Work overload induced changes in fast and slow skeletal muscle myosin heavy chain gene expression

Work induced hypertrophy of the slow postural soleus and the fast phasic plantaris muscles was produced by tenotomy of the synergistic gastrocnemius muscle. Increases in weight of both muscles were associated with proportionately even larger increases in total RNA and mRNA levels. Alterations in levels of specific myosin heavy chain (MHC) isoform mRNAs were measured using the slot blot procedure with radioactively labelled oligonucleotides as probes. Type 1 MHC gene expression was unaffected in both muscles by work overload, whereas type 2a was deinduced in the soleus and type 2b was deinduced in the plantaris. The neonatal MHC gene was transiently reinduced in the plantaris.     

Restriction fragment length polymorphism of cardiac myosin heavy chain gene in rats and its strain distribution

The distribution of an RFLP in EcoRI fragments of the cardiac myosin heavy chain gene among 29 strains of laboratory rats was examined. Southern blot hybridization of rat genomic DNAs with rat cardiac myosin heavy chain cDNA as a probe demonstrated an interstrain variation in one of eight EcoRI fragments. Of the 28 inbred strains examined, 10 had a fragment of 10 kbp, whereas 18 had a fragment of 7.5 kbp. The 15 samples of the remaining strain (Iar: WI outbred stock) had fragments of either 7.5 kbp or 10 and 7.5 kbp, indicating that this strain has maintained heterogeneity of these fragments.     

Sarcomeric myosin heavy chain is degraded by the proteasome

Cardiac myofibrillar proteins, like all other intracellular proteins, are in a dynamic state of continual degradation and resynthesis. The balance between these opposing metabolic processes ultimately determines the number of functional contractile units within each cardiac muscle cell. Although alterations in myofibrillar protein degradation have been shown to contribute to cardiac growth and remodeling, the intracellular proteolytic systems responsible for degrading myofibrillar proteins to their constitutive amino acids are currently unknown. Lactacystin, a recently developed, highly specific proteasome inhibitor, was used in this study to examine the role of the proteasome in myosin heavy chain (MHC) degradation in cultured neonatal rat ventricular myocytes. Cells were treated with growth medium alone or with lactacystin (1-50 microM) for up to 48 h. Lactacystin significantly increased the total protein/DNA ratio and markedly prolonged MHC half-life. Other proteasome inhibitors, namely carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (10 microM) and N-acetyl-L-leucyl-L-leucyl-norleucinal (100 microM), were also effective in suppressing MHC degradation. Lactacystin and other proteasome inhibitors also suppressed the markedly accelerated MHC degradation associated with Ca2+ channel blockade but did not prevent the disassembly and loss of myofibrils that accompanied contractile arrest. Thus, sarcomere disassembly precedes the degradation of MHC, which is at least in part mediated by the proteasome.     

Calcium channels and cation transport ATPases in cardiac hypertrophy induced by aortic constriction in newborn rats

In order to evaluate the contribution of pinocytosis to basal (no agonist) and lanthanide-insensitive store-activated Ca²⁺ inflow in freshly-isolated rat hepatocytes, the uptake of extracellular fluid by pinocytosis was measured at 20°C and used to predict the amount of extracellular Ca²⁺ taken up by pinocytosis. This was compared with the measured rate of Ca²⁺ uptake in the basal state, and with the measured lanthanide-insensitive component of divalent cation uptake stimulated by 2,5-di-tert-butylhydroquinone (DBHQ), an inhibitor of the smooth endoplasmic reticulum (Ca²⁺ + Mg²⁺)ATP-ase. Fluid uptake by pinocytosis was measured using [¹⁴C]sucrose. In hepatocytes incubated at 20°C, DBHQ increased the initial rate of sucrose uptake by about 35%. The data for sucrose uptake were used to calculate the volume of extracellular fluid taken up by pinocytosis which, in turn, was used to predict the amount of extracellular Ca ²⁺ taken up through pinocytosis in the basal and DBHQ-stimulated states. Rates of divalent cation inflow in the basal state were determined at 20°C by measuring the uptake of ⁴⁵Ca²⁺. The degree of stimulation of Ca²⁺ inflow by DBHQ and the lanthanide-insensitive component of DBHQ-stimulated divalent cation inflow were determined by measuring the rate of Mn²⁺-induced quenching of intracellular quin-2 in the absence of an agonist, and in the presence of DBHQ or DBHQ plus Gd³⁺. It was calculated that the process of pinocytosis accounts for at least 15% of Ca²⁺ uptake in the basal (no agonist) state, and for about 10% of DBHQ-stimulated lanthanide-insensitive Ca²⁺ uptake. It is concluded that in isolated hepatocytes (i) the release of Ca²⁺ from intracellular stores stimulates pinocytosis and (ii) the process of pinocytosis can account for a substantial proportion of basal Ca²⁺ inflow and a small proportion of DBHQ-stimulated lanthanide-insensitive Ca²⁺ inflow.Abbreviations RACC receptor-activated Ca²⁺ channel - DBHQ 2,5-di-tert-butylhydroquinone - [Ca²⁺] intracellular free Ca²⁺ concentration     

