Interleukin-6 promotes myogenic differentiation of mouse skeletal muscle cells: role of the STAT3 pathway.

Myogenic differentiation of skeletal muscle cells is characterized by a sequence of events that include activation of signal transducer and activator of transcription 3 (STAT3) and enhanced expression of its target gene Socs3. Autocrine effects of IL-6 may contribute to the activation of the STAT3-Socs3 cascade and thus to myogenic differentiation. The importance of IL-6 and STAT3 for the differentiation process was studied in C2C12 cells and in primary mouse wild-type and IL-6(-/-) skeletal muscle cells. In differentiating C2C12 myoblasts, the upregulation of IL-6 mRNA expression and protein secretion started after increased phosphorylation of STAT3 on tyrosine 705 and increased mRNA expression of Socs3 was observed. Knockdown of STAT3 and IL-6 mRNA in differentiating C2C12 myoblasts impaired the expression of the myogenic markers myogenin and MyHC IIb and subsequently myotube fusion. However, the knockdown of IL-6 did not prevent the induction of STAT3 tyrosine phosphorylation. The IL-6-independent activation of STAT3 was verified in differentiating primary IL-6(-/-) myoblasts. The phosphorylation of STAT3 and the expression levels of STAT3, Socs3, and myogenin during differentiation were comparable in the primary myoblasts independent of the genotype. However, IL-6(-/-) cells failed to induce MyHC IIb expression to the same level as in wild-type cells and showed reduced myotube formation. Supplementation of IL-6 could partially restore the fusion of IL-6(-/-) cells. These data demonstrate that IL-6 depletion during myogenic differentiation does not reduce the activation of the STAT3-Socs3 cascade, while IL-6 and STAT3 are both necessary to promote myotube fusion.

Therapeutic inhibition of miR-208a improves cardiac function and survival during heart failure.

BACKGROUND: Diastolic dysfunction in response to hypertrophy is a major clinical syndrome with few therapeutic options. MicroRNAs act as negative regulators of gene expression by inhibiting translation or promoting degradation of target mRNAs. Previously, we reported that genetic deletion of the cardiac-specific miR-208a prevents pathological cardiac remodeling and upregulation of Myh7 in response to pressure overload. Whether this miRNA might contribute to diastolic dysfunction or other forms of heart disease is currently unknown. METHODS AND RESULTS: Here, we show that systemic delivery of an antisense oligonucleotide induces potent and sustained silencing of miR-208a in the heart. Therapeutic inhibition of miR-208a by subcutaneous delivery of antimiR-208a during hypertension-induced heart failure in Dahl hypertensive rats dose-dependently prevents pathological myosin switching and cardiac remodeling while improving cardiac function, overall health, and survival. Transcriptional profiling indicates that antimiR-208a evokes prominent effects on cardiac gene expression; plasma analysis indicates significant changes in circulating levels of miRNAs on antimiR-208a treatment. CONCLUSIONS: These studies indicate the potential of oligonucleotide-based therapies for modulating cardiac miRNAs and validate miR-208 as a potent therapeutic target for the modulation of cardiac function and remodeling during heart disease progression.

Gene transfer, expression, and sarcomeric incorporation of a headless myosin molecule in cardiac myocytes: evidence for a reserve in myofilament motor function.

The purpose of this study was to implement a living myocyte in vitro model system to test whether a motor domain-deleted headless myosin construct could be incorporated into the sarcomere and affect contractility. To this end we used gene transfer to express a "headless" myosin heavy chain (headless-MHC) in complement with the native full-length myosin motors in the cardiac sarcomere. An NH2-terminal Flag epitope was used for unique detection of the motor domain-deleted headless-MHC. Total MHC content (i.e., headless-MHC+endogenous MHC) remained constant, while expression of the headless-MHC in transduced myocytes increased from 24 to 72 h after gene transfer until values leveled off at 96 h after gene transfer, at which time the headless-MHC comprised ∼20% of total MHC. Moreover, immunofluorescence labeling and confocal imaging confirmed expression and demonstrated incorporation of the headless-MHC in the A band of the cardiac sarcomere. Functional measurements in intact myocytes showed that headless-MHC modestly reduced amplitude of dynamic twitch contractions compared with controls (P<0.05). In chemically permeabilized myocytes, maximum steady-state isometric force and the tension-pCa relationship were unaltered by the headless-MHC. These data suggest that headless-MHC can express to 20% of total myosin and incorporate into the sarcomere yet have modest to no effects on dynamic and steady-state contractile function. This would indicate a degree of functional tolerance in the sarcomere for nonfunctional myosin molecules.

