Cholic acid and deoxycholic acid induce skeletal muscle atrophy through a mechanism dependent on TGR5 receptor.

Skeletal muscle atrophy is characterized by the degradation of myofibrillar proteins, such as myosin heavy chain or troponin. An increase in the expression of two muscle-specific E3 ligases, atrogin-1 and MuRF-1, and oxidative stress are involved in muscle atrophy. Patients with chronic liver diseases (CLD) develop muscle wasting. Several bile acids increase in plasma during cholestatic CLD, among them, cholic acid (CA) and deoxycholic acid (DCA). The receptor for bile acids, TGR5, is expressed in healthy skeletal muscles. TGR5 is involved in the regulation of muscle differentiation and metabolic changes. In this paper, we evaluated the participation of DCA and CA in the generation of an atrophic condition in myotubes and isolated fibers from the muscle extracted from wild-type (WT) and TGR5-deficient (TGR5-/- ) male mice. The results show that DCA and CA induce a decrease in diameter, and myosin heavy chain (MHC) protein levels, two typical atrophic features in C2 C12 myotubes. We also observed similar results when INT-777 agonists activated the TGR5 receptor. To evaluate the participation of TGR5 in muscle atrophy induced by DCA and CA, we used a culture of muscle fiber isolated from WT and TGR5-/- mice. Our results show that DCA and CA decrease the fiber diameter and MHC protein levels, and there is an increase in atrogin-1, MuRF-1, and oxidative stress in WT fibers. The absence of TGR5 in fibers abolished all these effects induced by DCA and CA. Thus, we demonstrated that CS and deoxycholic acid induce skeletal muscle atrophy through TGR5 receptor.

Effect of adeno-associated virus (AAV)-mediated overexpression of PEPCK-M (Pck2) on Clenbuterol-induced muscle growth.

We previously identified PEPCK-M (encoded by the Pck2 gene) to be highly up-regulated in skeletal muscle of pigs treated with Ractopamine, an anabolic beta-adrenergic receptor agonist. To determine whether PEPCK-M had a causative role in modulating the skeletal muscle growth response to Ractopamine, we used adeno-associated virus 1 (AAV1) to over-express Pck2 (AAV-Pck2) in murine skeletal muscle. A contralateral limb design was employed, such that each mouse served as its own control (injected with a GFP-only expressing AAV1, labelled AAV-GFP). Daily injections of Clenbuterol (1 mg/kg for 21 days) or vehicle control were also carried out to assess the effects of AAV-Pck2 overexpression on the anabolic response to a beta-adrenergic agonist. AAV-Pck2 overexpression in leg muscles of male C57BL6/J mice for 4 weeks (6-10 weeks of age) increased Pck2 mRNA (~100-fold), protein (not quantifiable) and enzyme activity (~3-fold). There was a trend (p = 0.0798) for AAV-Pck2 overexpression to reduce TA muscle weights, but there was no significant effect on muscle fibre diameters or myosin heavy chain isoform (MyHC) mRNA expression. When skeletal muscle growth was induced by daily administration of Clenbuterol (for 21 days), overexpression of AAV-Pck2 had no effect on the growth response, nor did it alter the expression of Phosphoserine Aminotransferase-1 (Psat1) or Asparagine Synthetase (Asns) mRNA or the Clenbuterol-induced decreases in MyHC IIa and IIx mRNA expression (p = 0.0065 and p = 0.0267 respectively). However AAV-Pck2 overexpression reduced TA muscle weights (p = 0.0434), particularly in the Control (vehicle treated) mice (p = 0.059 for AAV x Clenbuterol interaction) and increased the expression of Seryl-tRNA Synthetase (Sars) mRNA (p = 0.0477). Hence, contrary to the original hypothesis, AAV-Pck2 overexpression reduced TA muscle weights and did not mimic or alter the muscle hypertrophic effects of the beta-adrenergic agonist, Clenbuterol.

Butyrate promotes slow-twitch myofiber formation and mitochondrial biogenesis in finishing pigs via inducing specific microRNAs and PGC-1α expression1.

