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.     

Smooth muscle myosin isoform distribution and myosin ATPase in hypertrophied urinary bladder.

Hypertrophy of the urinary bladder was produced in rabbit by partial ligation of the urethra. Electrophoresis of the bladder smooth muscle myosin on highly porous (3.5-7% gradient) SDS-polyacrylamide gel revealed two heavy chain isoforms, SM-1 and SM-2 with approximate molecular weights of 204,000 and 200,000, respectively. The ratio of the SM-2 to SM-1 heavy chain is 3:1 for myosin isolated from normal bladder smooth muscle, and this ratio changes to about 1:1 in hypertrophied bladder. Despite a change in the ratio of SM-2 to SM-1, the myosin ATPase and the actin-activated ATPase activities are not altered in response to hypertrophy.     

Alterations in expression of myosin and myosin light chain kinases in response to vascular injury

Histochemical analysis ofballoon-injured rat carotid arteries revealed a coordinated expressionof nonmuscle myosin heavy chain-A and -B (NM-A and NM-B) in response toinjury. Expression of these nonmuscle myosin forms shifts from themedia to the adventitia and intima. In contrast, expression of smoothmuscle myosin heavy chain-1 (SM-1) within the media is not altered,whereas smooth muscle myosin heavy chain-2 (SM-2) expression declines.Western blotting shows a statistically significant increase inexpression of NM-A that occurs within 6 h in response to carotidinjury, suggesting this myosin form may be an appropriate experimental marker for proliferating, migrating cells in injured vessels. Nooverall change in the relative expression level of NM-B was detected,suggesting that compensatory declines in media expression are balancedby increases in the intima and adventitia. Expression of SM-1 did notchange in response to injury, whereas the expression of SM-2significantly declined between 24 h and 7 days. Expression ofmyosin light chain kinase is also negatively regulated, and the declinein its expression parallels downregulation of SM-2.

     

Nascent muscle fiber appearance in overloaded chicken slow-tonic muscle

The application of a weight overload to the humerus of chickens induces a hypertrophy of anterior latissimus dorsi (ALD) muscle fibers. This growth is accompanied by a rapid and almost complete replacement of one slow-tonic myosin isoform, SM-1, by another slow-tonic isoform, SM-2. In addition, a population of small fibers appears mainly in extrafascicular spaces and, concurrently, three additional myosin bands are detected by gel electrophoresis. Five antibodies against myosin heavy chain (MHC) isoforms were selected as immunocytochemical probes to determine the cellular location and nature of these myosins. The antibodies react with ventricular, fast skeletal muscle and either SM-1 or SM-2, or both the slow-tonic MHCs. The antifast and antiventricular antibodies react with myosin present in the 10-day embryonic ALD muscle but do not react with myosin in posthatch ALD muscle. The small fibers in overloaded muscle contain a myosin isoform characteristically expressed during the embryonic stage of ALD muscle development and therefore are named nascent myofibers. Some of the nascent myofibers do not react with the antibody to both slow-tonic MHCs, indicating the lack of the normal adult slow-tonic myosins which are expressed in 10-day embryos. In order to explore the origin of the nascent fibers, an electron microscopic study was performed. Stereological analysis of the existing fibers shows a stimulation of numbers and sizes of satellite cells. In addition, the volume occupied by nonmuscle and undifferentiated cells increases dramatically. Myotube formation with incipient myofibrils is seen in extrafascicular spaces. These data suggest that new muscle fiber formation accompanies hypertrophy in overloaded chicken ALD muscle and the process may involve satellite cell migration.     

The myosin converter domain modulates muscle performance

Myosin is the molecular motor that powers muscle contraction as a result of conformational changes during its mechanochemical cycle. We demonstrate that the converter, a compact structural domain that differs in sequence between Drosophila melanogaster myosin isoforms, dramatically influences the kinetic properties of myosin and muscle fibres. Transgenic replacement of the converter in the fast indirect flight muscle with the converter from an embryonic muscle slowed muscle kinetics, forcing a compensatory reduction in wing beat frequency to sustain flight. Conversely, replacing the embryonic converter with the flight muscle converter sped up muscle kinetics and increased maximum power twofold, compared to flight muscles expressing the embryonic myosin isoform. The substitutions also dramatically influenced in vitro actin sliding velocity, suggesting that the converter modulates a rate-limiting step preceding cross-bridge detachment. Our integrative analysis demonstrates that isoform-specific differences in the myosin converter allow different muscle types to meet their specific locomotion demands.     