Skeletal muscle cellularity and expression of myogenic regulatory factors and myosin heavy chains in rainbow trout (Oncorhynchus mykiss): effects of changes in dietary plant protein sources and amino acid profiles.

The nutritional regulation of skeletal muscle growth is very little documented in fish. The aim of the study presented here was to determine how changes in dietary plant protein sources and amino acid profiles affect the muscle growth processes of fish. Juvenile rainbow trout (Oncorhynchys mykiss) were fed two diets containing fish meal and a mixture of plant protein sources either low (control diet) or rich in soybean meal (diet S). Both diets were supplemented with crystalline indispensable amino acids (IAA) to match the rainbow trout muscle IAA profile. Diet S was also supplemented with glutamic acid, an AA present in high quantities in trout muscle. Rainbow trout fed diets C and S were not significantly different in terms of overall somatic growth or daily nitrogen gain, although their parameters of dietary protein utilisation differed. Distribution of skeletal white muscle fibre diameter and expression of certain selected muscle genes were also affected by dietary changes. In the white muscle, diet S led to a significant decrease (x0.9) in the mean and median diameters of muscle fibres, to a significant decrease (x0.6) in the expression of MyoD and to a significant increase (x1.7) in the expression of fast-MHC, with no significant changes in myogenin expression. There was no change in the expression of the genes analysed in lateral red muscle (MyoD, MyoD2, myogenin and slow-MHC). These results demonstrated that changes occurred in skeletal white muscle cellularity and expression of MyoD and fast-MHC, although overall growth and protein accretion were not modified, when a diet rich in soybean meal and glutamic acid was ingested. Present findings also indicated that the white and red muscles of rainbow trout are differently affected by nutritional changes.

Does the beta2-agonist clenbuterol help to maintain myocardial potential to recover during mechanical unloading?

OBJECTIVE: Chronic mechanical unloading induces left ventricular (LV) atrophy, which may impair functional recovery during support with an LV-assist device. Clenbuterol, a beta2-adrenergic receptor (AR) agonist, is known to induce myocardial hypertrophy and might prevent LV atrophy during LV unloading. Furthermore, beta2-AR stimulation is reported to improve Ca2+ handling and contribute to antiapoptosis. However, there is little information on the effects of clenbuterol during LV unloading. METHODS AND RESULTS: We investigated LV atrophy and function after LV unloading produced by heterotopic heart transplantation in isogenic rats. After transplantation, rats were randomized to 1 of 2 groups (n=10 each). The clenbuterol group received 2 mg.kg(-1).d(-1) of the drug for 2 weeks; the control group received normal saline. The weight of unloaded control hearts was 48% less than that of host hearts after 2 weeks of unloading. Clenbuterol significantly increased the weight of the host hearts but did not prevent unloading-induced LV atrophy. Papillary muscles were isolated and stimulated, and there was no difference in developed tension between the 2 groups. However, the inotropic response to the beta-AR agonist isoproterenol significantly improved in the clenbuterol group. The mRNA expression of myocardial sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) and fetal gene shift (myosin heavy chain [MHC] mRNA isozyme) was also significantly improved by clenbuterol treatment. There was no difference in beta1-AR mRNA expression between the 2 groups. In contrast, beta2-AR mRNA was significantly decreased in the clenbuterol-treated, unloaded heart. This indicates that clenbuterol may downregulate beta2-ARs. In the evaluation of apoptosis, mRNA expression of caspase-3, which is the central pathway for apoptosis, tended to be better in the clenbuterol group. CONCLUSIONS: During complete LV unloading, clenbuterol did not prevent myocardial atrophy but improved gene expression (SERCA2a, beta-MHC) and beta-adrenergic responsiveness and potentially prevented myocardial apoptosis. However, chronic administration of clenbuterol may be associated with downregulation of beta2-ARs.

