Drosophila indirect flight muscle specific Act88F actin mutants as a model system for studying congenital myopathies of the human ACTA1 skeletal muscle actin gene

Most human ACTA1 skeletal actin gene mutations cause dominant, congenital myopathies often with severely reduced muscle function and neonatal mortality. High sequence conservation of actin means many mutated ACTA1 residues are identical to those in the Drosophila Act88F, an indirect flight muscle specific sarcomeric actin. Four known Act88F mutations occur at the same actin residues mutated in ten ACTA1 nemaline mutations, A138D/P, R256H/L, G268C/D/R/S and R372C/S. These Act88F mutants were examined for similar muscle phenotypes. Mutant homozygotes show phenotypes ranging from a lack of myofibrils to almost normal sarcomeres at eclosion. Aberrant Z-disc-like structures and serial Z-disc arrays, ‘zebra bodies’, are observed in homozygotes and heterozygotes of all four Act88F mutants. These electron-dense structures show homologies to human nemaline bodies/rods, but are much smaller than those typically found in the human myopathy. We conclude that the Drosophila indirect flight muscles provide a good model system for studying ACTA1 mutations.

Mg(2+) Ions Bind at the C-Terminal Region of Skeletal Muscle alpha-Tropomyosin

Tropomyosin (Tm) is a dimeric coiled-coil protein that polymerizes through head-to-tail interactions. These polymers bind along actin filaments and play an important role in the regulation of muscle contraction. Analysis of its primary structure shows that Tm is rich in acidic residues, which are clustered along the molecule and may from sites for divalent cation binding. In a previous study, we showed that the Mg(2+)-induced increase in stability of the C-terminal half of Tin is sensitive to imitations near the C-terminus. In the present report, we study the interaction between Mg(2+) and full-length Tin and smaller fragments corresponding to the last 65 and 26 Tin residues. Although the smaller Tin peptide (Tm(259-284(W269))) is flexible and to large extent unstructured, the larger Tm(220-284(W269)) fragments forms a coiled coil in solution whose stability increases significantly in the presence of Mg(2+). NMR analysis shows thin Mg(2+) induces chemical shift perturbations in both Tm(220-284(W269)) and Tm(259-284(W269)) in the vicinity of His276, in which are located several negatively charged residues. (C) 2009 Wiley Periodicals, Inc. Biopolymers 91: 583-590, 2009.

NDRG2 promotes myoblast proliferation and caspase 3/7 activities during differentiation, and attenuates hydrogen peroxide - but not palmitate-induced toxicity

The function of the stress-responsive N-myc downstream-regulated gene 2 (NDRG2) in the control of myoblast growth, and the amino acids contributing to its function, are not well characterized. Here, we investigated the effect of increased NDRG2 levels on the proliferation, differentiation and apoptosis in skeletal muscle cells under basal and stress conditions. NDRG2 overexpression increased C2C12 myoblast proliferation and the expression of positive cell cycle regulators, cdk2, cyclin B and cyclin D, and phosphorylation of Rb, while the serine/threonine-deficient NDRG2, 3A-NDRG2, had less effect. The onset of differentiation was enhanced by NDRG2 as determined through the myogenic regulatory factor expression profiles and myocyte fusion index. However, the overall level of differentiation in myotubes was not different. While NDRG2 up-regulated caspase 3/7 activities during differentiation, no increase in apoptosis was measured by TUNEL assay or through cleavage of caspase 3 and PARP proteins. During H2O2 treatment to induce oxidative stress, NDRG2 helped protect against the loss of proliferation and ER stress as measured by GRP78 expression with 3A-NDRG2 displaying less protection. NDRG2 also attenuated apoptosis by reducing cleavage of PARP and caspase 3 and expression of pro-apoptotic Bax while enhancing the pro-survival Bcl-2 and Bcl-xL levels. In contrast, Mcl-1 was not altered, and NDRG2 did not protect against palmitate-induced lipotoxicity. Our findings show that NDRG2 overexpression increases myoblast proliferation and caspase 3/7 activities without increasing overall differentiation. Furthermore, NDRG2 attenuates H2O2-induced oxidative stress and specific serine and threonine amino acid residues appear to contribute to its function in muscle cells.