The present study aimed to investigate the influence of dietary butyrate supplementation on muscle fiber-type composition and mitochondrial biogenesis of finishing pigs, and the underlying mechanisms. Thirty-two LY (Landrace × Yorkshire) growing pigs with BW of 64.9 ± 5.7 kg were randomly allotted to either control (basal diet) or butyrate diets (0.3% butyrate sodium). Compared with the control group, diet supplemented with butyrate tended to increase average daily gain (P < 0.10). Pigs fed butyrate diet had higher intramuscular fat content, marbling score and pH24 h, and lower shear force and L*24 h in longissimus thoracis (LT) muscle than that fed control diet (P < 0.05). Interestingly, supplemented with butyrate increased (P < 0.05) the mRNA level of myosin heavy chain I (MyHC-I) and the percentage of slow-fibers, and decreased (P < 0.05) the mRNA level of MyHC-IIb in LT muscle. Meanwhile, pigs in butyrate group had an increase in mitochondrial DNA (mtDNA) copy number and the mRNA levels of mtDNA-encoded genes (P < 0.05). Moreover, feeding butyrate diet increased PGC-1α (PPAR γ coactivator 1α) level, decreased miR-133a-3p level and increased its target gene level (TEAD1, TEA domain transcription factor 1), increased miR-208b and miR-499-5p levels and decreased their target genes levels (Sp3 and Sox6, specificity protein 3 and SRY-box containing gene 6; P < 0.05) in the LT muscle. Collectively, these findings suggested that butyrate promoted slow-twitch myofiber formation and mitochondrial biogenesis, and the molecular mechanism may be via upgrading specific microRNAs and PGC-1α expression, finally improving meat quality.

Nandrolone administration with or without strenuous exercise increases cardiac fatal genes overexpression, calcium/calmodulin-dependent protein kinaseiiδ, and monoamine oxidase activities and enhances blood pressure in adult wistar rats.

Misuse of anabolic androgenic steroids (AAS) increases prevalence of cardiovascular abnormalities in athletes, and the underlying molecular mechanism involved in those abnormalities continues to be investigated. The aim of this study was to investigate the effect of chronic nandrolone exposure on alpha and beta-myosin heavy chain (MHC) isoforms gene expression transition, blood pressure related parameters, calcium/calmodulin-dependent protein kinaseIIδ (CaMKIIδ), and monoamine oxidase (MAO) activities in rats' hearts. It was also planned to evaluate the effect of strenuous exercise on cardiac abnormalities induced by nandrolone. Thirty-two male wistar rats were assigned into four groups, namely control, nandrolone, nandrolone with strenuous exercise, and strenuous exercise groups. Nandrolone consumption significantly increased systolic, diastolic, pulse and dicrotic pressure, mean arterial pressure, as well as the amplitude of first peak (H1). Moreover, exercise combined with nandrolone completely masked this effect. The mRNA expression of ß-MHC and the ratio of ß -MHC/α -MHC showed a significant increase in the nandrolone and nandrolone with strenuous exercise groups compared to those in the control group. The values of heart tissue calcium/calmoldulin-dependent protein kinase IIδ (CaMKIIδ), and monoamine oxidase (MAO) in the nandrolone, nandrolone with strenuous exercise and exercise groups were significantly higher than those values in the control group. These findings indicate that nandrolone-induced heart and hemodynamic abnormalities may in part be associated with MHC isoform changes and Ca2+ homeostasis changes mediated by increased CaMKIIδ and MAO activities and that these effects can be provoked via strenuous exercise.

All-trans retinoic acid increases the expression of oxidative myosin heavy chain through the PPARδ pathway in bovine muscle cells derived from satellite cells.

