Myosin Heavy Chain Protein Research Articles

Effect of diet-induced obesity and metabolic syndrome on skeletal muscles of Ossabaw miniature swine

Ossabaw swine fed excess kilocalorie diet develop metabolic syndrome (MS) characterized by obesity, hypertension, insulin resistance, and glucose intolerance with/without dyslipidemia. The purpose of this study was to test the hypothesis that MS would have a detrimental effect on skeletal muscle structure and cause changes in the expression of myosin heavy chains (MHCs). Adult male Ossabaw swine were fed for 24 wk high-fructose or high-fat/cholesterol/fructose diets to induce normolipidemic MS (MetS) or dyslipidemic MS (DMetS), respectively, and were compared with the lean swine on control diet. MetS swine showed mild MS, lacking increases in total and low density lipoprotein (LDL) cholesterol, both of which were highly upregulated in DMetS swine. There was an ∼1.2-fold increase in the cross-sectional areas of muscle fibers in MetS and DMetS groups compared with control for biceps femoris and plantaris muscles. In plantaris muscles, DMetS diet caused an ∼2-fold decrease in slow MHC mRNA and protein expression and an ∼1.2- to 1.8-fold increase in the number of intramyocellular lipid (IMCL) droplets without large changes in the size of the droplets. There was a trend to the decrease in slow MHC expression in muscles of swine on MetS diet. The number of IMCL droplets in muscle fibers of the MetS group was comparable to controls. These data correlate well with the data on total plasma cholesterol (control = 60, MetS = 70, and DMetS = 298 mg/dl) and LDL (control = 29, MetS = 30, and DMetS = 232 mg/dl). We conclude that structural changes observed in skeletal muscle of obese Ossabaw swine correlate with those previously reported for obese humans.

Mouse embryonic stem cell adherent cell differentiation and cytotoxicity (ACDC) assay

An adherent cell differentiation and cytotoxicity (ACDC) assay was developed using pluripotent J1 mouse embryonic stem cells (mESCs). Adherent mESCs were used to evaluate chemical-induced effects on both stem cell viability and differentiation using an in-cell western technique after a 9-day culture. DRAQ5/Sapphire700 stains were used to quantify cell number. Myosin heavy chain protein was used as a marker of cardiomyocyte differentiation and was corrected for cell number, thereby separating cytotoxicity and effects on differentiation. Acetic acid, 5-fluorouracil and bromochloroacetic acid were evaluated using the embryonic stem cell test and ACDC assay. Both systems distinguish the relative potencies of these compounds. TaqMan low-density arrays were used to characterize the time course of differentiation and effects of chemical exposure on multiple differentiation gene markers. The ACDC assay is a technique that can be used to evaluate the effects of xenobiotics on mESC differentiation and cell number using a single assay.

Changing pattern of gene expression is associated with ventricular myocyte dysfunction and altered mechanisms of Ca2+signalling in young type 2 Zucker diabetic fatty rat heart

The association between type 2 diabetes and obesity is very strong, and cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. The aim of this study was to investigate early changes in the pattern of genes encoding cardiac muscle regulatory proteins and associated changes in ventricular myocyte contraction and Ca(2+) transport in young (9- to 13-week-old) type 2 Zucker diabetic fatty (ZDF) rats. The amplitude of myocyte shortening was unaltered; however, time-to-peak shortening and time to half-relaxation of shortening were prolonged in ZDF myocytes (163 ± 5 and 127 ± 7 ms, respectively) compared with age-matched control rats (136 ± 5 and 103 ± 4 ms, respectively). The amplitude of the Ca(2+) transient was unaltered; however, time-to-peak Ca(2+) transient was prolonged in ZDF myocytes (66.9 ± 2.6 ms) compared with control myocytes (57.6 ± 2.3 ms). The L-type Ca(2+) current was reduced, and inactivation was prolonged over a range of test potentials in ZDF myocytes. At 0 mV, the density of L-type Ca(2+) current was 1.19 ± 0.28 pA pF(-1) in ZDF myocytes compared with 2.42 ± 0.40 pA pF(-1) in control myocytes. Sarcoplasmic reticulum Ca(2+) content, release and uptake and myofilament sensitivity to Ca(2+) were unaltered in ZDF myocytes compared with control myocytes. Expression of genes encoding various L-type Ca(2+) channel proteins (Cacna1c, Cacna1g, Cacna1h and Cacna2d1) and cardiac muscle proteins (Myh7) were upregulated, and genes encoding intracellular Ca(2+) transport regulatory proteins (Atp2a2 and Calm1) and some cardiac muscle proteins (Myh6, Myl2, Actc1, Tnni3, Tnn2, and Tnnc1) were downregulated in ZDF heart compared with control heart. A change in the expression of genes encoding myosin heavy chain and L-type Ca(2+) channel proteins might partly underlie alterations in the time course of contraction and Ca(2+) transients in ventricular myocytes from ZDF rats.