Inducible effects of icariin, icaritin, and desmethylicaritin on directional differentiation of embryonic stem cells into cardiomyocytes in vitro.

AIM: To investigate the possible inducible effects of icariin, icaritin, and desmethylicaritin on the directional differentiation of embryonic stem (ES) cells into cardiomyocytes in vitro. METHODS: ES cells were cultivated as embryoid bodies (EBs) in hanging drops with icariin, icaritin, or desmethylicaritin. ES cells treated with retinoic acid and with solvent were used as positive and negative controls, respectively. The cardiomyocytes derived from the ES cells were verified using immunocytochemistry. The expression of cardiac developmental-dependent genes was detected using the reverse transcription-polymerase chain reaction (RT-PCR) method. Cell cycle distribution and apoptosis were analyzed using flow cytometry to determine the partly inducible effect mechanisms involved. RESULTS: The total percentage of beating EBs treated with 10(-7) mol/L icariin, icaritin, or desmethylicaritin was 87% (P<0.01), 59% (P<0.01), and 49%, respectively. All the beating cardiomyocytes derived from the ES cells expressed cardiac-specific proteins for a-actinin and troponin T. Among them, 10(-7) mol/L icariin treatment resulted in a significantly advanced and increased mRNA level of a-cardiac major histocompatibility complex (MHC) and myosin light chain 2v (MLC-2v) in EBs in the early cardiac developmental stage. Before shifting to the cardiomyocyte phenotype, icariin could evoke the accumulation of ES cells in G0/G1 and accelerate apoptosis of the cell population (P<0.05). CONCLUSION: Icariin facilitated the directional differentiation of ES cells into cardiomyocytes at a concentration of 10(-7) mol/L. The promoting effect of icariin on cardiac differentiation was related to increasing and accelerating gene expression of a-cardiac MHC and MLC-2v, as well as regulating the cell cycles and inducing apoptosis.

Non-canonical Wnt signaling enhances differentiation of human circulating progenitor cells to cardiomyogenic cells.

Human endothelial circulating progenitor cells (CPCs) can differentiate to cardiomyogenic cells during co-culture with neonatal rat cardiomyocytes. Wnt proteins induce myogenic specification and cardiac myogenesis. Here, we elucidated the effect of Wnts on differentiation of CPCs to cardiomyogenic cells. CPCs from peripheral blood mononuclear cells were isolated from healthy volunteers and co-cultured with neonatal rat cardiomyocytes. 6-10 days after co-culture, cardiac differentiation was determined by alpha-sarcomeric actinin staining of human lymphocyte antigen-positive cells (fluorescence-activated cell-sorting analysis) and mRNA expression of human myosin heavy chain and atrial natriuretic peptide. Supplementation of co-cultures with Wnt11-conditioned medium significantly enhanced the differentiation of CPCs to cardiomyocytes (1.7+/-0.3-fold), whereas Wnt3A-conditioned medium showed no effect. Cell fusion was not affected by Wnt11-conditioned medium. Because Wnts inhibit glycogen synthase kinase-3beta, we further determined whether the glycogen synthase kinase-3beta inhibitor LiCl also enhanced cardiac differentiation of CPCs. However, LiCl (10 mM) did not affect CPC differentiation. In contrast, Wnt11-conditioned medium time-dependently activated protein kinase C (PKC). Moreover, the PKC inhibitors bisindolylmaleimide I and III significantly blocked differentiation of CPCs to cardiomyocytes. PKC activation by phorbol 12-myristate 13-acetate significantly increased CPC differentiation to a similar extent as compared with Wnt11-conditioned medium. Our data demonstrate that Wnt11, but not Wnt3A, augments cardiomyogenic differentiation of human CPCs. Wnt11 promotes cardiac differentiation via the non-canonical PKC-dependent signaling pathway.

Mitochondrial and sarcoplasmic proteins, but not myosin heavy chain, are sensitive to leucine supplementation in old rat skeletal muscle.

