α-cardiac-like myosin heavy chain MHCIα is not upregulated in transforming rat muscle

The expression of MHCI, an -cardiac-like myosin heavy chain isoform, was studied in extensor digitorum longus (EDL) and tibialis anterior (TA) rat muscles undergoing fast-to-slow transition by chronic low-frequency stimulation (CLFS), a condition inducing a transient upregulation of MHCI in rabbit muscle. In order to enhance the transformation process, CLFS was applied to hypothyroid rats. mRNA analyses were performed by RT-PCR, and studies at the protein level by immunoblotting and immunohistochemistry, using the F88 antibody (F88 12F8,1) demonstrated in the accompanying paper to be specific for MHCI. In total RNA preparations from slow- and fast-twitch muscles, MHCI mRNA was present at minute levels, at least three orders of magnitude lower than in cardiac atrium. As verified immunohistochemically, MHCI is present only in intrafusal fibres of rat muscle. Moreover, MHCI is not expressed in extrafusal fibres and, contrary to the rabbit, was not upregulated at both the mRNA and protein levels by CLFS. These results support our notion of species-specific responses to CLFS. Another antibody reported to be specific to MHCI, BA-G5, was also investigated by immunoblot and immunohistochemical analyses. Its specificity could not be validated for skeletal muscles of the rat. BG-A5 was shown to cross-react with MHCIIb and MHCI. These results question an upregulation of MHCI in transforming rat muscles as reported in studies based on the use of this antibody.     

Post-translational phosphorylation of the slow/β myosin heavy chain isoform in adult rabbit masseter muscle

Four different phenotypes of slow muscle fibers, characterized by differential epitope expression in the slow/ myosin heavy chain (MyHC) isoform, have been identified in adult rabbit masseter muscle. We investigated the role of post-translational phosphorylation in the expression of these four phenotypes. Serial cryostat sections were treated either with alkaline phosphatase to dephosphorylate proteins in the tissue, or with a brain kinase solution and ATP to phosphorylate them, and then stained, using four antibodies that bind specifically to the slow/ MyHC isoform. In sections pre-treated with phosphatase, immunoreactivity to antibody A4.840 was abolished, but it could be restored by subsequent kinase/ATP treatment or ATP alone, indicating that the expression of its epitope requires phosphorylation. Phosphatase treatment resulted in an exposure of the epitope for antibody A4.951 in cells that normally bind this antibody only weakly or not at all, but since heat treatment alone produced similar effects, the role of phosphorylation in this enhancement is less certain. Immunoreactivity to antibodies S58 and BA-D5 were not influenced by phosphatase pre-treatment. Kinase/ATP treatment was only effective in changing antibody binding when tissues already had been phosphatase treated. We interpret these results to mean that sites of potential phosphorylation may already be occupied by O-linked glycosylation.     

Functional properties of skinned rabbit skeletal and cardiac muscle preparations containing α-cardiac myosin heavy chain

Contractile properties of skinned muscle fibres from the masseter muscle and strips of heart atrium muscle from rabbits, both containing the -cardiac myosin heavy chain isoform (-cardiac MHC), were investigated and compared with those of other skeletal muscle fibre types. The stretch-induced delayed force increase (stretch activation) was investigated on maximally Ca²⁺-activated skinned preparations as an index of the kinetic properties of the myosin heads of various MHC isoforms. Skeletal muscle fibres containing exclusively -cardiac MHC (type ) and muscle strips of heart atrium showed specific kinetics of stretch activation intermediate between those of types IIA and I fibres. In agreement with available data the unloaded shortening velocity V _u of type fibres was also intermediate between that of types IIA and I fibres. Compared with skeletal muscle type fibres, muscle strips of heart atrium exhibited significantly (P<0.001) faster kinetics of both stretch activation and V _u. We conclude that type fibres have characteristic kinetic properties that fill the gap between the fast type II and the slow type I fibres, and the following order of velocity can be established: IIB>IID(X)>IIA>>I.     