High-level production of functional muscle alpha-tropomyosin in Pichia pastoris

Although numerous studies have reported the production of skeletal muscle alpha -tropomyosin in E. coli, the protein needs to be modified at the amino terminus in order to be active. Without these modifications the protein does not bind to actin, does not exhibit head-to-tail polymerization, and does not inhibit the actomyosin Mg2+-ATPase in the absence of troponin. on the other hand, the protein produced in insect cells using baculovirus as an expression vector (Urbancikova, M., and Hitchcock-DeGregori, S. E., J. Biol. Chem., 269, 24310-24315, 1994) is only partially acetylated at its amino terminal and therefore is not totally functional. In an attempt to produce an unmodified functional recombinant muscle alpha -tropomyosin for structure-function correlation studies we have expressed the chicken skeletal alpha -tropomyosin cDNA in the yeast Pichia pastoris. Recombinant protein was produced at a high level (20 mg/L) and was similar to the wild type muscle protein in its ability to polymerize, to bind to actin and to regulate the actomyosin S1 Mg2+-ATPase. (C) 2001 Academic Press.

STUDIES INTO THE MOLECULAR MECHANISMS REGULATING MUSCULAR ATROPHY RESULTING FROM HINDLIMB IMMOBILIZATION IN THE RAT (PROTEIN, SYNTHESIS, RNA)

The purpose of the work performed in this dissertation was to examine some of the possible regulatory mechanisms involved in the initiation of muscular atrophy during periods of decreased muscle utilization resulting from hindlimb immobilization in the rat. A 37% decrease in the rate of total muscle protein synthesis which has been observed to occur in the first 6 h of immobilization contributes significantly to the observed loss of protein during immobilization.^ The rates of cytochrome c and actin synthesis were determined in adult rat red vastus lateralis and gastrocnemius muscles, respectively, by the constant infusion and incorporation of ('3)H-tyrosine into protein. The fractional synthesis rates of both actin and cytochrome c were significantly decreased (P < 0.05) in the 6th h of hindlimb immobilization.^ RHA was extracted from adult rat gastrocnemius muscle by modification of the phenol: chloroform: SDS extraction procedures commonly used for preparation of RNA for hybridization analysis from other mammalian tissues. RNA content of rat gastrocnemius muscle, as determined by this method of extraction and its subsequent quantification by UV absorbance and orcinol assay, was significantly greater than the RNA content previously determined for adult rat gastrocnemius by other commonly employed methods.^ RNA extracted by this method from gastrocnemius muscles of control and 6h immobilized rats was subjected to "dot blot" hybridization to ('32)P-labelled probe from plasmid p749, containing a cDNA sequence complementary to (alpha)-actin mRNA and from rat skeletal muscle. (alpha)-Actin specific mRNA content as estimated by this procedure is not significantly decreased in rat gastrocnemius following 6h or hindlimb immobilization. However, (alpha)-actin specific mRNA content is significantly decreased (P < 0.05) in adult rat gastrocnemius (alpha)-actin specific mRNA is not decreased in adult rat gastrocnemius muscle following 6h of immobilization, a time when actin synthesis is significantly decreased, it is concluded that a change in (alpha)-actin specific mRNA content is not the initiating event responsible for the early decrease in actin synthesis observed in the 6th h of immobilization. ^

Upregulation of alpha-skeletal muscle actin and myosin heavy polypeptide gene products in degenerating rotator cuff muscles

Impaired function of shoulder muscles, resulting from rotator cuff tears, is associated with abnormal deposition of fat in muscle tissue, but corresponding cellular and molecular mechanisms, likely reflected by altered gene expression profiles, are largely unknown. Here, an analysis of muscle gene expression was carried out by semiquantitative RT-PCR in total RNA extracts of supraspinatus biopsies collected from 60 patients prior to shoulder surgery. A significant increase of alpha-skeletal muscle actin (p = 0.0115) and of myosin heavy polypeptide 1 (p = 0.0147) gene transcripts was observed in parallel with progressive fat deposition in the muscle, assessed on parasagittal T1-weighted turbo-spin-echo magnetic resonance images according to Goutallier. Upregulation of alpha-skeletal muscle actin and of myosin heavy polypeptide-1 has been reported to be associated with increased muscle tissue metabolism and oxidative stress. The findings of the present study, therefore, challenge the hypothesis that increased fat deposition in rotator cuff muscle after injury reflects muscle degeneration.