Novel mutation in MYH7 gene associated with distal myopathy and cardiomyopathy

A 25-year-old woman had childhood-onset muscle weakness and dilated cardiomyopathy. She exhibited predominantly distal weakness with early toe walking. Dilated cardiomyopathy required cardiac transplantation at age 15years. We identified a de-novo, heterozygous, missense mutation, c.2348G>C (p. Arg783Pro), in exon 21 of the MYH7 gene, which encodes slow skeletal muscle fiber/β-cardiac myosin heavy chain protein, that replaces a highly conserved arginine with a proline. This novel mutation that results in the unusual combined cardiac and skeletal muscle phenotype localizes to the essential light chain binding area, a region only previously shown to be mutated in hypertrophic cardiomyopathy.

Recombinant adenoviral microRNA-206 induces myogenesis in C2C12 cells

SummaryBackgroundThe expression of microRNA-206 (miR-206) is high in skeletal muscle but low in most other tissues. The expression of miR-206 is increased in muscular dystrophy, suggesting its involvement in the pathogenesis of muscle diseases. To determine the role of miR-206 in muscle cell differentiation and explore a possible gene therapy vector, we constructed a miR-206 adenoviral expression vector (AdvmiR-206) and tested for transfection into C2C12 stem cells.Material/MethodsA 355-bp PCR amplicon from C57B6 mouse skeletal muscle genomic DNA was inserted into the adenoviral shuttle vector pAdTrack-CMV, which was then co-transformed with the adenoviral backbone plasmid pAdEasy-1 into competent E. coli BJ5183 bacteria. The specificity and function of this recombinant adenoviral MiR-206 were studied in C2C12 cells by Northern blot, immunofluorescence, Western blot, and flow cytometry.ResultsIncreased expression of miR-206 in AdvmiR-206 transfected C2C12 cells (P<0.001) and resulted in morphological and biochemical changes over time that were similar to serum deprivation, including elongated cells and increased myosin heavy chain proteins. Even in the absence of serum deprivation, miR-206 overexpression accounted for a 50% reduction of S-phase cells (P<0.01). Moreover, in untransfected C2C12 cells, the introduction of miR-206-specific antisense oligoribonucleotides inhibited the normal response to serum deprivation. Twenty-four hours after lipofection of antisense oligoribonucleotides, the number of elongated cells was reduced by half (P<0.01).ConclusionsCollectively, these data support a role for miR-206 in myoblast differentiation. We foresee potential applications for the AdvmiR-206 vector in research and therapy.

Trans‐10, cis‐12 conjugated linoleic acid inhibits skeletal muscle differentiation and GLUT4 expression independently from NF‐κB activation