All-trans retinoic acid (ATRA) has been associated with various physiological phenomenon in mammalian adipose tissue and skeletal muscle. We hypothesized that ATRA may affect skeletal muscle fiber type in bovine satellite cell culture through various transcriptional processes. Bovine primary satellite cell (BSC) culture experiments were conducted to determine dose effects of ATRA on expression of genes and protein levels related to skeletal muscle fiber type and metabolism. The semimembranosus from crossbred steers (n = 2 steers), aged approximately 24 mo, were used to isolate BSC for 3 separate assays. Myogenic differentiation was induced using 3% horse serum upon cultured BSC with increasing doses (0, 1, 10, 100, and 1,000 nM) of ATRA. After 96 h of incubation, cells were harvested and used to measure the gene expression of protein kinase B (Akt), AMP-activated protein kinase alpha (AMPK), glucose transporter 4 (GLUT4), myogenin, lipoprotein lipase (LPL), myosin heavy chain (MHC) I, MHC IIA, MHC IIX, insulin like growth factor-1 (IGF-1), Peroxisome proliferator activated receptor gamma (PPARγ), PPARδ, and Smad transcription factor 3 (SMAD3) mRNA relative to ribosomal protein subunit 9 (RPS9). The mRNA expression of LPL was increased (P < 0.05) with 100 and 1,000 nM of ATRA. Expression of GLUT4 was altered (P < 0.05) by ATRA. The treatment of ATRA (1,000 nM) also increased (P < 0.05) mRNA gene expression of SMAD3. The gene expression of both PPARδ and PPARγ were increased (P < 0.05) with 1,000 nM of ATRA. Protein level of PPARδ was also affected (P < 0.05) by 1,000 nM of ATRA and resulted in a greater (P < 0.05) protein level of PPARδ compared to CON. All-trans retinoic acid (10 nM) increased gene expression of MHC I (P < 0.05) compared to CON. Expression of MHC IIA was also influenced (P < 0.05) by ATRA. The mRNA expression of MHC IIX was decreased (P < 0.05) with 100 and 1,000 nM of ATRA. In muscle cells, ATRA may cause muscle fibers to transition towards the MHC isoform that prefers oxidative metabolism, as evidenced by increased expression of genes associated with the MHC I isoform. These changes in MHC isoforms appeared to be brought about by changing PPARδ gene expression and protein levels.

A vitamin D analogue, eldecalcitol, enhances expression of fast myosin heavy chain subtypes in differentiated C2C12 myoblasts.

BACKGROUND: Several lines of evidence indicate that the active form of vitamin D has an anabolic effect on skeletal muscle. Eldecalcitol, an analogue of the active form of vitamin D, has the potential to increase bone density and decrease fracture risk. The objective of this study was to investigate the effect of eldecalcitol in C2C12 myogenic cells. METHODS: C2C12 cells were grown to confluency and the culture medium was replaced with low-glucose DMEM containing 2% horse serum. Eldecalcitol was added at a concentration of 1, 10 or 100 nM. Gene expression profiles of vitamin D receptor (VDR), MyoD, IGF-1, neonatal myosin heavy chain (MHC), and the fast MHC subtypes Ia, IIa, IIb and IId/x were analyzed by quantitative RT-PCR. Protein expression of MHC subtypes was evaluated by western blotting and immunostaining. RESULTS: Eldecalcitol upregulated gene expression of VDR, MyoD and IGF-1. Incubation with eldecalcitol in the absence of serum followed by the addition of serum after 1 h was associated with greater increases in the expression of these genes compared with co-incubation with eldecalcitol and serum. Gene expression of MHC subtypes IIa, IIb and IId/x was significantly increased by eldecalcitol. Protein expression of fast MHC subtypes was significantly increased by eldecalcitol at 1 and 10 nM. CONCLUSION: Similar to the active form of vitamin D, eldecalcitol had an anabolic effect on fast MHC subtypes. Taking into account its pharmacokinetic profile, eldecalcitol is expected to be beneficial for the maintenance and improvement of muscle function in elderly individuals.

Mitochondrial impairment induced by postnatal ActRIIB blockade does not alter function and energy status in exercising mouse glycolytic muscle in vivo.

Because it leads to a rapid and massive muscle hypertrophy, postnatal blockade of the activin type IIB receptor (ActRIIB) is a promising therapeutic strategy for counteracting muscle wasting. However, the functional consequences remain very poorly documented in vivo. Here, we have investigated the impact of 8-wk ActRIIB blockade with soluble receptor (sActRIIB-Fc) on gastrocnemius muscle anatomy, energy metabolism, and force-generating capacity in wild-type mice, using totally noninvasive magnetic resonance imaging (MRI) and dynamic(31)P-MRS. Compared with vehicle (PBS) control, sActRIIB-Fc treatment resulted in a dramatic increase in body weight (+29%) and muscle volume (+58%) calculated from hindlimb MR imaging, but did not alter fiber type distribution determined via myosin heavy chain isoform analysis. In resting muscle, sActRIIB-Fc treatment induced acidosis and PCr depletion, thereby suggesting reduced tissue oxygenation. During an in vivo fatiguing exercise (6-min repeated maximal isometric contraction electrically induced at 1.7 Hz), maximal and total absolute forces were larger in sActRIIB-Fc treated animals (+26 and +12%, respectively), whereas specific force and fatigue resistance were lower (-30 and -37%, respectively). Treatment with sActRIIB-Fc further decreased the maximal rate of oxidative ATP synthesis (-42%) and the oxidative capacity (-34%), but did not alter the bioenergetics status in contracting muscle. Our findings demonstrate in vivo that sActRIIB-Fc treatment increases absolute force-generating capacity and reduces mitochondrial function in glycolytic gastrocnemius muscle, but this reduction does not compromise energy status during sustained activity. Overall, these data support the clinical interest of postnatal ActRIIB blockade.

