavy chain (MyHC), myosin light chain (MyLC) isoforms' composition, their synthesis rate, as well as myosin binding C-protein synthesis rate, and muscle protein degradation rate were measured. In slow-twitch (ST) soleus (Sol) muscle MyHC I isoform relative content increased and MyHC IIa isoform decreased during excessive increase in the volume of endurance training (ET). In plantaris (Pla) muscle excessive increase in ET volume decreased MyHC I and IIb isoforms, and increased MyHC IIa and IId relative content. In extensor digitorum longus (EDL) muscle the relative content of MyHC IId isoform increased during ET, but excessive increase in training volume decreased it. In Pla muscle the relative content of MyLC 1 (slow) isoform decreased during ET, but excessive increase in ET volume decreased the relative content of MyLC 3 (fast) isoform in both fast-twitch (FT) muscles. Decrease in MyHC and myosin binding C-protein synthesis rate in Pla muscle had significant correlation with ET volume (r = - 0.537, p < 0.05 and r = - 0.727, p < 0.001 subsequently). MyHC I and IIb isoforms and MyLC 3 (fast) isoform in Pla muscle and MyHC IIb, IId and MyLC 3 (fast) isoforms in EDL muscle are the most sensitive to the increase in ET volume. Excessive increase in ET volume leads to a decrease in physical working capacity. The degradation of muscle protein increased during ET in all groups.

Control of cross-bridge cycling by myosin light chain phosphorylation in mammalian smooth muscle.

This review focuses on experiments in which the single turnover of myosin-bound ADP is used to characterize the regulation of the cross-bridge cycle by myosin light chain phosphorylation in mammalian smooth muscle. Under isometric conditions, at rest, when the myosin light chain is not phosphorylated, myosin cycles very slowly (about 0.004 s-1), while phosphorylation of the light chain results in a 50-fold increase in cycling rate of 0.2 s-1. Experiments consistently show that some myosin does not increase its cycling rate although its light chain is phosphorylated. Studies at low levels of myosin light chain phosphorylation show that phosphorylation also induces an increase in the cycling rate of unphosphorylated myosin. The fast cycling phosphorylated myosin is the main determinant of suprabasal myosin ATPase activity, while the cycling rate of cooperatively activated unphosphorylated myosin is slow and appears to depend on the extent of phosphorylation of the entire thick filament. Single turnover experiments measuring the rate of phosphorylation and dephosphorylation of myosin light chain show that the turnover of light chain phosphate can be very rapid (0.3-0.4 s-1) at suprabasal calcium concentrations. The expected effect of such a rapid turnover of light chain phosphorylation on the turnover of myosin-bound ADP is not observed. The effects of low levels of myosin light chain phosphorylation on the single turnover of myosin suggest that the same small pool of myosin remains phosphorylated for relatively long periods of time rather than the entire pool of myosin spending a small fraction of its cycle time in the phosphorylated state.

Increased K+ inhibits spontaneous contractions reduces myosin accumulation in cultured chick myotubes.

Increasing the K+ from 5.4 mM to 12 mM in the culture medium of developing chick myotubes causes an immediate cessation of spontaneous contractions and leads to an inhibition of myosin accumulation. The synthesis of myosin continues at the same rate in 12 mM K+ as in 5.4 mM K+ as measured by [3H]leucine incorporation into myosin corrected for differences in pool specific activity. Total protein synthesis and total protein accumulation are unaffected by growth in 12 mM K+. In addition, growth in 12 mM K+ did not alter the type of myosin heavy-chain isoform expression nor did it alter the pattern of myosin light-chain synthesis. However, the rate of myosin turnover increased threefold in cultures grown in 12 mM K+ compared to cultures grown in 5.4 mM K+, while total protein turnover was only marginally increased. We conclude that suppressed electrical or contractile activity of myotubes leads to an increased rate of myofibrillar protein turnover and that spontaneous mechanical and or electrical activity is required for continued myotube maturation in culture.