Regional differences in myosin heavy chain isoform expression and maximal shortening velocity of the rat vaginal wall smooth muscle

Contractility of the proximal and distal vaginal wall smooth muscle may play distinct roles in the female sexual response and pelvic support. The goal of this study was to determine whether differences in contractile characteristics of smooth muscle from these regions reside in differences in the expression of isoforms of myosin, the molecular motor for muscle contraction. Adult female Sprague-Dawley rats were killed on the day of estrus, and the vagina was dissected into proximal and distal segments. The Vmax at peak force was greater for tissue strips of the proximal vagina compared with that of distal (P < 0.01), although, at steady state, the Vmax for the muscle strips from the two regions was not different. Furthermore, at steady state, muscle stress was higher (P < 0.001) for distal vaginal strips (n = 5). Consistent with the high Vmax for the proximal vaginal strips, RT-PCR results revealed a higher %SM-B (P < 0.001) in the proximal vagina. A greater expression of SM-B protein (P < 0.001) was also detected by Western blotting (n = 4). Interestingly, there was no regional difference noted in SM-1/SM-2 isoforms (n = 6). The proximal vagina had a higher expression of myosin heavy chain protein (P < 0.01) and a greater percentage of smooth muscle bundles (P < 0.001). The results of this study are the first demonstration of a regional heterogeneity in Vmax and myosin isoform distribution in the vagina wall smooth muscle and confirm that the proximal vaginal smooth muscle exhibits phasic contractile characteristics compared with the distal vaginal smooth muscle, which is tonic.     

A variant derived from rabbit aortic smooth muscle: phenotype modulation and restoration of smooth muscle characteristics in cells in culture

We examined the relationship between growth arrest of smooth muscle cells and structural changes in microfilament bundles, and also that between the structural changes and the actions of contractile agonist using a multipassagable variant cell line (SM-3) derived from rabbit aortic smooth muscle cells. The content of smooth muscle type alpha-actin increased with density-dependent growth arrest of the SM-3 cells, but was attenuated in the logarithmically growing cultures. As assessed cytochemically, the growth-arrested cells contained longitudinally oriented bundles of actin-containing microfilament and myosin-based filaments visualized with rhodamine-phalloidin and antibody against myosin light chain 20, respectively, whereas both actin- and myosin-containing structures in logarithmically growing cells showed slight, shortened, or diffused patterns. Electron microscopic examination of the growth-arrested cells revealed that the cells contained numerous and conspicuous microfilament bundles associated with many compact electron-dense bodies. In addition, pinocytotic vesicles were often found near the plasma membrane in the growth-arrested cells. SM-3 cells in the growth-arrested phase responded to prostaglandin F2 alpha (3-30 microM) and rat endothelin (0.1-1.0 microM) with a reversible contractile response, in association with monophosphorylation and/or diphosphorylation of the myosin light chain 20. However, the influence of the contractile agonists was greatly reduced during logarithmic growth. These results suggest that in the SM-3 cells in the growth-arrested phase, there is a restoration of the contractile architecture and the myosin light chain phosphorylation system. Thus, this SM-3 cell line is expected to serve as a useful model for examining biochemical and physiological phenomena of smooth muscle.     

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.     