The capacity of skeletal muscle to contribute to glucose homeostasis depends on muscular insulin sensitivity. The expression of glucose transporter (GLUT)-4 is increased during myoblast differentiation, a process essential in maintenance of adult muscle. Therefore, processes that affect muscle differentiation may influence insulin dependent glucose homeostasis. Conjugated linoleic acids, and in particular trans-10, cis-12 CLA (t10, c12-CLA), are potent inducers of NF-kB in cultured skeletal myotubes, and NF-kB activation inhibits muscle differentiation. The aims of this study were to evaluate whether CLAs inhibit myogenic differentiation and lower GLUT4 mRNA expression and to address the involvement of NF-kB activation in potential effects of CLA on these processes. Incubation of C2C12 cells with t10, c12-CLA blocked the formation of myotubes, which was accompanied by reduced expression of the muscle specific genes creatine kinase, myogenin, myosin heavy chain perinatal and myosin heavy chain IIB, as well as decreased GLUT4 mRNA levels. However, genetic blockade of NF-kB was not sufficient to restore reduced myosin heavy chain protein expression following t10, c12-CLA treatment. Surprisingly, in contrast to myotubes, t10, c12-CLA was not able to activate NF-kB transcriptional activity in myoblasts. In conclusion, t10, c12-CLA inhibits myogenic differentiation and GLUT4 expression, independently from NF-kB activation.

Effects of Insulin-Like Growth Factor 1 on Muscle Atrophy and Motor Function in Rats with Brain Ischemia

Although insulin-like growth factor 1 (IGF 1) has been used in immobilizated muscles to prevent muscle atrophy, its effects on muscle atrophy after brain ischemia are not known. This study aimed to determine the effects of IGF 1 on preventing muscle atrophy in rats with brain ischemia. Middle cerebral artery occlusion (MCAO) was used to induce the brain ischemia. In the first part of the study, rats were assigned to sham control, ischemic control, and ischemia with different dosages of IGF 1 injection groups to determine the optimal dosage of IGF 1 on preventing muscle atrophy after brain ischemia. In the second part of the study, rats were assigned to sham control, ischemic control, ischemia with IGF 1, or with IGF 1 receptor inhibitor (AG1024) injection groups to determine the specificity of IGF 1 on preventing muscle atrophy after brain ischemia. IGF 1 or AG1024 was injected locally to calf muscles and anterior tibialis (TA) starting from one day after brain ischemia and injections were carried out every other day for 4 times. Muscle weight and myosin heavy chain (MHC) expression in both red (red gastrocnemius and soleus) and white (white gastrocnemius and TA) muscles were significantly decreased after brain ischemia. With at least moderate-dosage (200 ng/100 microl PBS) IGF 1 injection, the muscle weight and MHC protein could be restored in both red and white muscles resulting in better motor performance. However, the high-dose injection of IGF 1 (400 ng/100 microl PBS) did not result in further effects. IGF 1 increased the expression of p-Akt, but such effects were prevented by AG1024 resulting in muscle atrophy and poor motor function. In conclusion, peripheral application of IGF 1 not only prevented muscle atrophy but also enhanced motor function in rats with brain ischemia. The IGF 1-induced PI3K/Akt pathways are important for preventing muscle atrophy induced by brain ischemia.

Myosin heavy chain expression pattern as a marker for anabolic potency: desoxymethyltestosterone (madol), norandrostendione and testosterone repress MHC-IIb expression and stimulate MHC-IId/x expression in orchiectomized rat gastrocnemius muscle

Both 19-norandrostenedione (estr-4-ene-3,17-dione, NOR) and desoxymethyltestosterone (17alpha-methyl-5alpha-androst-2-en-17beta-ol, DMT or "madol") are 'designer steroids' misused for doping purposes in the bodybuilding scene. We have previously characterized the pharmacological profile of madol and identified potential adverse side effects. The aim of this study was to investigate the anabolic potency of NOR, madol and the reference substance testosterone propionate (TP). Besides wet weight of the M.levator ani (LA), we examined the effects on muscle fiber type composition and myosin heavy chain (MHC) expression in the M.gastrocnemius (Gas) muscle as additional markers for anabolic potency. A Hershberger assay was performed, where orchiectomized (orchi) male Wistar rats were treated subcutaneously with NOR, madol, TP or vehicle control (all 1 mg/kg BW/day) for 12 days. Wet weights of the Gas, LA, prostate and seminal vesicle were examined to determine anabolic and androgenic effects. Fiber type composition of the Gas muscle was analyzed using ATPase staining, and MHC protein profiles were determined by silver stain and Western blot analysis. NOR and madol exhibited strong anabolic and weak androgenic potency by stimulating growth of the LA but not the prostate and seminal vesicle. Skeletal muscle fiber type composition characterized by ATPase staining was not significantly altered between the treatment groups, although there was a tendency toward lower levels of type IIB and increased type IIA fibers in all treatment groups relative to orchi. MHC protein expression determined by Western blot and silver stain analysis revealed that MHC IId/x was significantly up-regulated, while MHC IIb was significantly down-regulated in NOR, madol and TP groups relative to orchi. There were no significant differences for MHC IIa and MHC I expression between groups. Results suggest that the observed MHC expression shift could serve as a molecular marker to determine anabolic activity of anabolic steroids at least in skeletal muscle of orchi rats. The molecular mechanisms as well as the androgen-dependent regulation of MHC expression in intact skeletal muscle remain to be further investigated.

