Exercise-induced alterations in skeletal muscle myosin heavy chain phenotype: dose-response relationship.

This study investigated the effects of exercise training duration on the myosin heavy chain (MHC) isoform distribution in rat locomotor muscles. Female Sprague-Dawley rats (120 days old) were assigned to either a sedentary control group or to one of three endurance exercise training groups. Trained animals ran on a treadmill at approximately 75% maximal O2 uptake for 10 wk (4-5 days/wk) at one of three different exercise durations (30, 60, or 90 min/day). Training resulted in increases (P < 0.05) in citrate synthase activity in the soleus and extensor digitorum longus in both the 60 and 90 min/day duration groups and in the plantaris (Pla) in all three exercise groups. All durations of training resulted in a reduction (P < 0.05) in the percentage of MHCIIb and an increase (P < 0.05) in the percentage of MHCIIa in the Pla. The magnitude of change in the percentage of MHCIIb in the Pla increased as a function of the training duration. In the extensor digitorum longus, 90 min of daily exercise promoted a decrease (P < 0.05) in percentage of MHCIIb and increases (P < 0.05) in the percentages of MHCI, MHCIIa, and MHCIId/x. Finally, training durations >/=60 min resulted in an increase (P < 0.05) in the percentage of MHCI and a concomitant decrease (P < 0.05) in the percentage of MHCIIa in the soleus. These results demonstrate that increasing the training duration elevates the magnitude of the fast-to-slow shift in MHC phenotype in rat hindlimb muscles.     

Stretch and force generation induce rapid hypertrophy and myosin isoform gene switching in adult skeletal muscle.

Using electrical stimulation to control force generation and limb immobilization to alter the degree of stretch, we have studied the role of mechanical activity in inducing hypertrophy and in determining fast and slow muscle fibre phenotype. Changes in gene expression were detected by analysing the RNA in hybridization studies employing cDNA probes specific for fast and slow myosin heavy chains and other genes. As a result of overload in the stretched position, the fast contracting tibialis anterior muscle in an adult rabbit is induced to synthesize much new protein and to grow by as much as 30% within a period as short as 4 days. This very rapid hypertrophy was found to be associated with an increase of up to 250% in the RNA content of the muscles and an abrupt change in the species of RNA produced. Both stretch alone and electrical stimulation alone caused repression of the fast-type genes and activation of the slow-type genes. it appears that the fast-type IIB genes are the default genes, but that the skeletal slow genes are expressed as a response to overload and stretch. These findings have implications as far as athletic training and rehabilitation are concerned.     

Aging-sensitive cellular and molecular mechanisms associated with skeletal muscle hypertrophy.

Sarcopenia is an age-related loss of muscle mass and strength. The aged can increase various measures of muscle size and strength in response to resistance exercise (RE), but this may not normalize specific tension. In rats, aging reduces the hypertrophy response and impairs regeneration. In this study, we measured cellular and molecular markers, indicative of muscle hypertrophy, that also respond to acute increases in loading. Comparing 6- and 30-mo-old rats, the aims were to 1) determine whether these markers are altered with age and 2) identify age-sensitive responses to acute RE. The muscles of old rats exhibited sarcopenia involving a deficit in contractile proteins and decreased force generation. The RNA-to-protein ratio was higher in the old muscles, suggesting a decrease in translational efficiency. There was evidence of reduced signaling via components downstream from the insulin/insulin-like growth factor (IGF)-I receptors in old muscles. The mRNA levels of myostatin and suppressor of cytokine signaling 2, negative regulators of muscle mass, were lower in old muscles but did not decrease following RE. RE induced increases in the mRNAs for IGF-I, mechano-growth factor, cyclin D1, and suppressor of cytokine signaling 3 were similar in old and young muscles. RE induced phosphorylation of the IGF-I receptor, and Akt increased in young but not old muscles, whereas that of S6K1 was similar for both. The results of this study indicate that a number of components of intracellular signaling pathways are sensitive to age. As a result, key anticatabolic responses appear to be refractory to the stimuli provided by RE.     

Dissociation and reassociation of rabbit skeletal muscle myosin.