Altered protein synthesis and creatine kinase in breast muscle cell cultures from dystrophic chick embryos: Corrective influence of leupeptin, pepstatin and diphenylhydantoin

The total protein synthesis (TPS), myosin synthesis (MS) and creatine kinase (CK) levels in muscle cell cultures obtained from 400 normal (strain 454) and 400 dystrophic chick embryos (strain 455) were investigated. The cultures were obtained from breast muscles of 12 day chick embryos by dissociation in 0.25% trypsin, preplating and plating of 5 × 10 5 floating cells on gelatin coated dishes in Minimal Essential Medium, 10% horse serum and 2% chick embryo extract. After 6 days, when electron-microscopic studies demonstrated good muscle differentiation, cell cultures were labeled with [ 3H]leucine. TPS and MS, respectively, showed 85% and 65% increases in breast muscle cell cultures from dystrophic chick embryos. The half-life times for total protein and myosin from dystrophics were 19 and 32 hr, respectively, as compared with 36 and 48 hr from controls. Noncollagen protein content (NCP) showed 27% decrease in postfusion stage (12 days) of cell cultures from dystrophics. The CK level showed 30% lower values in the cells from dystrophics but 50% higher values in their culture medium. The addition of leupeptin plus pepstatin (50 μg/ml) to these cultures restored NCP content, total protein and myosin turnover to normal values and significantly increased TPS and MS. The addition of diphenylhydantoin (DPH) (20 μg/ml) to cell cultures from dystrophics did not change the NCP content nor the turnover for total protein and myosin but significantly increased TPS, MS and CK while medium CK significantly decreased. The addition of leupeptin plus pepstatin or DPH to muscle cell cultures from normal chick embryos also significantly stimulated TPS and MS.

Myosin turnover in cultured muscle fibers relaxed by tetrodotoxin

It is possible to maintain chick embryo muscle fibers in culture for several weeks. During this time the fibers mature and undergo spontaneous contractions. Contractions may be abolished by tetrodotoxin. When normal and tetrodotoxin (TTX) cultures are compared for myosin accumulation it is found that the relaxed or tetrodotoxin-treated cultures fail to accumulate myosin after about 4 days. Rates of myosin synthesis in normal and relaxed cultures are, however, identical. Failure to accumulate myosin in relaxed cultures is associated with a 30–40% increase in the rate of turnover of myosin heavy chains compared with normal, contracting cultures. It is suggested that one basis for hypotrophy is to be found in a post-translational mechanism for regulating myosin heavy chain (MHC) turnover.

Coordinated synthesis and degradation of actin and myosin in a variety of myogenic and non-myogenic cells

The turnover of myosin and actin in both muscle and non-muscle cells in culture was investigated. By the double-label criterion, myosin and actin were coordinately synthesized and degraded in replicating, mononucleated fibroblasts, chondrocytes, BUdR-suppressed myogenic cells, and in post-mitotic, multinucleated myotubes. Myosin and actin were among the most stable proteins in each cell type. In single label ‘pulse-chase’ experiments, the half-lives of myosin and actin in all replicating, mononucleated cells were 2.5–3 days; in myotubes, however, they were approx. 6 days. Myosin and actin labelled in replicating presumptive myoblasts and chased until the cells ceased replicating and fused into multinucleated myotubes retained the degradation rate of 3 days; this differed from Jhe rate of 6 days shown for myosin and actin newly-synthesized in post-mitotic myotubes. The type of myosin synthesized in the mother presumptive myoblast, then, is transmitted to the postmitotic daughters. This myosin, however, is more rapidly degraded than the definitive myosin that is synthesized in the myotube.

Role of creatine in the regulation of cardiac protein synthesis.