TNF-α impairs regulation of muscle oxidative phenotype: implications for cachexia?

Chronic obstructive pulmonary disease (COPD) is characterized by weight loss, muscle wasting (in advanced disease ultimately resulting in cachexia), and loss of muscle oxidative phenotype (oxphen). This study investigates the effect of inflammation (as a determinant of muscle wasting) on muscle oxphen by using cell studies combined with analyses of muscle biopsies of patients with COPD and control participants. We analyzed markers (citrate synthase, β-hydroxyacyl-CoA dehydrogenase, and cytochrome c oxidase IV) and regulators (PGC-1α, PPAR-α, and Tfam) of oxphen in vastus lateralis muscle biopsies of patients with advanced COPD and healthy smoking control participants. Here 17 of 73 patients exhibited elevated muscle TNF-α mRNA levels. In these patients, significantly lower mRNA levels of all oxidative markers/regulators were found. Interestingly, these patients also had a significantly lower body mass index and tended to have less muscle mass. In cultured muscle cells, mitochondrial protein content and myosin heavy chain isoform I (but not II) protein and mRNA levels were reduced on chronic TNF-α stimulation. TNF-α also reduced mitochondrial respiration in a nuclear factor kappaB (NF-κB) -dependent manner. Importantly, TNF-α-induced NF-κB activation decreased promoter transactivation and transcriptional activity of regulators of mitochondrial biogenesis and muscle oxphen. In conclusion, these results demonstrate that TNF-α impairs muscle oxphen in a NF-κB-dependent manner.

Essential Role for p38α MAPK But Not p38γ MAPK in Igf2 Expression and Myoblast Differentiation

The muscle satellite cell is established as the major stem cell contributing to fiber growth and repair. p38 MAPK signaling is essential for myoblast differentiation and in particular for up-regulation of promyogenic Igf2 expression. p38 exists as four isoforms (alpha, beta, gamma, and delta), of which p38gamma is uniquely abundant in muscle. The aim of this study was to characterize p38 isoform expression and importance (using shRNA knockdown; demonstrated via both reduced protein and kinase activities) during myoblast differentiation. p38alpha and -gamma mRNA levels were most abundant in differentiating C2 cells with low/negligible contributions from p38beta and -delta, respectively. Increased phosphorylation of p38alpha and -gamma occurred during differentiation but via different mechanisms: p38alpha protein levels remained constant, whereas total p38gamma levels increased. Following shRNA knockdown of p38alpha, myoblast differentiation was dramatically inhibited [reduced myosin heavy chain (MHC), myogenin, pAkt protein levels]; significantly, Igf2 mRNA levels and promoter-reporter activities decreased. In contrast, knockdown of p38gamma induced a transient increase in both myogenin and MHC protein levels with no effect on Igf2 mRNA levels or promoter-reporter activity. Knockdown of p38alpha/beta markedly increased but that of p38gamma decreased caspase 3 activity, suggesting opposite actions on apoptosis. p38gamma was initially proposed to have a promyogenic function; however, p38gamma overexpression could not rescue reduced myoblast differentiation following p38alpha/beta inhibition. Therefore, p38alpha is essential for myoblast differentiation, and part of its action is to convert signals that indicate cell density into promyogenic gene expression in the form of the key peptide, IGF-II; p38gamma has a minor, yet opposing antimyogenic, function.