Mutation of a conserved glycine in the SH1-SH2 helix affects the load-dependent kinetics of myosin

The ATP hydrolysis rate and shortening velocity of muscle are load-dependent. At the molecular level, myosin generates force and motion by coupling ATP hydrolysis to lever arm rotation. When a laser trap was used to apply load to single heads of expressed smooth muscle myosin (S1), the ADP release kinetics accelerated with an assistive load and slowed with a resistive load; however, ATP binding was mostly unaffected. To investigate how load is communicated within the motor, a glycine located at the putative fulcrum of the lever arm was mutated to valine (G709V). In the absence of load, stopped-flow and laser trap studies showed that the mutation significantly slowed the rates of ADP release and ATP binding, accounting for the ~270-fold decrease in actin sliding velocity. The load dependence of the mutant's ADP release rate was the same as that of wild-type S1 (WT) despite the slower rate. In contrast, load accelerated ATP binding by ~20-fold, irrespective of loading direction. Imparting mechanical energy to the mutant motor partially reversed the slowed ATP binding by overcoming the elevated activation energy barrier. These results imply that conformational changes near the conserved G709 are critical for the transmission of mechanochemical information between myosin's active site and lever arm.     

Hereditary pituitary dwarfism in mice affects skeletal and cardiac myosin isozyme transitions differently

The dwarf mutation in mice interferes with the development of those anterior pituitary cells responsible for production of thyroid stimulating hormone, growth hormone, and prolactin. Myosin isozyme transitions in both cardiac and skeletal muscle were also found to be affected in this mutant. Electrophoresis of native myosins demonstrated that the fetal (V3) to adult (V1) ventricular cardiac isozyme transition was completely blocked in dwarf mice; in contrast, the neonatal to adult fast myosin transition in hind limb skeletal muscle was slowed but not totally inhibited. The persistence of neonatal myosin heavy chain for up to 55-75 d after birth in dwarf mice, as compared with 16 d in normal mice, was directly demonstrated by polypeptide and immunopolypeptide mapping. Morphological examination of 18-36-d-old dwarf skeletal muscles by optical and electron microscopy revealed a relative immaturity, but no signs of gross pathology were evident. Immunocytochemical analysis showed that the abnormal persistence of neonatal myosin occurs in most of the fibers. Multiple injections of thyroxine restored a normal isozyme complement to both cardiac and skeletal muscles within 11-15 d. Therefore, the effects of the dwarf mutation on myosin isozymes can be explained by the lack of thyroid hormone in these animals. Because the synthesis of growth hormone is not stimulated by thyroid hormone in dwarf mice as it would be in normal animals, these results demonstrate that thyroid hormone promotes myosin isozyme transitions independent of growth hormone production.     

Expression of SERCA2a is independent of innervation in regenerating soleus muscle

The speed of contraction of a skeletal muscle largely depends on the myosin heavy chain isoforms (MyHC), whereas the relaxation is initiated and maintained by the sarcoplasmic reticulum Ca²⁺-ATPases (SERCA). The expression of the slow muscle-type myosin heavy chain I (MyHCI) is entirely dependent on innervation, but, as we show here, innervation is not required for the expression of the slow-type sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a) in regenerating soleus muscles of the rat, although it can play a modulator role. Remarkably, the SERCA2a level is even higher in denervated than in innervated regenerating soleus muscles on day 7 when innervation is expected to resume. Later, the level of SERCA2a protein declines in denervated regenerated muscles but it remains expressed, whereas the corresponding mRNA level is still increasing. SERCA1 (i.e., the fast muscle-type isoform) expression shows only minor changes in denervated regenerating soleus muscles compared with innervated regenerating controls. When the soleus nerve was transected instead of the sciatic nerve, SERCA2a and MyHCI expressions were found to be even more uncoupled because the MyHCI nearly completely disappeared, whereas the SERCA2a mRNA and protein levels decreased much less. The transfection of regenerating muscles with constitutively active mutants of the Ras oncogene, known to mimic the effect of innervation on the expression of MyHCI, did not affect SERCA2a expression. These results demonstrate that the regulation of SERCA2a expression is clearly distinct from that of the slow myosin in the regenerating soleus muscle and that SERCA2a expression is modulated by neuronal activity but is not entirely dependent on it. slow type sarcoplasmic reticulum Ca²⁺ pump; MyHCI; nerve influence     

Heterogeneity in smooth muscle cell population accumulating in the neointimas and the media of poststenotic dilatation of the rabbit carotid artery.