Expression of an α-cardiac like myosin heavy chain in diaphragm,chronically stimulated,and denervated fast-twitch muscles of rabbit

An additional slow fibre type, type I, is detected in diaphragm and appears in fast-twitch hindlimb muscles of rabbit under the influence of altered neuromuscular activity. Type I fibres were delineated from fibres expressing myosin heavy chain I (type I) by immunohistochemistry with a monoclonal antibody raised against the -cardiac MHCI. When stained for mATPase after acid and alkaline preincubations, some type I fibres resembled type I and type IIA fibres, respectively. Some type I fibres displayed dissimilar mATPase staining, indicating heterogeneity of this fibre population. The appearance of numerous type I fibres in stimulated muscles, which in addition contain type IIA and type I fibres, suggested that they may be interspaced between types IIA and I. Electrophoresis under nondenaturing conditions disclosed an additional isomyosin both in normal diaphragm and stimulated muscles. This band displayed the same mobility as the slowest isomyosin in rabbit masseter muscle. It was recognized by the same monoclonal (anti-- cardiac MHC) antibody used for immunohistochemistry. Therefore, this isomyosin appeared to be very similar, but perhaps not identical to the -cardiac MHC-based isomyosin, probably resulting from discrete differences in the MHC complement. This assumption agrees with additional findings suggesting an even greater heterogeneity of the MHCs than generally assumed. In support of this, we show in atrium and masseter muscles the existence of an additional, electrophoretically distinct MHC isoform which migrates in close vicinity to MHCI     

Jaw-closing muscles of kangaroos express α-cardiac myosin heavy chain

The masseter muscle of eutherian grazing mammals typically express or slow myosin heavy chain (MyHC). Myosins in the masseter of 4 species of kangaroos and a slow limb muscle of one of them were compared with their cardiac myosin by pyrophosphate and sodium dodecyl sulphate (SDS) gel electrophoresis, immunoblotting and immunohistochemistry. It was found that ventricular muscle contains three isoforms homologous to V₁ (-MyHC homodimer), V₂ (heterodimer) and V₃ (-MyHC homodimer) of eutherian cardiac muscle, and that the masseter contained V₁, with traces of V₂ and V₃, in great contrast to eutherian ruminants, which express only V₃. A polyclonal antibody (anti-KJM) was raised in rabbits against red kangaroo masseter myosin. After cross-absorption against limb muscle myofibrils, anti-KJM specifically reacted in Westerns with MyHCs from masseter but not limb muscles, and immunohistochemically with masseter, but not limb muscle fibers. In pyrophosphate Western blots, anti-KJM reacted with V₁ but not with V₃. However, a monoclonal antibody specific for eutherian slow myosin stained all kangaroo slow muscle fibers but only weakly stained scattered fibers in the masseter. The SDS-PAGE revealed that light chain composition of masseter and ventricular myosins is identical, but isoforms of both light chains of kangaroo limb slow myosin were observed. These results confirm that kangaroo jaw muscle express -MyHC rather than -MyHC. The difference in MyHC gene expression between marsupial and eutherian grazers may be related to the fact that kangaroos are not ruminants, and have only a single chance to comminute food into fine particles, hence the need for the greater speed and power of muscle contraction associated with V₁ containing muscle fibers.     

Effect of glucocorticoïd receptor ligands on myosin heavy chains expression in rat skeletal muscles during controllable stress

The influence of agonist (dexamethasone) and antagonist (mifepristone) of glucocorticoïd receptor during controllable painless stress was evaluated on myosin heavy chains expression in three masticatory and two nape rat muscles: anterior digastric (AD), anterior temporalis (AT), masseter superficialis (MS), longissimus capitis (L) and rectus capitis dorsalis major (R). The relative amounts of myosin heavy chain (MHC) protein isoform contained were significantly affected in four muscles studied by dexamethasone and in three muscles studied under mifepristone, versus control during the stress procedure, after only 1 week of treatment. The control group AT muscles contained respectively 18.2% of MHC 2A, 34.5% of MHC 2X and 47.4% of MHC 2B. The effects of dexamethasone and mifepristone were opposite in this muscle: under dexamethasone, the relative proportions of the three isoforms were 14.2, 31.0 and 54.8%: consequently, MHC 2A and 2X decreased with the profit of 2B. Under mifepristone, the relative proportions were 21.1, 36.6 and 42.3% (MHC 2A and 2X increased to the detriment of 2B). The L muscle was not affected by the two treatments and MS muscle was only affected by dexamethasone. Dexamethasone increased MHC 2B to the detriment of MHC 2A in MS, AD and R. Mifepristone and dexamethasone induced the same changes in AD. The mifepristone treatment decreased the MHC 2X profile in R. Under dexamethasone, four muscles exhibited a significantly higher proportion of the more rapid isoforms than under mifepristone. A previous work showed that controllable stress induced a marked increase in the relative expression of MHC 2B in the same skeletal muscles (Martrette et al., 1998). Our results confirm then a significant participation of glucocorticoïd in MHC isoform expression during controllable stress.     