Differential effects of thin and thick filament disruption on zebrafish smooth muscle regulatory proteins

The smooth muscle actin binding proteins Caldesmon and Tropomyosin (Tm) promote thin filament assembly by stabilizing actin polymerization, however, whether filament assembly affects either the stability or activation of these and other smooth muscle regulatory proteins is not known. Measurement of smooth muscle regulatory protein levels in wild type zebrafish larvae following antisense knockdown of smooth muscle actin (Acta2) and myosin heavy chain (Myh11) proteins, and in colourless mutants that lack enteric nerves. Comparison of intestinal peristalsis in wild type and colourless larvae. Knockdown of Acta2 led to reduced levels of phospho-Caldesmon and Tm. Total Caldesmon and phospho-myosin light chain (p-Mlc) levels were unaffected. Knockdown of Myh11 had no effect on the levels of either of these proteins. Phospho-Caldesmon and p-Mlc levels were markedly reduced in colourless mutants that have intestinal motility comparable with wild type larvae. These in vivo findings provide new information regarding the activation and stability of smooth muscle regulatory proteins in zebrafish larvae and their role in intestinal peristalsis in this model organism.

Skeletal Muscle Signature of a Champion Sprint Runner

Human skeletal muscle contains three types of the main motor protein myosin heavy chain (MHC) and individual muscle fibers contain only one type of MHC (pure fiber types: I, IIa and IIx) or a mixture of MHC (hybrid fiber types: I/IIa and IIa/IIx). Each of these fiber types has a distinct phenotypic profile that contributes to human locomotion. The mixture of these fiber types within the muscle can have a profound impact on athletic performance. Our laboratory recently had the opportunity to examine the skeletal muscle profile of a World Record holder and World Champion sprint runner. PURPOSE: To examine single muscle fiber MHC composition (i.e. fiber type) and single muscle fiber contractile function (strength, speed and power) from this World Class sprinter. METHODS: A large number of isolated single muscle fibers from the vastus lateralis were analyzed for contractile function (n=53) and fiber type profile (n=329 fibers). RESULTS: Fiber type profile showed that 72% of the muscle contained fast motor proteins (MHC IIa and IIx). The novel finding was that the sprint champion's muscle contained 24% of the pure MHC IIx fiber type. Normal healthy human adult muscle contains less than 2% of the pure MHC IIx fiber type. Power output of the MHC IIx muscle fibers were the highest values recorded to date by a wide margin (35±4 W·l-1). Single fiber power output data from other athletic populations (swimmers and distance runners) and recreational athletes generally fall in the range of 5-10 W·l-1 and do not exceed 20 W·l-1. CONCLUSION: The unusually high abundance and high power output of the rare and powerful pure MHC IIx fiber type represent a unique muscle signature that provides a myocellular basis for the high level of athletic success achieved in sprinting events by this individual.

Electrical pulse stimulation induces contraction and oxidative modification of proteins in cultured C2C12 myotubes

Skeletal muscle cells produce free radicals which are believed necessary for effective muscle contraction. However, when a heightened free radical generation creates a redox imbalance, oxidative modification of contractile proteins may contribute to fatigue. We have pursued a cell culture model that allows us to quantify oxidative modification of contractile proteins. C2C12 myoblasts were grown to 90% confluence and differentiated into mature myotubes. Western blot analysis for myosin heavy chain (MHC) protein indicated expression by day five and maintenance of protein levels through day eight of differentiation. Electrical pulse stimulation (EPS) of myotubes on day five of differentiation produced visible, repetitive contraction of cells. A pulse frequency of 0.5 hz and duration of 24 ms for 90 minutes resulted in a 3.65 ± 0.74 fold increase in oxidative modification of MHC protein (six independent experiments). Oxidative modification of MHC protein was tempered (42 ± 15% of EPS alone) when cells were pre‐ and co‐incubated (during EPS) with 1mM Trolox (five independent experiments). This in vitro model shows promise for quantifying contraction induced oxidative modification of proteins in mature C2C12 myotubes. Further, this model could be used to develop knowledge of which nutritional antioxidants might prevent a redox imbalance in contracting skeletal muscle and thereby delay the onset of fatigue.