ROR alpha regulates the expression of genes involved in lipid homeostasis in skeletal muscle cells - Caveolin-3 and CPT-1 are direct targets of ROR

The staggerer mice carry a deletion in the RORalpha gene and have a prolonged humoral response, overproduce inflammatory cytokines, and are immunodeficient. Furthermore, the staggerer mice display lowered plasma apoA-I/-II, decreased plasma high density lipoprotein cholesterol and triglycerides, and develop hypo-alpha-lipoproteinemia and atherosclerosis. However, relatively little is known about RORalpha in the context of target tissues, target genes, and lipid homeostasis. For example, RORalpha is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for similar to40% of total body weight and 50% of energy expenditure. This lean tissue is a primary site of glucose disposal and fatty acid oxidation. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. In particular, the role of RORalpha in skeletal muscle metabolism has not been investigated, and the contribution of skeletal muscle to the ROR-/- phenotype has not been resolved. We utilize ectopic dominant negative RORalpha expression in skeletal muscle cells to understand the regulatory role of RORs in this major mass peripheral tissue. Exogenous dominant negative RORalpha expression in skeletal muscle cells represses the endogenous levels of RORalpha and -gamma mRNAs and ROR-dependent gene expression. Moreover, we observed attenuated expression of many genes involved in lipid homeostasis. Furthermore, we show that the muscle carnitine palmitoyltransferase-1 and caveolin-3 promoters are directly regulated by ROR and coactivated by p300 and PGC-1. This study implicates RORs in the control of lipid homeostasis in skeletal muscle. In conclusion, we speculate that ROR agonists would increase fatty acid catabolism in muscle and suggest selective activators of ROR may have therapeutic utility in the treatment of obesity and atherosclerosis.

Endurance training in humans leads to fiber type-specific increases in levels of peroxisome proliferator-activated receptor-[gamma] coactivator-1 and peroxisome proliferator-activated receptor-[alpha] in skeletal muscle

The peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1 (PGC-1) can induce mitochondria biogenesis and has been implicated in the development of oxidative type I muscle fibers. The PPAR isoforms α, β/δ, and γ control the transcription of genes involved in fatty acid and glucose metabolism. As endurance training increases skeletal muscle mitochondria and type I fiber content and fatty acid oxidative capacity, our aim was to determine whether these increases could be mediated by possible effects on PGC-1 or PPAR-α, -β/δ, and -γ. Seven healthy men performed 6 weeks of endurance training and the expression levels of PGC-1 and PPAR-α, -β/δ, and -γ mRNA as well as the fiber type distribution of the PGC-1 and PPAR-α proteins were measured in biopsies from their vastus lateralis muscle. PGC-1 and PPAR-α mRNA expression increased by 2.7- and 2.2-fold (P < 0.01), respectively, after endurance training. PGC-1 expression was 2.2- and 6-fold greater in the type IIa than in the type I and IIx fibers, respectively. It increased by 2.8-fold in the type IIa fibers and by 1.5-fold in both the type I and IIx fibers after endurance training (P < 0.015). PPAR-α was 1.9-fold greater in type I than in the II fibers and increased by 3.0-fold and 1.5-fold in these respective fibers after endurance training (P < 0.001). The increases in PGC-1 and PPAR-α levels reported in this study may play an important role in the changes in muscle mitochondria content, oxidative phenotype, and sensitivity to insulin known to be induced by endurance training.

17beta-estradiol upregulates the expression of peroxisome proliferator-activated receptor alpha and lipid oxidative genes in skeletal muscle

This study examined the actions of 17β-estradiol (E2) and progesterone on the regulation of the peroxisome proliferator-activated receptors (PPARα and PPARγ) family of nuclear transcription factors and the mRNA abundance of key enzymes involved in fat oxidation, in skeletal muscle. Specifically,
carnitine palmitoyltransferase I (CPT I), β-3-hydroxyacyl CoA dehydrogenase (β-HAD), and pyruvate dehydrogenase kinase 4 (PDK4) were examined. Sprague–Dawley rats were ovariectomized and treated with placebo (Ovx), E2, progesterone, or both hormones in combination (E+P). Additionally,
sham-operated rats were treated with placebo (Sham) to serve as controls. Hormone (or vehicle only) delivery was via time release pellets inserted at the time of surgery, 15 days prior to analysis. E2 treatment increased PPARα mRNA expression and protein content (P<0·05), compared with Ovx treatment. E2 also resulted in upregulated mRNA of CPT I and PDK4 (P<0·05). PPARγ mRNA expression was also increased (P<0·05) by E2 treatment, although protein content remained unaltered. These data
demonstrate the novel regulation of E2 on PPARα and genes encoding key proteins that are pivotal in regulating skeletal muscle lipid oxidative flux.