Expression of human β-myosin heavy chain fragments inEscherichia coli; localization of actin interfaces on cardiac myosin

Summary A cDNA clone coding for an internal fragment of slow-cardiac-myosin heavy chain was isolated from agt10 human skeletal muscle library. Six overlapping cDNA subclones, which span myosin heavy chain subregions and presumably interact with actin, were derived from this clone, fused to a-galactosidase vector and expressed inEscherichia coli. Three of the subclones were obtained by PCR (polymerase chain reaction) which enables gene or cDNA fragments to be amplified independently of preexisting restriction sites. Initially, various experiments were carried out using a long MHC (myosin heavy chain) fusion protein containing the 50 kDa-20 kDa connecting region, the whole 20 kDa region and the short subfragment 2 region. This MHC fusion protein was chemically or proteolytically cleaved in the same conditions as the native myosin molecule. Whole and truncated forms of the MHC fusion protein were separated on polyacrylamide gels, electroblotted on nitrocellulose sheets and renatured. They were then assayed in overlay experiments with F-actin and/or myosin light chains in solution. Specific antibodies were used to detect interactions between heavy chain fragments and F-actin or light chains. We thus observed that one long heavy chain fragment synthesized byE. coli behaved like proteolytic or chemical MHC preparations made from native myosin molecules. Two chymotryptic fragments of the MHC fusion protein, which are soluble at low ionic strength, cosedimented with F-actin in solution. Our results demonstrate that, in actin overlay experiments with whole fusion proteins, interactions seem to be due to the heavy chain fragment, not to the bacterial component. All interactions were non ATP-sensitive. We further investigated the possible participation of the six recombinant MHC fragments in contributing to the actomyosin interfaces on the 50 kDa-20 kDa regions of the human cardiac-MHC. The present procedure, which enables the synthesis of any MHC fragment independent of any protease site, is a powerful new tool for studying structure-function relationships within the myosin molecule family.     

The in vitro motility activity of β-cardiac myosin depends on the nature of the β-myosin heavy chain gene mutation in hypertrophic cardiomyopathy

Several mutations in the -myosin heavy chain gene cause hypertrophic cardiomyopathy. This study investigates (1) the in vitro velocities of translocation of fluorescently-labelled actin by -myosin purified from soleus muscle of 30 hypertrophic cardiomyopathy patients with seven distinct -myosin heavy chain gene mutations: Thr124Ile, Tyr162Cys, Gly256Glu, Arg403Gln, Val606Met, Arg870His, and Leu908Val mutations; and (2) motility activity of -myosin purified from cardiac and soleus muscle biopsies in the same patients. The velocity of translocation of actin by -myosin purified from soleus or cardiac muscle of 22 normal controls was 0.48 ± 0.09 m s–1. By comparison, the motility activity was reduced in all 30 patients with -myosin heavy chain gene mutations (range, 0.112 ± 0.041 to 0.292 ± 0.066 m s–1). Notably, the Tyr162Cys and Arg403Gln mutations demonstrated significantly lower actin sliding velocities: 0.123 ± 0.044, and 0.112 ± 0.041 m s–1, respectively. -myosin purified from soleus muscle from four patients with the Arg403Gln mutation had a similar actomyosin motility activity compared to -myosin purified from their cardiac biopsies (0.127 ± 0.045 m s–1 versus 0.119 ± 0.068 m s–1, respectively). Since these seven mutations lie in several distinct functional domains, it is likely that the mechanisms of their inhibitions of motility are different     

The expression of α-dystrobrevin and dystrophin during skeletal muscle regeneration

The expression of α-dystrobrevin and dystrophin in rat tibialis anterior muscles was chronologically evaluated during a cycle of regeneration after myonecrosis induced by the injection of cardiotoxin. In immunohistochemical studies, α-dystrobrevin and dystrophin were first stained weakly at the sarcolemma of some regenerating muscle fibers on day 5. On day 7, α-dystrobrevin was still stained weakly, whereas dystrophin was stained conspicuously. After day 10, α-dystrobrevin and dystrophin were both stained conspicuously on almost all regenerating muscle fibers. In the Western blot analysis, α-dystrobrevin and dystrophin were first detected as visible bands on days 5 and 7, respectively. The bands of α-dystrobrevin and dystrophin both darkened sequentially up to day 10. The protein levels based on the densitometrical analysis of the bands on each day were converted to the percentage of the protein level on day 28, which was taken as 100%. The sequential line based on these data showed that α-dystrobrevin and dystrophin reached 50% of the protein level on day 28 by 6.6 and 5.3 days, respectively. These data provide evidence that α-dystrobrevin regenerates more slowly than dystrophin in skeletal muscle. This revised version was published online in July 2006 with corrections to the Cover Date.     