Estradiol and genistein antagonize the ovariectomy effects on skeletal muscle myosin heavy chain expression via ER-β mediated pathways

The age-related decline in ovarian sex hormone production following the onset of menopause alters skeletal muscle metabolic, structural and functional characteristics. The myosin heavy chain (MHC) expression pattern defines skeletal muscle contraction velocity and is therefore an important factor in skeletal muscle function. The present study was designed to examine the effects of 17β estradiol (E2), estrogen receptor (ER) subtype selective agonists (ERα, ERβ) or genistein (Gen) following ovary removal (OVX) in female Wistar rats in combination with a high intensity treadmill-based exercise protocol (Ex) or normal cage-based activity (NoEx) on MHC protein expression patterns in the slow fiber type m.Soleus (Sol) and the fast fiber type m.Gastrocnemius (Gas). Gen and E2 in the Sol significantly stimulated MHC-I expression relative to OVX only in the absence of exercise (NoEx). MHC-IIb expression in the Gas was significantly increased relative to OVX in Gen Ex and E2 Ex and NoEx groups. The estrogenic effects in the Sol and Gas were both predominantly mediated via ERβ pathways, since the ERβ agonist induced greater MHC increases than OVX or ERα. We therefore propose that high intensity exercise in combination with exposure to E2, Gen, ERα or ERβ agonists in OVX rats exerts differential effects on MHC expression in skeletal muscles composed of mainly slow type I MHC (Sol) or fast type II MHC (Gas). In summary, the data shows that MHC composition is affected by estrogens and exercise in a fiber type specific manner and that these effects are mainly mediated by ER-β. This is of great importance with respect to skeletal muscle health and potential treatment with ER selective agonists.

Sepsis-induced myocardial depression is associated with transcriptional changes in energy metabolism and contractile related genes: A physiological and gene expression-based approach*

Increased nitric oxide production and altered mitochondrial function have been implicated in sepsis-induced cardiac dysfunction. The molecular mechanisms underlying myocardial depression in sepsis and the contribution of nitric oxide in this process however, are incompletely understood. To assess the transcriptional profile associated with sepsis-induced myocardial depression in a clinically relevant mouse model, and specifically test the hypothesis that critical transcriptional changes are inducible nitric oxide synthase-dependent. Laboratory investigation. University affiliated research laboratory. C57/BL6 wild type and congenic B6 129P2-Nos2tm1Lau/J (iNOS) mice. Assessment of myocardial function after 48 hrs of induction of polymicrobial sepsis by caecal ligation and perforation. We compared the myocardial transcriptional profile in C57/BL6 wild type mice and congenic B6 129P2-Nos2tm1Lau/J litter mates after 48 hrs of polymicrobial sepsis induced by caecal ligation and perforation. Profiling of 22,690 expressed sequence tags by gene set enrichment analysis demonstrated that inducible nitric oxide synthase -/- failed to down regulate critical bioenergy and metabolism related genes including the gene for peroxisome proliferator-activated receptor gamma coactivator 1. Bioinformatics analysis identified a striking concordance in down regulation of transcriptional activity of proliferator-activated receptor gamma coactivator 1-related transcription factors resulting in sepsis associated myocardial remodeling as shown by isoform switching in the expression of contractile protein myosin heavy chain. In inducible nitric oxide synthase -/- deficient mice, contractile depression was minimal, and the transcriptional switch was absent. Metabolic and myosin isoform gene expression switch in sepsis-induced myocardial depression is inducible nitric oxide synthase-dependent. Furthermore, we suggest that the molecular switch favoring the expression of fetal isoforms of contraction related proteins is associated with regulation of proliferator-activated receptor gamma coactivator 1 and related transcription factors in an inducible nitric oxide synthase-dependent manner.