Whereas dissociation of rabbit skeletal muscle myosin light chains occurs at an increased temperature (25 degrees) and in the absence of divalent cations, reassociation of the myosin oligomer requires a low temperature (4 degrees C) and the presence of divalent cations, thus resulting in the original light to heavy chain stoichiometry. With a 5-10 per cent release of alkali light chains, LC1 and LC3, and a 50 per cent dissociation of the Ca2+ binding light chain, LC2, there is no significant decrease in myosin ATPase activity irrespective of the cation activator, however, there is an approximate 15-20 per cent decrease in actomyosin ATPase activity. With reassociation of the myosin oligomer, actomyosin ATPase activity is partially restored as well as the original number of Ca2+ binding sites.     

Kinetic effects of myosin regulatory light chain phosphorylation on skeletal muscle contraction

Kinetic analysis of contracting fast and slow rabbit muscle fibers in the presence of the tension inhibitor 2,3-butanedione monoxime suggests that regulatory light chain (RLC) phosphorylation up-regulates the flux of weakly attached cross-bridges entering the contractile cycle by increasing the actin-catalyzed release of phosphate from myosin. This step appears to be separate from earlier Ca(2+) regulated steps. Small step-stretches of single skinned fibers were used to study the effect of phosphorylation on fiber mechanics. Subdivision of the resultant tension transients into the Huxley-Simmons phases 1, 2(fast), 2(slow), 3, and 4 reveals that phosphorylation reduces the normalized amplitude of the delayed rise in tension (stretch activation response) by decreasing the amplitudes of phase 3 and, to a lesser extent, phase 2(slow). In slow fibers, the RLC P1 isoform phosphorylates at least 4-fold faster than the P2 isoform, complicating the role of RLC phosphorylation in heart and slow muscle. We discuss the functional relevance of the regulation of stretch activation by RLC phosphorylation for cardiac and other oscillating muscles and speculate how the interaction of the two heads of myosin could account for the inverse effect of Ca(2+) levels on isometric tension and rate of force redevelopment (k(TR)).     

The influence of ethylenediaminetetraacetate on white skeletal muscle myosin.

Myosin from rabbit white skeletal muscle was treated with 10 mM EDTA in 150 mM phosphate buffer. After precipitation of myosin by dialysis against a 14-fold volume of water, EDTA-treated myosin, myosin before treatment and the supernatant from the treatment of myosin with EDTA were examined on sodium dodecyl sulphate-polyacrylamide gels by electrophoresis. It has been found that the quantity of LC2 light chains diminished after treatment with EDTA, and the supernatant contained the LC2 light chains. Treatment of myosin with EDTA in the presence of Mg2+ does not change the stoichiometry of the LC2 light chain and the supernatant is free from LC2 light chains. The treatment of myosin with p-chloromercuri-benzoate leads to dissociation of the same amount of LC2 light chains. It is suggested that divalent cations and thiol groups are engaged in the attachment of LC2 light chain to the myosin molecule.     

Studies on the subunit interactions of skeletal muscle myosin subfragment 1. Evidence for subunit exchange between isozymes under physiological ionic strength and temperature

Evidence is presented that under physiological conditions of ionic strength and temperature, where myosin Subfragment 1 is hydrolyzing MgATP, the interaction between its subunits is extremely labile. Incubation of [3H]N-ethylmaleimide-SF1(A1) with N-ethylmaleimide-SF1(A2) in the presence of 10 mM MgATP at 37 degrees C resulted in the exchange of subunits between these isozymes. This is readily discernible from the subunit composition and distribution of the 3H label after separation of the isozymes by ion exchange chromatography. Moreover, incubation of unmodified SF1(A1) or SF1(A2) with the free Alkali light chains A2 and A1, respectively, under the same conditions led to the formation of significant amounts of the hybrid species. These findings suggest that in vivo the Alkali light chain-heavy chain interaction of Subfragment 1 is in a state of dynamic equilibrium between associated and dissociated states.     