Creatine loading elevates the intracellular phosphorylation potential and alters adaptive responses of rat fast-twitch muscle to chronic low-frequency stimulation.

This study tested the hypothesis that elevating the intracellular phosphorylation potential (IPP = [ATP]/[ADP]free) within rat fast-twitch tibialis anterior muscles by creatine (Cr) loading would prevent fast-to-slow fibre transitions induced by chronic low-frequency electrical stimulation (CLFS, 10 Hz, 12 h/day). Creatine-control and creatine-CLFS groups drank a solution of 1% Cr + 5% dextrose, ad libitum, for 10 days before and during 10 days of CLFS; dextrose-control and dextrose-CLFS groups drank 5% dextrose. Cr loading increased total Cr (P < 0.025), phosphocreatine (PCr) (P < 0.003), and the IPP (P < 0.0008) by 34%, 45%, and 64%, respectively. PCr and IPP were 46% (P < 0.002) and 76% (P < 0.02) greater in creatine-CLFS than in dextrose-CLFS. Higher IPP was confirmed by a 58% reduction in phospho-AMP-activated protein kinase α (Thr172) (P < 0.006). In dextrose-CLFS, myosin heavy chain (MyHC) I and IIa transcripts increased 32- and 38-fold (P < 0.006), respectively, whereas MyHC-IIb mRNA decreased by 75% (P < 0.03); the corresponding MyHC-I and MyHC-IIa protein contents increased by 2.0- (P < 0.03) and 2.7-fold (P < 0.05), respectively, and MyHC-IIb decreased by 30% (P < 0.03). In contrast, within creatine-CLFS, MyHC-I and MyHC-IIa mRNA were unchanged and MyHC-IIb mRNA decreased by 75% (P < 0.003); the corresponding MyHC isoform contents were not altered. Oxidative reference enzymes were similarly elevated (P < 0.01) in dextrose-CLFS and creatine-CLFS, but reciprocal reductions in glycolytic reference enzymes occurred only in dextrose-CLFS (P < 0.02). Preservation of the glycolytic potential and greater SERCA2 and parvalbumin contents in creatine-CLFS coincided with prolonged time to peak tension and half-rise time (P < 0.01). These results highlight the IPP as an important physiological regulator of muscle fibre plasticity and demonstrate that training-induced changes typically associated with improvements in muscular endurance or increased power output are not mutually exclusive in Cr-loaded muscles.

Leucine supplementation accelerates connective tissue repair of injured tibialis anterior muscle.

This study investigated the effect of leucine supplementation on the skeletal muscle regenerative process, focusing on the remodeling of connective tissue of the fast twitch muscle tibialis anterior (TA). Young male Wistar rats were supplemented with leucine (1.35 g/kg per day); then, TA muscles from the left hind limb were cryolesioned and examined after 10 days. Although leucine supplementation induced increased protein synthesis, it was not sufficient to promote an increase in the cross-sectional area (CSA) of regenerating myofibers (p > 0.05) from TA muscles. However, leucine supplementation reduced the amount of collagen and the activation of phosphorylated transforming growth factor-ß receptor type I (TßR-I) and Smad2/3 in regenerating muscles (p < 0.05). Leucine also reduced neonatal myosin heavy chain (MyHC-n) (p < 0.05), increased adult MyHC-II expression (p < 0.05) and prevented the decrease in maximum tetanic strength in regenerating TA muscles (p < 0.05). Our results suggest that leucine supplementation accelerates connective tissue repair and consequent function of regenerating TA through the attenuation of TßR-I and Smad2/3 activation. Therefore, future studies are warranted to investigate leucine supplementation as a nutritional strategy to prevent or attenuate muscle fibrosis in patients with several muscle diseases.

Prevention of liver cancer cachexia-induced cardiac wasting and heart failure.