Evidence for a role of immunoproteasomes in regulating cardiac muscle mass in diabetic mice

The ubiquitin–proteasome system plays an important role in regulating muscle mass. Inducible immunoproteasome subunits LMP-2 and LMP-7 are constitutively expressed in the heart; however, their regulation and functions are poorly understood. We here investigated the hypothesis that immunoproteasomes regulate cardiac muscle mass in diabetic mice. Type 1 diabetes was induced in wildtype mice by streptozotocin. After hyperglycemia developed, insulin and the proteasome inhibitor epoxomicin were used to treat diabetic mice for 6weeks. Isolated mouse hearts were perfused with control or high glucose solution. Catalytic proteasome β-subunits and proteolytic activities were analyzed in the heart by immunoblotting and fluorogenic peptide degradation assays, respectively. Insulin and epoxomicin blocked loss of heart weight and improved cardiac function in diabetic mice. LMP-7 and its corresponding chymotryptic-like proteasome activity were increased in diabetic hearts and high glucose-treated hearts. Myosin heavy chain protein was decreased in diabetic hearts, which was largely reversed by epoxomicin. High glucose decreased LMP-2 protein levels in perfused hearts. In diabetic hearts, LMP-2 expression was downregulated whereas expression of the phosphatase and tensin homologue deleted on chromosome ten (PTEN) and the muscle atrophy F-box were upregulated. Moreover, mice with muscle-specific knockout of PTEN gene demonstrated increased cardiac muscle mass, while mice with LMP-2 deficiency demonstrated PTEN accumulation, muscle mass loss, and contractile impairment in the heart. Therefore, we concluded that high glucose regulates immunoproteasome subunits and modifies proteasome activities in the heart, and that dysregulated immunoproteasome subunits may mediate loss of cardiac muscle mass in experimental diabetic mice.

Contractile Properties of Human Fetal Skeletal Muscle Proteins

Congenital contracture syndromes associated with myofilament proteins are present in 1 out of every 1000 live births. Because the myosin heavy chain protein family has several isoforms unique to the pre-natal development of muscle, it is important to know the contractile properties of this tissue to determine how mutations affect performance and development. However, information on human fetal muscle contractile properties is lacking. We are beginning to characterize the contractile properties of this tissue using in vitro motility assays, where we can deconstruct which proteins of developing muscle can be attributed to differences seen in patients with congenital contracture syndromes. Embryonic myosin was isolated from human fetal muscle samples at 15.4 weeks gestation. An in vitro motility assay was performed at 30 degrees C with an ionic strength of 0.17 to determine the predominant myosin's enzymatic properties. The max speed of filaments on human fetal skeletal myosin (1.9 um/s) was significantly lower than filaments on adult rat skeletal myosin (4.8 um/s) under the same conditions. The Km values were similar between the human fetal (0.02 mM) and adult rat (0.03mM) myosins. Ongoing work includes studying the effects of increased ADP, substitution of ATP with dATP, studying Ca2+ regulation of in vitro motility assays using actin and thin filament regulatory proteins purified from fetal muscle of approximately the same gestational period, and skinned muscle cell experiments. By utilizing these assays, we can develop a more mature understanding of muscle contraction during development and form better hypotheses about the mechanism of how specific mutations lead to these contracture syndromes. Establishing these contractile properties will lead to better hypotheses regarding the development of contractures in utero and how it is affected by mutations associated with congenital contracture syndromes. Supported by HL65497 (Regnier) and HD48895 (Bamshad).