The biochemical kinetics underlying actin movement generated by one and many skeletal muscle myosin molecules

To better understand how skeletal muscle myosin molecules move actin filaments, we determine the motion-generating biochemistry of a single myosin molecule and study how it scales with the motion-generating biochemistry of an ensemble of myosin molecules. First, by measuring the effects of various ligands (ATP, ADP, and P(i)) on event lifetimes, tau(on), in a laser trap, we determine the biochemical kinetics underlying the stepwise movement of an actin filament generated by a single myosin molecule. Next, by measuring the effects of these same ligands on actin velocities, V, in an in vitro motility assay, we determine the biochemistry underlying the continuous movement of an actin filament generated by an ensemble of myosin molecules. The observed effects of P(i) on single molecule mechanochemistry indicate that motion generation by a single myosin molecule is closely associated with actin-induced P(i) dissociation. We obtain additional evidence for this relationship by measuring changes in single molecule mechanochemistry caused by a smooth muscle HMM mutation that results in a reduced P(i)-release rate. In contrast, we observe that motion generation by an ensemble of myosin molecules is limited by ATP-induced actin dissociation (i.e., V varies as 1/tau(on)) at low [ATP], but deviates from this relationship at high [ATP]. The single-molecule data uniquely provide a direct measure of the fundamental mechanochemistry of the actomyosin ATPase reaction under a minimal load and serve as a clear basis for a model of ensemble motility in which actin-attached myosin molecules impose a load.     

The mechanism of skeletal muscle myosin ATPase. Interaction of myosin active center with ATP and with ADP

The kinetics of the fluorescence enhancement and the transient release of H+ caused by the binding of ADP to the active center of myosin has been compared to that caused by myosin-ATP interaction. The results show that both the time courses of the fluorescence enhancement and the transient H+ release caused by ADP binding, like that caused by ATP hydrolysis in the initial burst, are monophasic exponential processes. The fact that the rates of these two processes are also equal suggests that they both reflect the same mechanistic event in the mechanism of ADP binding. The kinetics of ADP binding as measured by the fluorescence enhancement and the H+ release is different from that of ATP. This is in agreement with our previous finding that the enhancement of fluorescence and the transient release of H+, in the case of ATP, reflect the initial burst of ATP hydrolysis, whereas in the case of ADP, they represent a conformational change in the myosin-ADP complex. The magnitude of the H+ transient caused by the initial burst is approximately equal to that caused by ADP binding. The amplitude of the fluorescence enhancement caused by ADP binding is equal to one-third of that caused by the initial burst.     

Fibrinoligase-catalyzed cross-linking of myosin from platelet and skeletal muscle.

Fibrinoligase (thrombin- and calcium-activated Factor XIII) from human plasma catalyzes the incorporation of dansylcadaverine and [¹⁴C]putrescine into myosin, prepared from either human platelets or rabbit skeletal muscle. At least 9 mol of amine is incorporated per mole of myosin of either type when the enzyme is used under saturating conditions. Both heavy and light chains of the platelet and muscle myosins incorporate dansylcadaverine and [ ¹⁴C]putrescine. However, in quantitative terms, the incorporation into the light chains of either type is much less than into the heavy chains. Profound fluorescent changes occurred when dansylcadaverine was bound to myosin. Highly cross-linked platelet and muscle myosin polymers form in the absence of added amines, indicating the presence of both acceptor and donor sites. ATPase activity was not altered by cross-linking of 50–60% of myosin. The nature of the cross-link in myosin was found to be a γ-glutamyl-?-lysine bond, with an average of 19 mol of dipeptide per mole of platelet myosin.     

Comparative effects of hindlimb suspension and exercise on skeletal muscle myosin isozymes in rats

The purpose of this study was to ascertain the time course of changes, whilst suspending the hindlimb and physical exercise training, of myosin light chain (LC) isoform expression in rat soleus and vastus lateralis muscles. Two groups of six rats were suspended by their tails for 1 or 2 weeks, two other groups of ten rats each were subjected to exercise training on a treadmill for 9 weeks, one to an endurance training programme (1-h running at 20 m.min-1 5 days.week-1), and the other to a sprint programme (30-s bouts of running at 60 m.min-1 with rest periods of 5 min). At the end of these experimental procedures, soleus and vastus lateralis superficialis muscles were removed for myosin LC isoform determination by two-dimensional gel electrophoresis. Hindlimb suspension for 2 weeks significantly increased the proportion of fast myosin LC and decreased slow myosin LC expression in the soleus muscle. The pattern of myosin LC was unchanged in the vastus lateralis muscle. Sprint training or endurance training for 9 weeks increased the percentage of slow myosin LC in vastus lateralis muscle, whereas soleus muscle myosin LC was not modified. These data indicate that hindlimb suspension influences myosin LC expression in postural muscle, whereas physical training acts essentially on phasic muscle. There were no differences in myosin LC observed under the influence of sprint- or endurance-training programme.     