AIMS: Symptoms of cancer cachexia (CC) include fatigue, shortness of breath, and impaired exercise capacity, which are also hallmark symptoms of heart failure (HF). Herein, we evaluate the effects of drugs commonly used to treat HF (bisoprolol, imidapril, spironolactone) on development of cardiac wasting, HF, and death in the rat hepatoma CC model (AH-130). METHODS AND RESULTS: Tumour-bearing rats showed a progressive loss of body weight and left-ventricular (LV) mass that was associated with a progressive deterioration in cardiac function. Strikingly, bisoprolol and spironolactone significantly reduced wasting of LV mass, attenuated cardiac dysfunction, and improved survival. In contrast, imidapril had no beneficial effect. Several key anabolic and catabolic pathways were dysregulated in the cachectic hearts and, in addition, we found enhanced fibrosis that was corrected by treatment with spironolactone. Finally, we found cardiac wasting and fibrotic remodelling in patients who died as a result of CC. In living cancer patients, with and without cachexia, serum levels of brain natriuretic peptide and aldosterone were elevated. CONCLUSION: Systemic effects of tumours lead not only to CC but also to cardiac wasting, associated with LV-dysfunction, fibrotic remodelling, and increased mortality. These adverse effects of the tumour on the heart and on survival can be mitigated by treatment with either the ß-blocker bisoprolol or the aldosterone antagonist spironolactone. We suggest that clinical trials employing these agents be considered to attempt to limit this devastating complication of cancer.

Extensive impact of saturated fatty acids on metabolic and cardiovascular profile in rats with diet-induced obesity: a canonical analysis.

BACKGROUND: Although hypercaloric interventions are associated with nutritional, endocrine, metabolic, and cardiovascular disorders in obesity experiments, a rational distinction between the effects of excess adiposity and the individual roles of dietary macronutrients in relation to these disturbances has not previously been studied. This investigation analyzed the correlation between ingested macronutrients (including sucrose and saturated and unsaturated fatty acids) plus body adiposity and metabolic, hormonal, and cardiovascular effects in rats with diet-induced obesity. METHODS: Normotensive Wistar-Kyoto rats were submitted to Control (CD; 3.2 Kcal/g) and Hypercaloric (HD; 4.6 Kcal/g) diets for 20 weeks followed by nutritional evaluation involving body weight and adiposity measurement. Metabolic and hormonal parameters included glycemia, insulin, insulin resistance, and leptin. Cardiovascular analysis included systolic blood pressure profile, echocardiography, morphometric study of myocardial morphology, and myosin heavy chain (MHC) protein expression. Canonical correlation analysis was used to evaluate the relationships between dietary macronutrients plus adiposity and metabolic, hormonal, and cardiovascular parameters. RESULTS: Although final group body weights did not differ, HD presented higher adiposity than CD. Diet induced hyperglycemia while insulin and leptin levels remained unchanged. In a cardiovascular context, systolic blood pressure increased with time only in HD. Additionally, in vivo echocardiography revealed cardiac hypertrophy and improved systolic performance in HD compared to CD; and while cardiomyocyte size was unchanged by diet, nuclear volume and collagen interstitial fraction both increased in HD. Also HD exhibited higher relative ß-MHC content and ß/α-MHC ratio than their Control counterparts. Importantly, body adiposity was weakly associated with cardiovascular effects, as saturated fatty acid intake was directly associated with most cardiac remodeling measurements while unsaturated lipid consumption was inversely correlated with these effects. CONCLUSION: Hypercaloric diet was associated with glycemic metabolism and systolic blood pressure disorders and cardiac remodeling. These effects directly and inversely correlated with saturated and unsaturated lipid consumption, respectively.

Effects of genistein and estrogen on the genioglossus in rats exposed to chronic intermittent hypoxia may be HIF-1α dependent.