Alterations in the proportions of skeletal muscle proteins following a unilateral lesion to the substantia nigra pars compacta of rats

It is well established that mammalian skeletal muscles exhibit a considerable degree of plasticity and one of the main determining factors of this plasticity is the activity pattern and duration of motoneurone discharge. Lesions to the right substantia nigra pars compacta (SNpc) of six adult rats were made to determine whether altered output from the SNpc ultimately leads to a change in the expression of proteins in contralateral skeletal muscles. After 4 months, altered motor performance was identified by the administration of amphetamine. After 7 months, 30–70% of dopaminergic cells in the SNpc had been destroyed. The protein content of muscles was then quantified from densitometric scans of gels, and expressed as a % of the amount of actin (the protein used as a reference in this study). The lesion affected the expression of different protein isoforms in the fast- and slow-twitch muscles. In slow-twitch soleus muscles, the lesion decreased the proportion of α-tropomyosin and increased the proportion of β-tropomyosin. In the fast-twitch extensor digitorum longus muscles, the lesion increased the proportion of the fast isoform of troponin-T_1f, and decreased the proportions of the two isoforms of myosin light chain. This study establishes a connection between the chronic effects of a lesion to the SNpc, with a loss of dopaminergic neurones, impaired motor performance, and altered expression of proteins in skeletal muscle. The implication of these results is that the altered motor function observed in Parkinson’s disease may be associated with alterations to the expression of skeletal muscle proteins.

The STARS signaling pathway: a key regulator of skeletal muscle function

During the last decade, the striated muscle activator of Rho signaling (STARS), a muscle-specific protein, has been proposed to play an increasingly important role in skeletal muscle growth, metabolism, regeneration and stress adaptation. STARS influences actin dynamics and, as a consequence, regulates the myocardin-related transcription factor A/serum response factor (MRTF-A/SRF) transcriptional program, a well-known pathway controlling skeletal muscle development and function. Muscle-specific stress conditions, such as exercise, positively regulates, while disuse and degenerative muscle diseases are associated with a downregulation of STARS and its downstream partners, suggesting a pivotal role for STARS in skeletal muscle health. This review provides a comprehensive overview of the known role and regulation of STARS and the members of its signaling pathway, RhoA, MRTF-A and SRF, in skeletal muscle.

A non-cross-bridge, static tension is present in permeabilized skeletal muscle fibers after active force inhibition or actin extraction

Cornachione AS, Rassier DE. A non-cross-bridge, static tension is present in permeabilized skeletal muscle fibers after active force inhibition or actin extraction. Am J Physiol Cell Physiol 302: C566-C574, 2012. First published November 16, 2011; doi: 10.1152/ajpcell.00355.2011.-When activated muscle fibers are stretched, there is a long-lasting increase in the force. This phenomenon, referred to as "residual force enhancement," has characteristics similar to those of the " static tension," a long-lasting increase in force observed when muscles are stretched in the presence of Ca2+ but in the absence of myosin-actin interaction. Independent studies have suggested that these two phenomena have a common mechanism and are caused either by 1) a Ca2+-induced stiffening of titin or by 2) promoting titin binding to actin. In this study, we performed two sets of experiments in which activated fibers (pCa(2+) 4.5) treated with the myosin inhibitor blebbistatin were stretched from 2.7 to 2.8 mu m at a speed of 40 L-o/s, first, after partial extraction of TnC, which inhibits myosin-actin interactions, or, second, after treatment with gelsolin, which leads to the depletion of thin (actin) filaments. We observed that the static tension, directly related with the residual force enhancement, was not changed after treatments that inhibit myosin-actin interactions or that deplete fibers from troponin C and actin filaments. The results suggest that the residual force enhancement is caused by a stiffening of titin upon muscle activation but not with titin binding to actin. This finding indicates the existence of a Ca2+-regulated, titin-based stiffness in skeletal muscles.

