Altered expression of myosin light-chain isoforms in chronically stimulated fast-twitch muscle of the rat

Fast-twitch tibialis anterior muscle of the rat was chronically stimulated for periods of 18 days, 28 days and 56 days. Changes in the myosin light-chain (LC) pattern consisted in an increase in LC1f, concomitant with a decrease in LC3f. In contrast to previous findings in chronically stimulated fast-twitch tibialis anterior muscle of the rabbit, no substantial increases occurred in the slow myosin light-chain isoforms. In vivo labeling using [35S]methionine incorporation revealed differences in relative turnover between the fast myosin light chains. The relative turnover of the fast myosin light chains appeared to increase in normal muscle in the order LC2f less than LC1f less than LC3f. As judged from [35S]methionine incorporation, the changes in light-chain tissue content mainly resulted from altered synthesis rates. However, in the case of LC3f the decrease in protein content could not only be explained by a reduced synthesis, but, additionally, appeared to be due to enhanced degradation. Parvalbumin, which was included in the present study, was also found to decrease in the stimulated muscle. However, its decrease appeared to result primarily from reduced synthesis.     

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.     

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.

     

Antagonistic effects of chronic low frequency stimulation and thyroid hormone on myosin expression in rat fast-twitch muscle

This study investigates effects of chronic low frequency stimulation (CLFS) on myosin heavy (MHC) and light chain (MLC) expression in fast-twitch muscles in hypothyroid, euthyroid, and hyperthyroid rats. The changes at both the mRNA and protein level indicated antagonistic effects of thyroid hormone and CLFS: under euthyroid conditions, CLFS mainly elicited a MHCIIb----MCHIId----MHCIIa transition. Whereas CLFS did not induce the slow MHCI in the euthyroid state, this isoform was present in the hypothyroid state and was further enhanced with CLFS indicating the suppressive effect of thyroid hormone to be stronger than the inductive influence of CLFS. Hyperthyroidism alone suppressed the expression MHCIIa and enhanced a MHCIId to MHCIIb transition. This shift to the faster MHC isoforms was only partially counteracted by CLFS. Thus, it appeared that thyroid hormone had a graded suppressive effect on the expression of MHC isoforms in the order MHCIId less than MHCIIa less than MHCI. Elevated neuromuscular activity partially counteracted these hormone effects. Changes in MLC mRNAs were consistent with those in the MHC pattern, i.e. increases or decreases in MHCIIb led to corresponding changes in the expression of MLC3f. A similar relationship existed for the slow MHCI and the slow MLC isoforms.     

Developmental progression of myosin gene expression in cultured muscle cells

Myosin heavy chains are encoded by distinct members of a multigene family at different stages of muscle development. Study of the underlying regulatory mechanisms has been hindered because transitions in myosin expression have not been readily attained in tissue culture. Here we show a transition from early (fetal) to late (perinatal/adult) myosins defined by two monoclonal antibodies, F1.652 and N3.36, in the myotubes of mouse C2C12 cells. On day 1 of differentiation, essentially all myosin was early myosin. By day 8, early myosin dropped to 25% of its day 1 value and was replaced by late myosin. The transition occurred without neural contact, connective tissue components, or complex substrates, suggesting that its regulation may be intrinsic to the muscle cell. Our results demonstrate that a developmental progression in myosin gene expression, which occurs rapidly, with high frequency, and under relatively simple conditions, is now amenable to molecular analysis in cultured muscle cells.     

The expression of myosin heavy chain isoforms in normal and hypertrophied chicken slow muscle

Hypertrophy was produced in the anterior latissimus dorsi (ALD) muscle of 5-wk-old chickens by application of a load to the humerus. After 4 wk, hypertrophied ALD muscles were greater than 2.5 times heavier than contralateral control ALD muscles. Two isomyosins are distinguishable in normal ALD muscles by their different electrophoretic mobilities. It is shown here that the faster migrating SM-1 isomyosin decreases in abundance with age and that the application of an overload enhances both the rate and extent of this process. Monoclonal antibodies were selected by an immunotransfer technique that were specific for the heavy chains associated with either SM-1 or SM-2, or cross-reacted with both isoforms. The cellular distribution of the SM-1 and SM-2 isomyosins was analyzed by immunofluorescent technique using these antibodies. Anti-SM-1 and anti-SM-2 antibodies reacted with separate populations of cells, whereas the third antibody reacted with all myocytes in the normal ALD muscle. These data suggest that there is an exclusive cellular distribution of myosin heavy chains associated with SM-1 and SM-2 proteins. Immunofluorescent analysis of hypertrophied muscle showed the anti-SM-2-specific antibody reacting with all myocytes, whereas the anti-SM-1-specific antibody reacted with none. This is consistent with the elimination of the SM-1 isoform in hypertrophied muscles.     

Changes in myosin heavy-chain isoform synthesis of chronically stimulated rat fast-twitch muscle.