Leucine has a major anabolic impact on muscle protein synthesis in young as in old animals. However, myosin heavy chain (MHC), sarcoplasmic and mitochondrial proteins may differently respond to anabolic factors, especially during aging. To test this hypothesis, fractional synthesis rates (FSR) of the three muscle protein fractions were measured using a flooding dose of [1-(13)C] phenylalanine, in gastrocnemius muscle of adult (8 months) and old (22 months) rats, either in postabsorptive state (PA), or 90-120 min after ingestion of a alanine-supplemented meal (PP+A) or a leucine-supplemented meal (PP+L). In adult and old rats, in comparison with PA, leucine stimulated mitochondrial (adult: 0.260+/-0.011 vs 0.238+/-0.012%h(-1); old: 0.289+/-0.010 vs 0.250+/-0.010%h(-1); PP+L vs PA, P<0.05) and sarcoplasmic (adult: 0.182+/-0.011 vs 0.143+/-0.006%h(-1); old: 0.195+/-0.010 vs 0.149+/-0.008%h(-1); PP+L vs PA, P<0.05) protein FSR, but not MHC synthesis in old rats (0.101+/-0.009 vs 0.137+/-0.018%h(-1); PP+L vs PA, P=NS). In conclusion, synthesis of specific muscle protein is activated by leucine supplementation, but MHC may be less sensitive to anabolic factors with aging.

Differential gene expression in infarct scar and viable myocardium from rat heart following coronary ligation.

Post-myocardial infarction (MI) remodeling of cardiac myocytes and the myocardial interstitium results in alteration of gross ventricular geometry and ventricular dysfunction. To investigate the mechanisms of the remodeling process of the heart after large MI, the expression of various genes in viable left ventricle and infarct scar tissue were examined at 16 weeks post-MI. Steady-state expression of Na(+)-K+ ATPase alpha-1 and -2, phospholamban (PLB), alpha-myosin heavy chain (alpha-MHC), ryanodine receptor (Rya) and Ca2+ ATPase (Serca2) mRNAs were decreased in the infarct scar vs noninfarcted sham-operated controls (P < 0.05). On the other hand, Gialpha2 and beta-MHC mRNAs were upregulated (P < 0.05, respectively) in the infarct scar whereas Na(+)-K+ ATPase-beta, Na(+)-Ca2+ exchanger and Gs mRNAs were not altered vs control values. In viable left ventricle, the alpha-1 subunit of Na(+)-K+ ATPase, alpha-3, beta-isoforms, Rya, beta-MHC, Gialpha2, Gs and Na(+)-Ca2+ exchanger were significantly elevated while expression of the alpha-2 subunit of Na(+)-K+ ATPase, PLB and Serca2 were significantly decreased compared to controls. Expression of CK2alpha mRNA was elevated in noninfarcted heart (145 +/- 15%) and diminished in the infarct scar (66 +/- 13%) vs controls. Expression of beta-MHC mRNA was elevated in both viable and infarct scar tissues of experimental hearts (140 +/- 31% and 183 +/- 30% vs. controls, respectively). These results suggest that cardiac genes in the infarcted tissue and viable left ventricle following MI are differentially regulated.

Inhibition of muscle differentiation by the novel muscleblind-related protein CHCR.

Growth factor withdrawal from proliferating myoblasts induces the expression of muscle-specific genes essential for myogenesis. By suppression subtractive hybridization (SSH), we have cloned a novel human cDNA that encodes a Cys3His zinc finger protein named CHCR (Cys3His CCG1-Required). CHCR is related to Muscleblind (Mbl), a Drosophila melanogaster protein required for terminal muscle differentiation. It also displays sequence similarity to EXP/MBNL, a human Mbl protein that interacts with CUG expansions associated with the degenerative muscular disease, myotonic dystrophy (DM1). This relationship with EXP/MBNL and Mbl suggests that CHCR also functions during muscle differentiation. We have found that CHCR mRNA and protein levels decrease upon differentiation of mouse myoblast cells. Constitutive expression of CHCR in C2C12 cells inhibits the induction of sarcomeric myosin heavy chain (MyHC) upon serum deprivation. Induction of myogenin, an earlier marker of muscle differentiation, is inhibited to a lesser extent, while expression of the cell cycle inhibitor, p21, remains unaffected. Loss of CHCR function by morpholino antisense oligonucleotide treatment accelerates MyHC induction during differentiation of myoblast cells. These complementary gain- and loss-of-function results suggest that CHCR is an inhibitor of myogenesis. CHCR represents the first muscleblind-related protein that antagonizes, instead of promotes, muscle differentiation.