The expression of dystrophin and α1-syntrophin during skeletal muscle regeneration

The expression of dystrophin and 1-syntrophin in rat tibialis anterior muscles were evaluated during a cycle of regeneration after myonecrosis induced by the injection of cardiotoxin. Immunohistochemical studies were performed in cryosections of muscles on days 1, 3, 5, 7, 10, 14, 21 and 28 after injection of cardiotoxin. Western blot analysis was also examined in muscle on days 1, 3, 5, 7, 10, 14, 21 and 28. In immunohistochemical studies, dystrophin was stained weakly at the sarcolemma of some regenerating muscle fibers on day 3, and by day 10 it was stained strongly on almost all regenerating muscle fibers. 1-syntrophin was stained weakly at the sarcolemma of some regenerating fibers on day 5, and by day 14 it was detected on all regenerating muscle fibers. In Western blot analysis, dystrophin (DYS1) and 1-syntrophin (1S) were completely absent on day 1. Re-expression of DYS1 and 1S was visible by day 5 and accelerated thereafter. The Western blots of DYS1 and 1S were densitometrically analyzed on each day. The protein levels on each day were converted to the percentage of the protein level on day 28, which was taken as 100%. From the sequential line based on these data, the following results were obtained on the chronological course of DYS1 and 1S. DYS1: 25% of the protein level on day 28 was reached by 3.5 days, 50% was reached by 5.3 days, and 90% was reached by 6.9 days. 1S: 25% of the protein level on day 28 was reached by 4.6 days, 50% was reached by 6.0 days, and 90% was reached by 12.5 days. In this study, DYS1 regenerated earlier than 1S at the sarcolemma of regenerating muscle fibers.     

Effects of β-guanidinopropionic acid-feeding on the patterns of myosin isoforms in rat fast-twitch muscle

Administration of -guanidinopropionic acid (-GPA) to rats as 1% of their diet for 6 weeks led to an accumulation of -GPA and -GPA-phosphate and to a depletion of creatine and phosphocreatine in the fast-twitch plantaris muscle. Adenosine triphosphate concentration was also decreased. Electrophoretic analyses were performed to investigate the effects of -GPA on the patterns of fast (FM) and slow (SM) isomyosins, myosin heavy chain (HC) isoforms and myosin light chain (LC) isoforms. The relative concentrations of fast isomyosins FM1 and FM2 decreased, whereas slow isomyosin SM increased. The increase in slow isomyosin corresponded to an increase in the relative concentration of the slow myosin HCI. The changes of the myosin light chain pattern consisted of increases in the relative concentrations of the two slow isoforms, LC1sb and LC2s, and decreases in the fast isoforms LC2f and LC3f. These results demonstrate that -GPA administration, leading to a depletion in energy-rich phosphates and a reduced phosphorylation potential, has an impact on myosin isoform expression in rat fast-twitch skeletal muscle.     

Exercise with low glycogen increases PGC-1α gene expression in human skeletal muscle

Recent studies suggest that carbohydrate restriction can improve the training-induced adaptation of muscle oxidative capacity. However, the importance of low muscle glycogen on the molecular signaling of mitochondrial biogenesis remains unclear. Here, we compare the effects of exercise with low (LG) and normal (NG) glycogen on different molecular factors involved in the regulation of mitochondrial biogenesis. Ten highly trained cyclists (VO_2max 65 ± 1 ml/kg/min, W _max 387 ± 8 W) exercised for 60 min at approximately 64 % VO_2max with either low [166 ± 21 mmol/kg dry weight (dw)] or normal (478 ± 33 mmol/kg dw) muscle glycogen levels achieved by prior exercise/diet intervention. Muscle biopsies were taken before, and 3 h after, exercise. The mRNA of peroxisome proliferator-activated receptor-γ coactivator-1 was enhanced to a greater extent when exercise was performed with low compared with normal glycogen levels (8.1-fold vs. 2.5-fold increase). Cytochrome c oxidase subunit I and pyruvate dehydrogenase kinase isozyme 4 mRNA were increased after LG (1.3- and 114-fold increase, respectively), but not after NG. Phosphorylation of AMP-activated protein kinase, p38 mitogen-activated protein kinases and acetyl-CoA carboxylase was not changed 3 h post-exercise. Mitochondrial reactive oxygen species production and glutathione oxidative status tended to be reduced 3 h post-exercise. We conclude that exercise with low glycogen levels amplifies the expression of the major genetic marker for mitochondrial biogenesis in highly trained cyclists. The results suggest that low glycogen exercise may be beneficial for improving muscle oxidative capacity.     