Chronic low-frequency stimulation was used for studying the adaptive potential of rat fast-twitch muscle to increased neuromuscular activity. The sequential exchange of myosin heavy chain isoforms HCIIb with HCIId and HCIIa was studied at the translational level using an in-vivo-labeling technique with [35S]methionine. Alterations in heavy chain isoform synthesis, i.e. a decrease in the labeling of HCIIb concomitant with an enhanced labeling of HCIId/IIa, were detectable already two days after the onset of stimulation. This time course corresponds to the previously observed alterations in the amounts of HCIIb and HCIIa mRNAs. However, significant changes in the relative protein amounts of HCIIb and HCIId/IIa were recorded only after an 8-day stimulation period. This delay at the protein level was interpreted to relate to the slow turnover of HCIIb which was estimated from its decay in long-term stimulated muscles with an approximate value of 14.7 days. Therefore, protein degradation seems to be an important post-translational regulatory step in the remodeling process of the thick filament.     

Rapid and reversible changes in myosin heavy chain expression in response to increased neuromuscular activity of rat fast-twitch muscle

Chronic 10 Hz stimulation of rat fast-twitch muscle induced rapid and reversible changes in the tissue levels of fast myosin heavy chain (HC) mRNA isoforms. These changes consisted of a rapid decrease in HCIIb mRNA and a progressive increase in HCIIa mRNA. After 15 days, the HCIIb mRNA normally amounting to approximately 80%, had decreased to less than 5% of the sum of the two HC mRNA isoforms. HCIIb mRNA was again detectable one day after cessation of stimulation and progressively increased at the expense of HCIIa mRNA with ongoing recovery. These results point to a down-regulation of the HCIIb gene by the applied stimulus pattern which, conversely, enhances the expression of the HCIIa gene.     

Effect of hormonal and neurotrophic factors on the expression of fast myosin in slow muscle

Myosin ATPase and succinic dehydrogenase activity and fast myosin (indirect immunochemical test) were assayed in m. soleus of guinea-pigs after the administration of T4 (200 micrograms/kg) to animals every day for 3 weeks. This was followed by the application of 10 mM colchicine solution to sciatic nerve for 6 min. Fast muscle fibers and the line of precipitation with antiserum to fast myosin were revealed in soleus muscle of experimental animals after the application of colchicine and T4 injection.     

Activity-dependent gene regulation in conditionally-immortalized muscle precursor cell lines

Skeletal muscle contractile activity has been implicated in many aspects of muscle cell differentiation and maturation. Much of the research in this area has depended upon costly and labor-intensive cultures of isolated primary muscle cells because widely available immortalized muscle cell lines often do not display a high level of either spontaneous or stimulated contractile activity. We sought to develop conditionally-immortalized skeletal muscle cell lines that would provide a source of myofibers that exhibit robust spontaneous contractile activity similar to primary muscle cultures. Using a tetracycline-regulated retroviral vector expressing a temperature-sensitive T-antigen to infect primary myoblasts, we isolated individual clonal muscle precursor cell lines that have characteristics of activated satellite cells during growth and rapidly differentiate into mature myotubes with spontaneous contractile activity after culture in non-transformation-permissive conditions. Comparison of these cell lines (known as rat myoblast-like tetracycline (RMT) cell lines) to primary cell cultures revealed that they share a wide variety of morphological, physiological, and biochemical characteristics. Most importantly, the time-course and extent of activity-dependent gene regulation observed in primary cell culture for all genes tested, including subunits of the nicotinic acetylcholine receptor (nAChR), muscle specific kinase (MuSK), and myogenin, is reproduced in RMT lines. These immortalized cell lines are a useful alternative to primary cultures for studying muscle differentiation and molecular and physiological aspects of electrical activity in muscle fibers.     

The myosin alkali light chains of mouse ventricular and slow skeletal muscle are indistinguishable and are encoded by the same gene

We have isolated a cDNA recombinant plasmid (pA29) identified as encoding part of the ventricular muscle myosin light chain MLC1v. This cDNA contains a 300-base pair fragment which under conditions of moderate stringency shows specific hybridization to MLC1v mRNA with no detectable cross-hybridization with the mRNAs encoding the fast skeletal muscle isoforms MLC1F and MLC3F, or the atrial muscle isoform MLC1A. Under these conditions hybridization is seen with an abundant mRNA present in slow skeletal muscle (soleus) which is indistinguishable from ventricular MLC1V mRNA on the basis of size and of thermal stability of hybrids formed with plasmid pA29. The mouse MLC1V and MLC1S proteins are found to co-migrate on two-dimensional gels. We therefore conclude that these isoforms are the same and are encoded by the same mRNA. Analysis of mouse DNA has identified a single region of the genome which hybridizes to this same fragment of pA29. This region has been isolated in a recombinant phage and has been shown to contain a single gene showing homology with MLC1V mRNA by R-loop analysis. We therefore conclude that MLC1V and MLC1S are encoded by a single gene. The pattern of segregation of a restriction fragment length polymorphism identified for this gene between Mus musculus and Mus spretus has been followed in an F1 backcross between these two mouse species. The results show the MLC1V/MLC1S gene to be closely linked to a marker at the distal end of mouse chromosome 9.