OBJECTIVES: Chronic intermittent hypoxia (CIH) is a frequent feature of OSAHS. The present study was designed to evaluate the effects of genistein and estrogen on genioglossus contractile and regeneration properties in CIH rats and investigate the involvement of HIF-1α. METHODS: Ovariectomized female rats were exposed to CIH for 5 weeks. Genistein and estrogen were administered by intraperitoneal injection. The genioglossus myoblasts of rat were also isolated and cultured in vitro, and the HIF-1α shRNA lentivirus was used. RESULTS: Muscle fatigue resistance and myogenic regeneration were significantly decreased after CIH but were partially reversed by estrogen and genistein treatment. The effect of estrogen was more powerful than that of genistein. Compared with control group, RT-PCR and western blotting showed higher levels of HIF-1α mRNA and protein in the CIH group, but estrogen and genistein treatment reduced the levels of HIF-1α mRNA and protein in rats exposed to CIH. In genioglossus myoblasts, the expression of HIF-1α was up-regulated under hypoxia rather than normoxia and decreased over time under both hypoxia and normoxia during myogenic differentiation. HIF-1α knockdown relieved myogenesis inhibition under hypoxia. CONCLUSION: We concluded that genistein and estrogen may inhibit the overexpression of HIF-1α induced by CIH and improve the endurance and regeneration of the genioglossus muscle.

Histological changes and changes in the myosin mRNA content of the porcine masticatory muscles after masseter treatment with botulinum toxin A.

OBJECTIVES: Botulinum toxin A (Botox) is increasingly used for treatment of muscle hyperfunction. For a better understanding of the possible morphologic and chewing changes in patients induced by a therapy with Botox, muscle fiber and myosin heavy chain (MyHC) mRNA alterations were examined in this animal study. MATERIALS AND METHODS: The investigation was carried out on 14-week-old pigs (seven treated animals, eight controls; calculated animal size with a power of 0.5). To initialise the total immobilisation of the right masseter, the Botox injection was distributed into ten areas. After a 56-day period, muscle tissue was taken from the left and right side of the masseter (three regions), temporal (two regions), medial pterygoid and geniohyoid muscles using a standardized method. The muscle fiber cross sections were examined immunohistochemically. Fiber staining was accomplished with antibodies to specific MyHC isoforms. The MyHC mRNA changes were analysed using real-time RT-PCR. RESULTS: Muscles adapt to such stress by changing fiber types and MyHC mRNA content. Paralysed masseters display atrophic changes while other masticatory muscles show hypertrophic changes. The results indicated that the typical distributions of type IIa und IIb fiber types in masticatory muscles were increased in the masseter muscles due to Botox application. On the other hand, the masseters without Botox in the treated group showed a significant increase of type I MyHC. CONCLUSIONS: Application of Botox may lead to uncontrolled structural changes in affected and unaffected muscles. CLINICAL RELEVANCE: Treatment of muscle hypertrophy with Botox may cause muscle imbalance.

Peripheral benzodiazepine receptor ligand Ro5-4864 inhibits isoprenaline-induced cardiac hypertrophy in rats.

Oxidative stress plays a significant role in the pathogenesis of cardiac hypertrophy. Peripheral benzodiazepine receptors are ubiquitously expressed in various tissues, including the heart. Peripheral benzodiazepine receptors have been reported to be involved in the protection of cells against oxygen radical damage. The present study was designed to determine whether Ro5-4864 (a peripheral benzodiazepine receptor ligand) can inhibit isoprenaline-induced cardiac hypertrophy. Male Wistar rats (body weight 150-200g) were administered, isoprenaline (5mg/kg, body weight, subcutaneously) alone or along with Ro5-4864 (0.1 and 0.5mg/kg, body weight, intraperitoneally) once daily for 14days. Control rats received normal saline subcutaneously (1.0ml/kg). Isoprenaline-induced changes in heart weight to body weight ratio, left ventricular wall thickness (M-mode echocardiography and gross morphometry) and myocyte size were significantly prevented by both the doses of Ro5-4864. Ro5-4864 also attenuated isoprenaline-induced increase in interstitial fibrosis, lipid peroxidation and changes in endogenous antioxidants (glutathione, superoxide dismutase and catalase). Isoprenaline-induced cardiac hypertrophy was associated with increased expression of beta myosin heavy chain, which was also prevented by Ro5-4864. This is the first study to demonstrate a salutary effect of Ro5-4864 in experimental cardiac hypertrophy.

SB431542 treatment promotes the hypertrophy of skeletal muscle fibers but decreases specific force.