Mutation of the IIB myosin heavy chain gene results in muscle fiber loss and compensatory hypertrophy

The fast skeletal IIb gene is the source of most myosin heavy chain (MyHC) in adult mouse skeletal muscle. We have examined the effects of a null mutation in the IIb MyHC gene on the growth and morphology of mouse skeletal muscle. Loss in muscle mass of several head and hindlimb muscles correlated with amounts of IIb MyHC expressed in that muscle in wild types. Decreased mass was accompanied by decreases in mean fiber number, and immunological and ultrastructural studies revealed fiber pathology. However, mean cross-sectional area was increased in all fiber types, suggesting compensatory hypertrophy. Loss of muscle and body mass was not attributable to impaired chewing, and decreased food intake as a softer diet did not prevent the decrease in body mass. Thus loss of the major MyHC isoform produces fiber loss and fiber pathology reminiscent of muscle disease.     

Spatial and temporal changes in myosin heavy chain gene expression in skeletal muscle development

Seven myosin heavy chains (MyHC) are expressed in mammalian skeletal muscle in spatially and temporally regulated patterns. The timing, distribution, and quantitation of MyHC expression during development and early postnatal life of the mouse are reported here. The three adult fast MyHC RNAs (IIa, IIb, and IId/x) are expressed in the mouse embryo and each mRNA has a distinct temporal and spatial distribution. In situ hybridization analysis demonstrates expression of IIb mRNA by 14.5 dpc, which proceeds developmentally in a rostral to caudal pattern. IId/x and IIa mRNAs are detectable 2 days later. Ribonuclease protection assays demonstrate that the three adult fast genes are expressed at approximately equal levels relative to each other in the embryo but at quite low levels relative to the two developmental isoforms, embryonic and perinatal. Just after birth major changes in the relative proportions of different MyHC RNAs and protein occur. In all cases, RNA expression and protein expression appear coincident. The changes in MyHC RNA and protein expression are distinct in different muscles and are restricted in some cases to particular regions of the muscle and do not always reflect their distribution in the adult.     

Age-dependent changes in contraction and regional myocardial myosin heavy chain isoform expression in rats.

The goals of this study were to measure the relative levels of the alpha- and beta-isoforms of myosin heavy chain (MHC-alpha and MHC-beta, respectively) in multiple, specific regions of the adult rat heart and to determine whether age-dependent changes in isoform levels in different regions are uniform. Relative amounts of MHC-alpha and MHC-beta were determined in right and left atria and left ventricular (LV) Purkinje fibers (PF), papillary muscles, trabeculae, and endo-, mid-, and epicardial regions at 2, 5, 10, 16, and 21 mo. PFs contained substantial amounts of myosin and were striated and capable of generating force and shortening on activation. Levels of MHC-beta increased in all LV compartments with age, especially between 2 and 5 mo. There was more MHC-beta in PFs than other LV sites. There were regional differences in the level of MHC-beta throughout the LV at all ages, and the rates of change within regions differed. Ca(2+)-activated tension in PFs and trabeculae was compared at 2 and 22 mo. PF tension was less than trabecula tension, and this difference may be explained by differences in MHC content. V(max) and tension-generating ability in PFs decreased with age. Maximal tension generated by trabeculae did not change during aging. A large proportion of the increase in the level of MHC-beta that is normally associated with aging occurs at a relatively early age in rat LV. PFs, with their small diameters and short diffusion distance, should be considered for skinned multicellular myocardial studies.     

Endurance training affects myosin heavy chain phenotype in regenerating fast-twitch muscle

Bigard, Xavier A., Chantal Janmot, Danièle Merino,Françoise Lienhard, Yannick C. Guezennec, and Anne D'Albis.Endurance training affects myosin heavy chain phenotype inregenerating fast-twitch muscle. J. Appl.Physiol. 81(6): 2658-2665, 1996.The aim of thisstudy was to analyze the effects of treadmill training (2 h/day, 5 days/wk, 30 m/min, 7% grade for 5 wk) on the expression of myosinheavy chain (MHC) isoforms during and after regeneration of afast-twitch white muscle [extensor digitorum longus (EDL)]. Male Wistar rats were randomly assigned to a sedentary(n = 10) or an endurance-trained (ET;n = 10) group. EDL muscle degeneration and regeneration were induced by two subcutaneous injections of a snaketoxin. Five days after induction of muscle injury, animals were trainedover a 5-wk period. It was verified that ~40 days after venomtreatment, central nuclei were present in the treated EDL muscles fromsedentary and ET rats. The changes in the expression of MHCs in EDLmuscles were detected by using a combination of biochemical andimmunocytochemical approaches. Compared with contralateral nondegenerated muscles, relative concentrations of types I, IIa, andIIx MHC isoforms in ET rats were greater in regenerated EDL muscles(146%, P < 0.05; 76%,P < 0.01; 87%,P < 0.01, respectively). Their elevation corresponded to a decreasein the relative concentration of type IIb MHC (36%,P < 0.01). Although type I accountedfor only 3.2% of total myosin in regenerated muscles from the ETgroup, the cytochemical analysis showed that the proportion of positive staining with the slow MHC antibody was markedly greater in regenerated muscles than in contralateral ones. Collectively, these results demonstrate that the regenerated EDL muscle is sensitive to endurance training and suggest that the training-induced shift in MHC isoforms observed in these muscles resulted from an additive effect of regeneration and repeated exercise.