Blunting effect of hypoxia on the proliferation and differentiation of human primary and rat L6 myoblasts is not counteracted by Epo

The aim of this study was to evaluate whether hypoxia and/or erythropoietin would be able to modulate proliferation/differentiation processes of rat and human myoblasts. Rat L6 and primary human myoblasts were grown in 21% or 1% O(2) in the presence or absence of recombinant human erythropoietin (RhEpo). Presence of erythropoietin receptors (EpoR) was assayed using RT-PCR and Western blotting techniques. Cell proliferation was evaluated by determining the doubling time and kinetics of cultures by counting cells. Cell differentiation was analysed by determining myogenic fusion index using antibodies against the myosin heavy chain. Expression of myogenin and myosin heavy chain (MHC) proteins were evaluated using the Western blotting technique. After 96 h culture in growth medium for 2.5 and 9 h, doubling time of L6 and human primary myoblasts respectively, had increased in 1% O(2) conditions (P < 0.01). Kinetics of culture showed alteration in proliferation at 72 h in L6 myoblast cultures and at 4 days in human primary myoblasts. The myogenic fusion index had reduced by 30% in L6 myoblasts and by 20% in human myoblasts (P < 0.01). Expression of myogenin and MHC had reduced by around 50%. Despite presence of EpoR mRNA and protein, RhEpo did not counteract the effects of hypoxia either in L6 cells or in human myoblasts. The data show that exposure to hypoxic conditions (1% O(2)) of rat and human myoblasts altered their proliferation and differentiation processes. They also show that Epo is not an efficient growth factor to counteract this deleterious effect.

β2-Adrenergic agonist-induced hypertrophy of the quadriceps skeletal muscle does not modulate disease severity in the rodent meniscectomy model of osteoarthritis

SummaryObjectiveTo examine whether β2-adrenergic agonist-induced hypertrophy of the quadriceps skeletal muscle can modulate the severity of osteoarthritis (OA) in the rodent meniscectomy (MNX) model.MethodsMale Lewis rats were subcutaneously administered with 1.5 mg/kg/day clenbuterol hydrochloride (n = 15) or saline vehicle (n = 20) for 14 days. Following pre-treatment, five animals from each group were sacrificed to assess the immediate effects of clenbuterol. The remaining animals underwent either invasive knee surgery (clenbuterol pre-treated n = 10; saline pre-treated n = 10) or a sham control surgical procedure (saline pre-treated n = 5). During disease initiation and progression, weight bearing was assessed by hindlimb loading. Myosin heavy chain (MHC) protein isoforms were quantified by silver stained SDS PAGE. OA severity was graded by assessment of toluidine blue stained step coronal sections of the total knee joint.ResultsClenbuterol treatment resulted in an increase in total bodyweight, growth rate and in quadriceps skeletal muscle mass. Meniscal surgery resulted in the development of OA-like lesions, changes to weight bearing, and changes in MHC protein expression in the quadriceps. Clenbuterol-induced skeletal muscle hypertrophy had no effect on either weight bearing or articular pathology following MNX surgery.ConclusionsOur data reveal that clenbuterol-induced skeletal muscle hypertrophy is unable to mimic the beneficial clinical effects of increased musculature derived through targeted strength training in humans, in a rodent model of MNX-induced OA. In addition we observed fibre-type switching to “slow twitch” in the quadriceps muscle during the induction of OA that warrants further investigation as to its relationship to joint stability.

Pax‐3 and Pax‐7 Label Muscle Progenitor Cells During Myotomal Myogenesis in Coregonus lavaretus (Teleostei: Coregonidae)

In Coregonus lavaretus, prior the mesoderm segmentation, in cells adjacent to the notochord called adaxial cells MyoD and slow myosin heavy chain (MyHC-slow) proteins were observed. After somite formation, adaxial cells migrate towards the lateral part of the myotome and form a layer of red muscles. Deeper cells differentiate into white muscle fibres. In situ hybridization using Pax-3 molecular probe revealed, that after somitogenesis, Pax-3 is expressed in a layer of cells superficial to the myotome resembling the "external cells" (found in many teleosts species) or dermomyotome described in Amniota. During later developmental stages Pax-3 gene is expressed in cells in intermyotomal space and then in myoblasts between myotubes. In these cells Pax-7 protein was also observed. Pax-3/7 positive cells which have migrated into the myotomes differentiate into satellite cells/secondary myoblasts and participate in hypertrophic and hyperplastic growth of muscles.