Effects of myostatin deficiency on PGC-1α and FNDC5 expression in three different murine muscle types

Myostatin (MSTN) is a key negative regulator of muscle growth and development. Skeletal, cardiac, and smooth muscles were isolated from MSTN knockout (MSTN?∕?) and control mice to investigate the effect of knocking out MSTN on peroxisome proliferator-activated receptor 1 coactivator (PGC-1α)-III and fibronectin domain 5 (FNDC5) expression. Various molecular biology techniques were used to analyze the changes in PGC-1α-FNDC5 in different muscle types from MSTN?∕? mice. The expression levels of PGC-1α and FNDC5 in the skeletal, cardiac, and smooth muscles of MSTN?∕? mice differed from those in the skeletal, cardiac, and smooth muscles of normal mice. This study revealed that knocking out MSTN resulted in inconsistent PGC-1α and FNDC5 expression in specific muscles. It proved for the first time that MSTN deletion attenuated the expression of PGC-1α and FNDC5 in three different murine muscle types. MSTN deletion may have additional effects on the status ofFNDC5 expression. Further research, however, is needed to confirm this conclusion.     

No effect of twitchin phosphorylation on the rate of myosin head detachment in molluscan catch muscle: are myosin heads involved in the catch state?

Phosphorylation of twitchin is known to abolish the catch state of anterior byssus retractor muscle (ABRM) of the bivalve mollusc Mytilus edulis. To investigate the role of myosin head involvement in force maintenance during catch, the effect of twitchin phosphorylation on myosin head detachment was studied in saponin-skinned fibre bundles of ABRM. The detachment rate of myosin heads was deduced from two types of experiments: (1) force decay after stepwise stretch of maximally Ca²⁺-activated fibre bundles (pCa 4.5) and (2) force decay from high-force rigor, the former induced by a stepwise increase in ATP concentration elicited by photolysis of caged ATP (pCa<8). The rate of detachment was not affected by thiophosphorylation or phosphorylation of twitchin by 0.12 mM cAMP in the presence of the phosphatase inhibitor cyclosporine A (1 M). Conversely, measurements of the rate of stretch-induced delayed force increase (stretch activation) and of the force increase following an ATP step in low-force rigor (pCa 4.5) suggest that the rate of myosin head attachment decreases after twitchin phosphorylation. We conclude that catch is not due to myosin heads remaining attached to actin filaments, but depends on myofilament interconnections that break down when twitchin is phosphorylated.     

Cloning of a phospholipase C-δ1 of rabbit skeletal muscle

Summary The phospholipase C isoform responsible for the increase in the total myoplasmic inositol 1,4,5-trisphosphate concentration during tetanic contraction of isolated skeletal muscle and its mechanism of activation is not known. We have cloned and sequenced a phospholipase C cDNA of rabbit skeletal muscle coding for a protein of 745 amino acids with a molecular mass of 84 440 kDa. The deduced amino acid sequence exhibits the phospholipase C-specific domains X and Y which according to current knowledge very likely represent the catalytic centre of the enzyme. An overall sequence homology of 88% to the phospholipase C-1 of rat brain suggests that the encoded protein represents a phospholipase C-1 isoform of rabbit skeletal muscle. Northern blot analysis shows, that this phospholipase C- is dominantly expressed in skeletal muscle, less strongly in smooth muscle (uterus) and lung and weakly in heart, kidney and brain. In the N-terminal part of the primary structure a consensus sequence for a canonical EF-hand Ca²⁺ binding domain can be identified together with a short positively charged motif which recently has been suggested to be essential for the binding of phosphatidylinositol 4,5-bisphosphate. If these two domains which are unique for phospholipase C- are sufficient in establishing a mechanism for the activation of the enzyme, inositol 1,4,5-trisphosphate formation in skeletal muscle could be the consequence of an increase in myoplasmic Ca²⁺.     

Study of hypoxia-induced expression of HIF-1α in retina pigment epithelium

It was shown that the expression of HIF-1α in retinal pigment epithelium increased under hypoxic conditions. Eight hours after the start of hypoxic exposure, the expression of HIF-1α reached the peak and sustained after 24-hour hypoxia. However, the morphology of PRE cells began to change and the expression of HIF-1α decreased after long-term (48-hour) hypoxia. Hypoxia-induced increase in the level of HIF-1α in RPE. It can be an important step in choriodal neovascularization. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 3, pp. 293–297, March, 2007