Analysis of altered gene expression in rat soleus muscle atrophied by disuse.

The present study involved a global analysis of genes whose expression was modified in rat soleus muscle atrophied after hindlimb suspension (HS). HS muscle unloading is a common model for muscle disuse that especially affects antigravity slow-twitch muscles such as the soleus muscle. A cDNA cloning strategy, based on suppression subtractive hybridization technology, led to the construction of two normalized soleus muscle cDNA libraries that were subtracted in opposite directions, i.e., atrophied soleus muscle cDNAs subtracted by control cDNAs and vice versa. Differential screening of the two libraries revealed 34 genes with altered expression in HS soleus muscle, including 11 novel cDNAs, in addition to the 2X and 2B myosin heavy chain genes expressed only in soleus muscles after HS. Gene up- and down-regulations were quantified by reverse Northern blot and classical Northern blot analysis. The 25 genes with known functions fell into seven important functional categories. The homogeneity of gene alterations within each category gave several clues for unraveling the interplay of cellular events implied in the muscle atrophy phenotype. In particular, our results indicate that modulations in slow- and fast-twitch-muscle component balance, the protein synthesis/secretion pathway, and the extracellular matrix/cytoskeleton axis are likely to be key molecular mechanisms of muscle atrophy. In addition, the cloning of novel cDNAs underlined the efficiency of the chosen technical approach and gave novel possibilities to further decipher the molecular mechanisms of muscle atrophy.

Nitric oxide regulates smooth-muscle-specific myosin heavy chain gene expression at the transcriptional level-possible role of SRF and YY1 through CArG element.

Nitric oxide (NO) plays an important role in vascular regulation through its vasodilatory, antiatherogenic, and antithrombotic properties. NO inhibits platelet adhesion and aggregation and modulates smooth muscle cell (SMC) proliferation and migration. In animals with experimentally induced vascular injury, ec-NOS gene transfection not only restored NO production to normal levels but also increased vascular reactivity of the injured vessels. However, it is unclear whether NO regulates smooth-muscle-specific gene expression. We report here that addition of PDGF-BB to vascular smooth muscle cells suppressed SM-MHC expression but treatment with the NO donors FK409 and SNAP restored SM-MHC mRNA/protein expression. In vitro transfection and subsequent CAT assays demonstrated that exogenous NO can restore PDGF-BB-induced suppression of SM-MHC promoter activity. Promoter deletion analysis revealed that a CArG-3 box located at -1276 bp in the SM-MHC promoter was important for NO-dependent promoter regulation and as well as high level promoter activity. Gel mobility shift assays showed that CArG-3 contained the SRF binding site and a binding site for YY1, a nuclear factor which acts as a negative regulator on muscle-specific promoters. Interestingly, NO donor FK409 reduced YY1 binding to the CArG-3 element but increased SRF binding, suggesting that these two factors bind competitively to the overlapping sites. We also found that mutation to the YY1 binding site in the CArG-3 element resulted in a loss of PDGF-BB-induced suppression of the SM-MHC promoter activity. These findings indicate that NO regulates SM-MHC gene expression at the transcriptional level at least partially through the regulation of transcription factor binding activities on the CArG element. Thus we propose that NO plays a positive role in maintaining the differentiated state of VSMCs.

Effects of endurance training on skeletal muscle oxidative capacities with and without selenium supplementation.

The purpose of this study was to examine the changes induced by endurance training, with or without selenium (Se) supplementation on: 1) mitochondrial activity of succinate dehydrogenase (SDH) and cytochrome c oxidase (Cyt Ox),2) the myosin heavy chain (MHC) expression in muscle fibers and 3) their association with aerobic performance. Twenty-four male students volunteered to participate in this double blind study: selenium (Sel, N = 12) vs placebo (Pla, N = 12). During a 10-wk endurance training program, the Sel group received a daily Se supplementation containing 180 micrograms of organic selenium (selenomethionine), while the Pla group received a placebo. Before (Pre) and after (Post) the program (3 sessions wk-1) an endurance exercise (Capmax) was performed in order to determine the aerobic endurance capacity assessed by the total oxygen uptake during the running test (VO2tot). All parameters of aerobic performance were increased in both groups, concomitantly to a rise in mitochondrial Cyt Ox activity. Two positive relationships were found: 1) between type I MHC and VO2tot increments (r = 0.65, P < 0.05), 2) between training volumes and VO2tot increments (r = 0.53, P < 0.05; N = 23). The training program produced an 8.2% significant increase in type I MHC (P < 0.05) while type II MHC decrease was not significant (-4.4%). Although they were almost non-existent before the program, muscle fibers which co-expressed type I and II MHC displayed a marked increase afterwards (4.9 +/- 5.7 vs 1.1 +/- 2.1%, P < 0.05). Muscle GSH-Px activity, at rest, did not respond to endurance training or Se supplementation. The results suggest that the neuromuscular system is still in an evolutive state after 10 weeks of endurance training, and that selenium supplementation has no effect on endurance training-induced adaptations.