Peroxisome proliferator-activated receptor {gamma} coactivator 1{alpha} (PGC-1{alpha}) promotes skeletal muscle lipid refueling in vivo by activating de novo lipogenesis and the pentose phosphate pathway

Exercise induces a pleiotropic adaptive response in skeletal muscle, largely through peroxisome proliferator-activated receptor coactivator 1 (PGC-1 ). PGC-1 enhances lipid oxidation and thereby provides energy for sustained muscle contraction. Its potential implication in promoting muscle refueling remains unresolved, however. Here, we investigated a possible role of elevated PGC-1 levels in skeletal muscle lipogenesis in vivo and the molecular mechanisms that underlie PGC-1 -mediated de novo lipogenesis. To this end, we studied transgenic mice with physiological overexpression of PGC-1 and human muscle biopsies pre- and post-exercise. We demonstrate that PGC-1 enhances lipogenesis in skeletal muscle through liver X receptor -dependent activation of the fatty acid synthase (FAS) promoter and by increasing FAS activity. Using chromatin immunoprecipitation, we establish a direct interaction between PGC-1 and the liver X receptor-responsive element in the FAS promoter. Moreover, we show for the first time that increased glucose uptake and activation of the pentose phosphate pathway provide substrates for RNA synthesis and cofactors for de novo lipogenesis. Similarly, we observed increased lipogenesis and lipid levels in human muscle biopsies that were obtained post-exercise. Our findings suggest that PGC-1 coordinates lipogenesis, intramyocellular lipid accumulation, and substrate oxidation in exercised skeletal muscle in vivo.

Studies on regulation of skeletal muscle differentiation by growth factors

Skeletal muscle differentiation involves sequential events in which proliferating undifferentiated myoblasts withdraw from the cell cycle and fuse to form multinucleated myotubes. The process of fusion is accompanied by the disappearance of proteins associated with cell proliferation and the coordinate induction of a battery of muscle-specific gene products, which includes the muscle isoenzyme of creatine kinase, nicotinic acetylcholine receptor, and contractile proteins such as alpha-actin. The molecular events associated with myogenesis are particularly amenable to experimental analysis because the events which occur in vivo can be recapitulated in vitro using established muscle cell lines. Initiation of myogenic differentiation in vitro can be achieved by removing serum from the culture medium. Myogenesis, therefore, can be considered to be regulated through a repression-type of mechanism by components in serum. The objectives of this project were to identify the components involved in regulation of myogenesis and approach the mechanism(s) whereby these components achieve their regulatory function. Initially, the effects of a series of polypeptide growth factors on myogenesis were examined. Among them TGF$\beta$ and FGF were found to be potent inhibitors of myogenic differentiation which did not affect cell proliferation. The inhibitory effects of these growth factors on differentiation requires their persistent presence in the culture medium. After myoblasts have undergone fusion, they become refractory to the inhibitory effects of TGF$\beta$, FGF, and serum. When fusion is inhibited by the presence of EGTA, a Ca$\sp{2+}$ chelator, muscle-specific genes are expressed reversibly upon removal of inhibitory growth factors. Subsequent exposure of biochemically differentiated cells to serum or TGF$\beta$ leads to down-regulation of muscle-specific genes. Stimulation with serum also leads to reentry of myocytes into the cell cycle, whereas fused myotubes are irreversibly and terminally differentiated. Measurement of levels of TGF$\beta$ receptors reveals that under non-fusing conditions, TGF$\beta$ receptor levels in biochemically differentiated myocytes remained as high as in undifferentiated myoblasts, while during terminal differentiation, TGF$\beta$ receptors decreased at least five-fold. Thus, down-regulation of TGF$\beta$ receptors is coupled to irreversible differentiation, but not reversible differentiation in the absence of fusion. The possible involvement of second messenger systems, such as cAMP and protein kinase C, in the pathway(s) by which TGF$\beta$, FGF, or serum factors transduce their signals from the cell surface to the nucleus was also examined. The results showed that myogenic differentiation is subject to negative regulation through cAMP elevation-dependent and cAMP elevation-independent pathways and that serum mitogens, TGF$\beta$ and FGF inhibit differentiation through a mechanism independent of cAMP-elevation or protein kinase C activation. ^