Rabbit smooth muscles contain at least three types of myosin heavy chain (MHC) isoforms; SM1, SM2 and SMemb (NMHC-B), the expression of which is developmentally regulated. We have recently reported that smooth muscles with the embryonic phenotype accumulate in the neointimas produced by endothelial denudation or high-cholesterol feeding. In this study, we examined MHC isoform expression in the neointimas and the media of poststenotic dilatation of the rabbit carotid artery, and determined the phenotype of the smooth muscle cell in the dilated segment. We report here that neointimal cells in the dilated segment are smooth muscle cells with the embryonic phenotype as previously reported in our ballooning-injury study. The medial smooth muscles, however, are composed of heterogeneous population of smooth muscles which differ in stage of differentiation as determined by the MHC isoform expression. These results indicate that MHC isoforms are useful molecular markers to identify abnormally proliferating smooth muscles in diseased arteries and to understand the process of atherogenesis occurring following vascular injury.     

Effects of downstream migration on myosin heavy chain expression and dihydropyridine receptor density in farmed smolt of Atlantic salmon

The expression of myosin heavy chain (MHC) subunits and dihydropyridine receptors (DHPRs) from red and white tail muscles of cultured smolts of Atlantic salmon Salmo salar was analysed from samples taken: (1) before the fish were transferred to the river and (2) after the migration distance of c . 50 km. The relative work load of migrating fish, estimated on the basis of swimming speed of Atlantic salmon and discharge rate of the River Simojoki, Finland, was maximally 1700 times higher than that of fish in culture. The data show that after five migration days the density of DHPRs in the muscles of the smolts was increased already. Furthermore, the results indicated a transition from the fast‐to‐slow MHC isoform. Transfer of fish to a natural environment and downstream migration thus had a significant effect on the expression of muscle proteins.     

Competitive control of myosin expression: hypertrophy vs. hyperthyroidism

The competition between two opposing influences on the phenotypic expression of skeletal muscle myosin were studied to determine which was the dominant regulator. Experimental hyperthyroidism, which induces fast myosin expression, was produced by subcutaneous implantation of a 40-day constant-time-release triiodothyronine pellet. Compensatory hypertrophy, which induces slow myosin expression, was produced by surgical removal of a synergistic hindlimb muscle. Hyperthyroidism increased the percentage of type II fibers and the fast myosin isoforms in both the plantaris and soleus muscles. Hypertrophy significantly increased the percentage of type I fibers and the slow myosin type in the plantaris and soleus muscles. However, with the simultaneous introduction of hyperthyroidism and hypertrophy, only the hyperthyroid effects were observed. Hyperthyroidism and not physiological demand was found to be the dominant regulator of skeletal muscle myosin expression.     

Neural regulation of mammalian fast and slow muscle myosins: an electrophoretic analysis.

Mammalian nerves to fast and slow muscles have the remarkable property of changing the speed of contraction of muscles following cross-reinnervation. The biochemical basis of speed transformation is the change in myosin in ATPase activity. This paper provides electrophoretic evidence for structural changes in myosin from cross-reinnervated muscles. A method is described for the separation of intact fast and slow muscle myosins by polyacrylamide gel electrophoresis. This method utilizes the fact that ATP and its analogs prevent the formation of myosin polymers in low ionic strength buffers. In this system, normal fast muscle myosin has a higher electrophoretic mobility than slow muscle myosin. Normal rat soleus myosin has a major slow and a minor fast component due to two populations of muscle fibers. The same muscle cross-reinnervated by a fast muscle nerve shows only the fast component, The normal, homogeneous fast extensor digitorum longus muscle has only the electrophoretically fast myosin, but following cross-reinnervation it shows both fast and slow components. These results suggest that mammalian motor nerves can induce or suppress the expression of genes that code for fast and slow skeletal muscle myosins.     

Cyr61 mediates the expression of VEGF, alphav-integrin, and alpha-actin genes through cytoskeletally based mechanotransduction mechanisms in bladder smooth muscle cells.