The small molecule inhibitor SB431542 inhibits activin type I receptors. The muscle growth-inhibitor myostatin binds to and signals via these receptors. The aim of this study was to test the hypothesis that SB431542 can inhibit myostatin-related Smad signaling and induce muscle growth in cultured C2C12 myotubes and increase growth and specific force in cultured Xenopus muscle fibers. The effect of SB431542 was assessed in vitro on C2C12 myotubes and ex vivo using mature Xenopus muscle fibers. SB431542 treatment reduced myostatin-induced C-terminal Smad2 phosphorylation and resulted in the formation of enlarged myotubes. However myogenin expression was unchanged, while p70 S6k phosphorylation at Thr389, total myosin heavy chain, and the rate of protein synthesis were all reduced. Mature Xenopus muscle fibers that were treated with SB431542 had a higher fiber cross-sectional area but decreased specific force production than control. SB431542 can initially antagonize myostatin signaling, but long-term unexpected signaling effects occur. Muscle fibers hypertrophy, but their specific force decreases compared to control.

Dexamethasone induces dysferlin in myoblasts and enhances their myogenic differentiation.

Glucocorticoids are beneficial in many muscular dystrophies but they are ineffective in treating dysferlinopathy, a rare muscular dystrophy caused by loss of dysferlin. We sought to understand the molecular basis for this disparity by studying the effects of a glucocorticoid on differentiation of the myoblast cell line, C2C12, and dysferlin-deficient C2C12s. We found that pharmacologic doses of dexamethasone enhanced the myogenic fusion efficiency of C2C12s and increased the induction of dysferlin, along with specific myogenic transcription factors, sarcolemmal and structural proteins. In contrast, the dysferlin-deficient C2C12 cell line demonstrated a reduction in long myotubes and early induction of particular muscle differentiation proteins, most notably, myosin heavy chain. Dexamethasone partially reversed the defect in myogenic fusion in the dysferlin-deficient C2C12 cells. We hypothesize that a key therapeutic benefit of glucocorticoids may be the up-regulation of dysferlin as an important component of glucocorticoid-enhanced myogenic differentiation.

Effects of creatine supplementation during resistance training on myosin heavy chain (MHC) expression in rat skeletal muscle fibers.

The purpose of this study was to utilize a rodent model to test the hypothesis that creatine (Cr) supplementation during resistance training would influence the pattern of slow-twitch muscle myosin heavy chain (MHC) isoforms expression. Male Wistar rats (2-3 months old, 250-300 g) were divided into 4 groups: Nontrained without creatine supplementation (CO), nontrained with creatine supplementation (CR), trained without creatine supplementation (TR), and trained with creatine supplementation (TRCR). TR and TRCR groups were submitted to a resistance training program for 5 weeks (5 days/week) for morphological and biochemical analysis of the soleus muscle. Weightlifting exercise involved jump sessions into water, carrying progressive overload equivalent to percentage of body weight. CR and TRCR groups were given creatine at 0.5 g/kg(-1)/d(-1). Both Cr supplementation and resistance training alone or associated did not result in significant alterations (p > 0.05) in body weight gain, food intake, and muscle weight in the CR, TR and TRCR groups compared to the CO group. Also compared to the CO group, the CR group showed a significant (p < 0.02) increase in MHCI content and a reduction in MHCII; inversely, the TR group increased the MHCII content and reduced MHCI (p < 0.02). When combined, both creatine and resistance training did not promote significant (p > 0.05) changes in MHC content of the TRCR group compared to the CO group. The data show that Cr supplementation provides a potential action to abolish the exercise-induced MHC isoform transitions from slow to fast in slow-twitch muscle. Thus, Cr supplementation might be a suitable strategy to maintaining a slow phenotype in slow muscle during resistance training, which may be favorable to maintenance of muscle oxidative capacity of endurance athletes.

Decreased muscle ACE activity enhances functional response to endurance training in rats, without change in muscle oxidative capacity or contractile phenotype.