Endothelin-1 is involved in norepinephrine-induced ventricular hypertrophy in vivo. Acute effects of bosentan, an orally active, mixed endothelin ETA and ETB receptor antagonist.

BACKGROUND: Endothelin-1 (ET-1) has potent effects on cell growth and induces hypertrophy of cultured ventricular myocytes. Catecholamines increase expression of ET-1 mRNA by cultured myocytes. We investigated the role of endogenous ET-1 in catecholamine-induced hypertrophy in vivo by studying the effects of continuous norepinephrine infusion on physical and molecular markers of ventricular hypertrophy, ventricular and noncardiac expression of ET-1 mRNA, and the acute effects of bosentan, an orally active ETA and ETB receptor antagonist. METHODS AND RESULTS: Seventy male Sprague-Dawley rats (175 to 200 g) were divided into four groups: (1) sham-operated rats, (2) norepinephrine-infused rats (600 micrograms.kg-1.h-1 by subcutaneous osmotic pump, up to 7 days), (3) sham-operated rats given bosentan, and (4) norepinephrine-infused rats given bosentan. Bosentan (100 mg/kg once daily) was administered by gavage for 6 days starting 1 day before operation. Norepinephrine caused increases in absolute ventricular weight and ratios of ventricular weight to body weight and ventricular RNA to protein. Ventricular expression of mRNAs for atrial natriuretic factor, skeletal alpha-actin, and beta-myosin heavy chain, which in adult rat ventricle are indicators of hypertrophy, also increased. Ventricular expression of ET-1 mRNA was elevated in the norepinephrine group at 1, 2, and 3 days. By 5 days, this had fallen to control levels. In lung, kidney, and skeletal muscle, norepinephrine did not significantly increase expression of ET-1 mRNA. Bosentan attenuated norepinephrine-induced increases in ventricular weight, ratio of RNA to protein, and expression of skeletal alpha-actin mRNA and beta-myosin heavy chain mRNA at 5 days, but it did not attenuate increased ventricular expression of atrial natriuretic factor mRNA. CONCLUSIONS: These data suggest that endogenous ET-1 plays a direct role in mediating norepinephrine-induced ventricular hypertrophy in vivo.

Recombinant expression of the brush border myosin I heavy chain.

Although the specific functions of myosin I motors are not known, their localization to membrane structures suggests a function in membrane motility. Different myosin I isoforms in the same cell or in different cells can possess different localizations. To determine if the localization and biochemical activity of the best-characterized mammalian myosin I, chicken intestinal epithelium brush border myosin I, was dependent on determinants of the membrane or actin cytoskeleton specific to epithelial cells, we transfected the cDNA for the heavy chain of this myosin into COS cells. Transient transfection of COS cells with the chicken brush border myosin heavy chain resulted in the production of recombinant myosin I. Recombinant brush border myosin I localized to protrusions of the plasma membrane, particularly at spreading edges, and also to unknown cytoplasmic structures. Some cells expressing particularly high levels of brush border myosin I possessed a highly irregular surface. Recombinant brush border myosin I purified from COS cells bound to actin filaments in an ATP-dependent manner and decorated actin filaments to form a characteristic appearance. The recombinant myosin also catalyzed calcium-sensitive, actin-activated MgATPase activity similar to that of the native enzyme. Thus, any cellular factor required for the general membrane localization or biochemical activity of brush border myosin I is present in COS cells as well as intestinal epithelium.