Application of cyclic strain to bladder smooth muscle (SM) cells results in profound alterations of the histomorphometry, phenotype, and function of the cells. The onset of this process is characterized by the activation of a cascade of signaling events coupled to progressive and, perhaps, interdependent changes of gene expression. In particular, externally applied cyclic stretch to cultured bladder SM cells results in the transient expression of the Cyr61 gene that encodes a cysteine-rich heparin-binding protein originally described as a proangiogenic factor capable of altering the gene programs for angiogenesis, adhesion, and extracellular matrix synthesis. In this study, we investigated the effects of mechanical stretch-induced Cyr61 on the expression of potential mechanosensitive Cyr61 target genes and the signaling pathways involved. We showed that suppression of Cyr61 expression with an adenoviral vector encoding an antisense oligonucleotide reduced mechanical strain-induced VEGF, alpha(v)-integrin, and SM alpha-actin gene expression but had no effect on the myosin heavy chain isoforms SM-1 and SM-2. Signaling pathways involving RhoA GTPase, phosphatidyl inositol 3-kinase, and cytoskeletal actin dynamics altered stretch-induced Cyr61 and Cyr61 target genes. Reciprocally, adenovirus-mediated overexpression of Cyr61 in cells cultured under static conditions increased the expression of VEGF, alpha(v)-integrin, and SM alpha-actin, as well as that of SM-1 and SM-2 isoforms, suggesting that the effects of a sustained expression of Cyr61 extend to SM specific contractile function. These effects were dependent on integrity of the actin cytoskeleton. Together, these results indicate that Cyr61 is an important determinant of the genetic reprogramming that occurs in mechanically challenged cells.     

Regenerated rat fast muscle transplanted to the slow muscle bed and innervated by the slow nerve,exhibits an identical myosin heavy chain repertoire to that of the slow muscle

 The hypothesis that the limited adaptive range observed in fast rat muscles in regard to expression of the slow myosin is due to intrinsic properties of their myogenic stem cells was tested by examining myosin heavy chain (MHC) expression in regenerated rat extensor digitorum longus (EDL) and soleus (SOL) muscles. The muscles were injured by bupivacaine, transplanted to the SOL muscle bed and innervated by the SOL nerve. Three months later, muscle fibre types were determined. MHC expression in muscle fibres was demonstrated immunohistochemically and analysed by SDS-glycerol gel electrophoresis. Regenerated EDL transplants became very similar to the control SOL muscles and indistinguishable from the SOL transplants. Slow type 1 fibres predominated and the slow MHC-1 isoform was present in more than 90% of all muscle fibres. It contributed more than 80% of total MHC content in the EDL transplants. About 7% of fibres exhibited MHC-2a and about 7% of fibres coexpressed MHC-1 and MHC-2a. MHC-2x/d contributed about 5–10% of the whole MHCs in regenerated EDL and SOL transplants. The restricted adaptive range of adult rat EDL muscle in regard to the synthesis of MHC-1 is not rooted in muscle progenitor cells; it is probably due to an irreversible maturation-related change switching off the gene for the slow MHC isoform. Accepted: 11 June 1996     

Alternative N-terminal regions of Drosophila myosin heavy chain tune muscle kinetics for optimal power output

We assessed the influence of alternative versions of a region near the N-terminus of Drosophila myosin heavy chain on muscle mechanical properties. Previously, we exchanged N-terminal regions (encoded by alternative exon 3s) between an embryonic (EMB) isoform and the indirect flight muscle isoform (IFI) of myosin, and demonstrated that it influences solution ATPase rates and in vitro actin sliding velocity. Because each myosin is expressed in Drosophila indirect flight muscle, in the absence of other myosin isoforms, this allows for muscle mechanical and whole organism locomotion assays. We found that exchanging the flight muscle specific exon 3 region into the embryonic isoform (EMB-3b) increased maximum power generation (P(max)) and optimal frequency of power generation (f(max)) threefold and twofold compared to fibers expressing EMB, whereas exchanging the embryonic exon 3 region into the flight muscle isoform (IFI-3a) decreased P(max) and f(max) to approximately 80% of IFI fiber values. Drosophila expressing IFI-3a exhibited a reduced wing beat frequency compared to flies expressing IFI, which optimized power generation from their kinetically slowed flight muscle. However, the slower wing beat frequency resulted in a substantial loss of aerodynamic power as manifest in decreased flight performance of IFI-3a compared to IFI. Thus the N-terminal region is important in tuning myosin kinetics to match muscle speed for optimal locomotory performance.