In the present study, we tested the hypothesis that chronic ANG I-converting enzyme (ACE) inhibition could improve the training-induced improvement in endurance exercise performance and that this could be related to enhanced skeletal muscle metabolic efficiency. Female Wistar rats were assigned to four groups comprising animals either maintained sedentary or endurance trained (Sed and Tr, respectively), and treated or not for 10 wk with an ACE inhibitor, perindopril (2 mg.kg(-1).day(-1)) (Per and Ct, respectively) (n = 8 each). Trained rats underwent an 8-wk treadmill training protocol that consisted of 2 h/day running at 30 m/min on a 8% decline. Before the start of and 1 wk before the end of experimental conditioning, the running time to exhaustion of rats was measured on a treadmill. The training program led to an increase in endurance time, higher in Tr-Per than in Tr-Ct group (125% in Tr-Ct vs. 183% in Tr-Per groups, P < 0.05). Oxidative capacity, measured in saponin-permeabilized fibers of slow soleus and fast plantaris muscles, increased with training, but less in Tr-Per than in Tr-Ct rats. The training-induced increase in citrate synthase activity also was less in soleus from Tr-Per than Tr-Ct rats. The training-induced increase in the percentage of the type IIa isoform of myosin heavy chain (MHC) (45%, P < 0.05) and type IIx MHC (25%, P < 0.05) associated with decreased type IIb MHC (34%, P < 0.05) was minimized by perindopril administration. These findings demonstrate that the enhancement in physical performance observed in perindopril-treated animals cannot be explained by changes in mitochondrial respiration and/or MHC distribution within muscles involved in running exercise.

Dehydroepiandrosterone replacement therapy in hypoadrenal women: protein anabolism and skeletal muscle function.

OBJECTIVE: To determine whether dehydroepiandrosterone (DHEA) replacement therapy in hypoadrenal women improves performance, muscle protein accretion, and mitochondrial functions. PARTICIPANTS AND METHODS: Thirty-three hypoadrenal women were enrolled in the study from May 1, 2002, through May 31, 2003. Twenty-eight completed a 12-week, prospective, randomized, placebo-controlled, crossover study with either daily placebo or 50 mg of DHEA with a 2-week washout period and then crossed over to the other treatment. Body composition, physical performance, whole-body and muscle protein metabolism, and mitochondrial functions were determined. RESULTS: Administration of DHEA significantly increased plasma levels of DHEA sulfate, testosterone, and androstenedione but did not change body composition, muscle strength, peak aerobic capacity, and whole-body protein turnover or synthesis rates of mitochondrial, sarcoplasmic, or mixed muscle proteins. Muscle mitochondrial oxidative enzymes and messenger RNA (mRNA) levels of genes encoding mitochondrial proteins and nuclear transcription factors did not change after DHEA administration. However, mRNA levels of muscle myosin heavy chain 1 (P=.004), which determines muscle fiber type, and those of insulinlike growth factor binding proteins 4 and 5 significantly decreased (P=.02 and P=.03, respectively). CONCLUSION: Three months of DHEA administration increased DHEA sulfate and androgen levels but had no effect on physical performance, body composition, protein metabolism, or muscle mitochondrial biogenesis in hypoadrenal women. However, lowering of mRNA levels of binding proteins of insulinlike growth factor 1 and myosin heavy chain 1 suggests potential effects of longterm treatment with DHEA on muscle fiber type.

Oral amino acid supplementation counteracts age-induced sarcopenia in elderly rats.

We investigated the effects of a specific mixture of amino acid (AA) supplements on the adaptation changes induced by aging in the soleus muscle of rats. Male Wistar rats were divided into 3 groups (n = 5 each): young control (YO), 3 months of age; elderly control (EL), 18 months of age; and elderly orally supplemented with an AA mixture (EL-AA), 18 months of age, given as 0.1 g/kg per day in drinking water for 8 weeks. Myosin heavy chain (MHC) composition was analyzed in all muscles. The total fiber number and fiber cross-sectional area of types 1 and 2A fibers were also measured in immunostained sections of the soleus muscle. The ratios between the sarcomere volume (Vsar) and the total volume (Vtot) and single muscle fibers were studied by electron microscopy. The expression of total and phosphorylated serine/threonine protein kinase mammalian target of rapamycin (mTOR), a potent regulator of messenger RNA translation initiation, was also determined in all groups. Aging was associated with an overall shift toward the expression of a slower MHC phenotype, atrophy of fast and slow fibers, a significant decrease in Vtot/Vsar, and no changes in total fiber number. AA supplementation antagonized the effects of aging. A shift toward the expression of faster MHC isoforms was observed. Fiber atrophy appeared to be partly counteracted by the AA supplements; we noted an increase in cross-sectional area fibers and Vtot/Vsar in EL-AAs. Total and phosphorylated mTOR expression appeared to decrease in EL and was restored by the AA supplements. Collectively, these results suggest that aging-induced muscle adaptations can be partly restored by AA supplementation. An mTOR signal pathway may mediate the effects on fiber trophism.