     

Effects of voluntary wheel running on cardiac function and myosin heavy chain in chemically gonadectomized rats

Reducing testosterone and estrogen levels with a luteinizing hormone-releasing hormone agonist such as Zoladex (i.e., chemical gonadectomy) is a common treatment for many prostate and breast cancer patients, respectively. There are reports of surgical gonadectomy inducing cardiac dysfunction, and exercise has been shown to be cardioprotective under these circumstances. Minimal research has been done investigating the effects of chemical gonadectomy and increased physical activity on cardiac function. The purpose of this investigation was to examine the effects of chemical gonadectomy and physical activity on cardiac function. Male (M) and female (F) Sprague-Dawley rats received either Zoladex treatment (Zol) that suppressed gonadal function for 8 wk or control implants (Con) and either were allowed unlimited access to voluntary running wheels (WR) or remained sedentary (Sed) throughout the treatment period. In vivo and ex vivo left ventricle (LV) function were then assessed, and myosin heavy chain (MHC) expression was analyzed to help explain LV functional differences. Hearts from M Sed+Zol exhibited significantly lower aortic blood flow velocity, developed pressure, and maximal rate of pressure development and higher beta-MHC expression than M Sed+Con. Hearts from F Sed+Zol exhibited significantly lower LV wall thicknesses, fractional shortening, and developed pressure and higher beta-MHC expression than F Sed+Con. This cardiac dysfunction was not evident in hearts from M or F WR+Zol, and this was associated with a preservation of the MHC isoform distribution. Thus an 8-wk chemical gonadectomy with Zoladex promoted cardiac dysfunction in male and female rats, and voluntary wheel running protected against this cardiac dysfunction.     

Training affects myosin heavy chain phenotype in the trapezius muscle of women

The aim of this investigation was to determine whether 10 weeks of three different types of training can alter the myosin heavy chain (MyHC) composition of the trapezius muscle. Twenty-one women were randomly assigned to three training groups that performed strength (n=9), endurance (n=7) or coordination training (n=5). Pre and post biopsies were taken from the upper part of the descending trapezius muscle and were analysed for MyHC isoform content using 5% gel electrophoresis. In addition, we have studied the expression of embryonic and neonatal MyHCs using double-immunofluorescence staining. In the strength-trained group, there was a significant increase in the amount of MyHC IIA and a significant decrease in the amount of MyHC IIB and MyHC I. In the endurance group, there was a significant decrease in the amount of MyHC IIB. MyHC composition in the coordination group was not altered. Following the training period, myotubes and individual small-sized muscle fibres were observed in the strength and endurance trained groups. These structures were stained with the markers for early myogenesis (MyHC embryonic and neonatal). These data suggest that specific shifts in MyHC isoforms occur in the trapezius muscle following strength and endurance training. The presence of small-sized muscle fibres expressing the developmental isoforms of MyHC suggests that strength and endurance training induced the formation of new muscle fibres. Accepted: 31 March 1999     

Acanthamoeba myosin I heavy chain kinase is activated by phosphatidylserine-enhanced phosphorylation

The actin-activated Mg2(+)-ATPase activities of myosins I from Acanthamoeba castellanii are fully expressed only when a single amino acid on their heavy chain is phosphorylated by myosin I heavy chain kinase. Here we show that kinase isolated by a procedure designed to minimize its phosphorylation during purification can incorporate up to 7.5 mol of phosphate/mol of enzyme when incubated with ATP, possibly by autophosphorylation. The rate of phosphorylation is enhanced about 20-fold by phosphatidylserine but is unaffected by calcium ions. Phosphorylation increases the rate at which the kinase phosphorylates the regulatory site of myosin I by about 50-fold. These results suggest that (auto?)phosphorylation may regulate the activity of myosin I heavy chain kinase in vivo. The stimulation of kinase phosphorylation by phosphatidylserine (other phospholipids have not yet been tested) is of particular interest because myosin I has been shown to be tightly associated with membranes, especially the plasma membrane.