The observation that increased muscular activity leads to muscle hypertrophy is well known, but identification of the biochemical and physiological mechanisms by which this occurs remains an important problem. Experiments have been described (5, 6) which suggest that creatine, an end product of contraction, is involved in the control of contractile protein synthesis in differentiating skeletal muscle cells and may be the chemical signal coupling increased muscular activity and the increased muscular mass. During contraction, the creatine concentration in muscle transiently increases as creatine phosphate is hydrolyzed to regenerate ATP. In isometric contraction in skeletal muscle for example, Edwards and colleagues (3) have found that nearly all of the creatine phosphate is hydrolyzed. In this case, the creatine concentration is increased about twofold, and it is this transient change in creatine concentration which is postulated to lead to increased contractile protein synthesis. If creatine is found in several intracellular compartments, as suggested by Lee and Vissher (7), local changes in concentration may be greater then twofold. A specific effect on contractile protein synthesis seems reasonable in light of the work of Rabinowitz (13) and of Page et al. (11), among others, showing disproportionate accumulation of myofibrillar and mitochondrial proteins in response to work-induced hypertrophy and thyroxin-stimulated growth. Previous experiments (5, 6) have shown that skeletal muscles cells which have differentiated in vitro or in vivo synthesize myosin heavy-chain and actin, the major myofibrillar polypeptides, faster when supplied creatine in vitro. The stimulation is specific for contractile protein synthesis since neither the rate of myosin turnover nor the rates of synthesis of noncontractile protein and DNA are affected by creatine. The experiments reported in this communication were undertaken to test whether creatine selectively stimulates contractile protein synthesis in heart as it does in skeletal muscle.

Metabolism of 3-methylhistidine in man

The metabolism of L-3-methylhistidine was studied in man using intravenously administered ( 14C)3-methylhistidine. Analysis for expired 14CO 2 for periods up to 8 hr following a single intravenous injection revealed no radioactivity, indicating that this compound is not oxidized in man. Analysis of urine samples for total radioactivity showed that 75% of the administered dose was excreted in 24 hr and 95% in 48hr. Ion-exchange chromatography of urine samples with monitoring of the column eluate by a flow liquid-scintillation technique showed the presence of only two radioactive peaks. The time taken to elute these peaks was compatible with the major excretory component (95.5%) being ( 14C)3-methylhistidine, accompanied by a small amount (4.5%) in the form of N-acetyl-( 14C)3-methylhistidine. The plasma disappearance curves of ( 14C)3-methylhistidine suggested a half-life of approximately 130 min. The inability to oxidize 3-methylhistidine and its quantitative excretion as the original compound as well as its N-acetyl derivative is similar to its metabolic fate in the rat and therefore suggests that 3-methylhistidine excretion may provide a reliable measure of actin and myosin turnover in the whole animal or in human subjects.

In Vitro Studies of Regeneration and Innervation of Muscle from Dystrophic (dy2J) Mutant Mice

Publisher Summary This chapter focuses on the in vitro studies of regeneration and innervation of muscle from dystrophic (dy 2 j) mutant mice. In the present study, a modified neuromuscular culture model is utilized in which adult, instead of the usual embryonic, muscle tissue is coupled with fetal spinal cord explants. There is a bewildering array of evidence of deranged metabolism in dystrophic mice. Creatinine balance is abnormal, cholesterol and total lipid levels are increased, distribution of lipoproteins and glycoproteins, deranged turnover of myosin increased, and levels of activity of many enzymes altered. This study was undertaken to reevaluate earlier concepts of the primary causes of muscular dystrophy. The experimental setup relied essentially on a tissue culture system in which spinal cord and muscle coupling between tissue explanted from normal and dystrophic mice provided a system for studying the mechanism underlying nerve-muscle interaction. The study has yielded the following observations. Recent observations have demonstrated that significant neuromuscular relationships can develop in vitro between spinal cord and embryonic muscle tissues.

In vitro studies of beating heart cells in culture : XV. Myosin turnover and the effect of serum

Myosin turnover in rat heart cells in culture was determined from measurements of the rate of degradation by following loss of radioactivity from “pulse” -labeled cells, and by measuring the rate of myosin synthesis directly as rate of incorporation of radioactive amino acids into myosin or indirectly by calculation from degradation rates. The half-life of myosin in complete growth medium (containing serum) was about 4 days, sometimes ranging up to 8 days. Cells in medium without serum do not divide and show an increased degradation and decreased rate of synthesis. Nondividing cells in complete growth medium turn over myosin at a normal rate. Serum thus appears to contain factors which protect myosin against degradation and also which stimulate synthesis. Myosin synthesis seems to be dependent on RNA synthesis, since myosin levels and synthesis decrease occurs as quickly as 4 hr after treatment with actinomycin D.

Proposed mechanism for the turnover of myosin and its relation to muscular dystrophy.

Proposed mechanism for the turnover of myosin and its relation to muscular dystrophy.