Depolymerization of brain microtubules by skeletal muscle myosin.

Brain microtubules are found to disperse rods of skeletal muscle myosin and become decorated with amorphous aggregates of myosin. Then microtubules are partially depolymerized by myosin. Myosin shows high Mg2+-GTPase activity which is not influenced by microtubules, and induces the partial depolymerization of microtubules by exhaustion of GTP in the solution. H-meromyosin depolymerizes microtubules like myosin does. H-meromyosin is, however, contaminated with a trace amount of trypsin, which irreversibly depolymerizes microtubules.     

Autophosphorylation of skeletal muscle myosin light chain kinase.

Ca2+/calmodulin-dependent myosin light chain kinase phosphorylates the regulatory light chain of myosin. Rabbit skeletal muscle myosin light chain kinase also catalyzes a Ca2+/calmodulin-dependent autophosphorylation with a rapid rate of incorporation of 1 mol of 32P/mol of kinase and a slower rate of incorporation up to 1.52 mol of 32P/mol. Autophosphorylation was inhibited by a peptide substrate that has a low Km value for myosin light chain kinase. Autophosphorylation at both rates was concentration-independent, indicating an intramolecular mechanism. There were no significant changes in catalytic properties toward light chain and MgATP substrates or in calmodulin activation properties upon autophosphorylation. After digestion with V8 protease, phosphopeptides were purified and sequenced. Two phosphorylation sites were identified, Ser 160 and Ser 234, with the former associated with the rapid rate of phosphorylation. Both sites are located amino terminal of the catalytic domain. These results indicate that the extended "tail" region of the enzyme can fold into the active site of the kinase.     

Interaction of bacterial flagellum filaments with skeletal muscle myosin

Interaction of isolated bacterial flagellum filaments (BFF) and intact flagella from E. coli MS 1350 and B. brevis G.-B.p+ with rabbit skeletal myosin was studied. BFF were shown to coprecipitate with myosin (but not with isolated myosin rod) at low ionic strength, that is, under conditions of myosin aggregation. The data of electron microscopy indicate that filaments of intact bacterial flagella interact with isolated myosin heads (myosin subfragment 1, S1), and this interaction is fully prevented by addition of Mg2+ -ATP. Addition of BFF inhibited both K+ -EDTA- and Ca2+ -ATPase activity of skeletal muscle myosin, but had no effect on its Mg2+ -ATPase activity. Monomeric flagellin did not coprecipitate with myosin and had no effect on its ATPase activities. BFF were shown to compete with F-actin in myosin binding. It is concluded that BFF interact with myosin heads and affect their ATPase activity. Thus, BFF composed of a single protein flagellin are in many respects similar to actin filaments. Common origin of actin and flagellin may be a reason for this similarity.     

Dense precipitate of brain tubulin with skeletal muscle myosin

Purified tubulin from porcine brain formed a dense precipitate at 37 degrees C with muscle myosin filaments from rabbit skeletal muscle; this effect was greater than that with partially purified tubulin. ATP or GTP, which prevented the myosin filaments from precipitating, inhibited the formation of the dense precipitate, but did not dissociate the dense precipitate once formed. The dense precipitate was found by thin-section electron microscopy to be composed to side-by-side aggregates of myosin filaments whose projections might be decorated by tubulin. The decoration was also seen by negative-stain electron microscopy. The binding of tubulin to myosin filaments decreased the Mg2+- and Ca2+-GTPase activity of the myosin by about half, but did not affect either Mg2+- or Ca2+-ATPase activity. The binding ratio of tubulin to myosin in the presence of 5 mM MgCl2 was 2.2 mol/mol using purified tubulin and 1.8 mol/mol using partially purified tubulin. Five mM ATP and GTP in the presence of 5 mM MgCl2 decreased the tubulin binding by 1.6-2.0 and 1.1-1.3 mol/mol, respectively, when added before an encounter of tubulin with myosin filaments, but did not cause any decrease when added after such an encounter.