Myostatin as a Biomarker of Muscle Wasting and other Pathologies-State of the Art and Knowledge Gaps.

Sarcopenia is a geriatric syndrome with a significant impact on older patients' quality of life, morbidity and mortality. Despite the new available criteria, its early diagnosis remains difficult, highlighting the necessity of looking for a valid muscle wasting biomarker. Myostatin, a muscle mass negative regulator, is one of the potential candidates. The aim of this work is to point out various factors affecting the potential of myostatin as a biomarker of muscle wasting. Based on the literature review, we can say that recent studies produced conflicting results and revealed a number of potential confounding factors influencing their use in sarcopenia diagnosing. These factors include physiological variables (such as age, sex and physical activity) as well as a variety of disorders (including heart failure, metabolic syndrome, kidney failure and inflammatory diseases) and differences in laboratory measurement methodology. Our conclusion is that although myostatin alone might not prove to be a feasible biomarker, it could become an important part of a recently proposed panel of muscle wasting biomarkers. However, a thorough understanding of the interrelationship of these markers, as well as establishing a valid measurement methodology for myostatin and revising current research data in the light of new criteria of sarcopenia, is needed.

Alterations in activin A-myostatin-follistatin system associate with disease activity in inflammatory myopathies.

OBJECTIVES: The aim of this study was to investigate the systemic and skeletal muscle levels of atrophy-associated myokines in patients with idiopathic inflammatory myopathies (IIM) and their association with clinical characteristics of myositis. METHODS: A total of 94 IIM patients and 162 healthy controls were recruited. Of those, 20 IIM patients and 28 healthy controls underwent a muscle biopsy. Circulating concentrations of myostatin, follistatin, activin A and TGF-ß1 were assessed by ELISA. The expression of myokines and associated genes involved in the myostatin signalling pathway in muscle tissue was determined by real-time PCR. RESULTS: We report decreased levels of circulating myostatin (median 1817 vs 2659 pg/ml; P = 0.003) and increased follistatin (1319 vs 1055 pg/ml; P = 0.028) in IIM compared with healthy controls. Activin A levels were also higher in IIM (414 vs 309 pg/ml; P = 0.0005) compared with controls. Myostatin was negatively correlated to muscle disease activity assessed by physician on visual analogue scale (MDA) (r = -0.289, P = 0.015) and positively to manual muscle testing of eight muscles (r = 0.366, P = 0.002). On the other hand, follistatin correlated positively with MDA (r = 0.235, P = 0.047). Gene expression analysis showed higher follistatin (P = 0.003) and myostatin inhibitor follistatin-like 3 protein (FSTL3) (P = 0.008) and lower expression of activin receptor type 1B (ALK4) (P = 0.034), signal transducer SMAD3 (P = 0.023) and atrophy marker atrogin-1 (P = 0.0009) in IIM muscle tissue compared with controls. CONCLUSION: This study shows lower myostatin and higher follistatin levels in circulation and attenuated expression of myostatin pathway signalling components in skeletal muscle of patients with myositis, a newly emerging pattern of the activin A-myostatin-follistatin system in muscle wasting diseases.

Targeting Unique Epitopes on Highly Similar Proteins GDF-11 and GDF-8 with Modified DNA Aptamers.

The mature forms of the TGF-ß family members GDF-11 and GDF-8 are highly similar 25 kDa homodimers with 90% amino acid sequence identity and 99% similarity. Cross-reactivity of GDF-11 and GDF-8 binding reagents is common, making it difficult to attribute distinct roles of these two proteins in biology. We report the selection of GDF-11 and GDF-8 specific SOMAmer (Slow Off-rate Modified Aptamer) reagents aided by a combination of positive selection for one protein coupled with counter-selection against the other. We identified GDF-11 specific SOMAmer reagents from four modified DNA libraries that showed a high affinity (Kd range 0.05-1.2 nM) for GDF-11 but did not bind to GDF-8 (Kd > 1 µM). Conversely, we identified one SOMAmer reagent for GDF-8 from one of the modified libraries that demonstrated excellent affinity (Kd = 0.23 nM) and specificity. In contrast, standard protocols that utilized only positive selection produced binding reagents with similar affinity for both proteins. High affinity and specificity were efficiently encoded in minimal sequences of 21 nucleotides for GDF-11 and 24 nucleotides for GDF-8. Further characterization in pull-down, competition, sandwich-binding, and kinetic studies revealed robust binding under a wide range of buffer and assay conditions. For highly similar proteins like GDF-11 and GDF-8, our method of selection coupled with counter-selection was essential for identification of high-affinity, specific reagents that have the potential to elucidate the fundamental distinction of these growth factors in biology.

Myostatin promotes the epithelial-to-mesenchymal transition of the dermomyotome during somitogenesis.

BACKGROUND: Myostatin (MSTN), a member of the transforming growth factor-ß (TGF-ß) superfamily, has been implicated in the negative regulation of skeletal myogenesis. However, the molecular mechanism through which MSTN regulates early embryonic myogenesis is not well understood. RESULTS: We demonstrate that MSTN regulates early embryonic myogenesis by promoting the epithelial-to-mesenchymal transition (EMT) of the dermomyotome during somitogenesis in chicks. We show that the MSTN gene is first expressed at the center of the dermomyotome. As somitogenesis progresses, its expression extends dorsally and ventrally along the plane of the dermomyotome. By combining in situ hybridization and immunofluorescence assays, we demonstrate that the expression pattern of MSTN is spatiotemporally well correlated with EMT of the dermomyotome. Our gain- and loss-of-function experiments further reveal that MSTN can induce EMT of the chick dermomyotome. We also show that MSTN induces EMT of a nonsmall cell lung carcinoma cell line (A549) and Madin-Darby canine kidney cells in vitro. CONCLUSIONS: Our experimental data suggest that MSTN regulates myogenesis by promoting EMT during somitogenesis. These findings provide novel insights into the functions of MSTN during early embryonic myogenesis. Developmental Dynamics 247:1241-1252, 2018. © 2018 Wiley Periodicals, Inc.

Cardiokines as Modulators of Stress-Induced Cardiac Disorders.

Almost 30 years ago, the protein, atrial natriuretic peptide, was identified as a heart-secreted hormone that provides a peripheral signal from the myocardium that communicates to the rest of the organism to modify blood pressure and volume under conditions of heart failure. Since then, additional peripheral factors secreted by the heart, termed cardiokines, have been identified and shown to coordinate this interorgan cross talk. In addition to this interorgan communication, cardiokines also act in an autocrine/paracrine manner to play a role in intercellular communication within the myocardium. This review focuses on the roles of newly emerging cardiokines that are mainly increased in stress-induced cardiac diseases. The potential of these cardiokines as clinical biomarkers for diagnosis and prognosis of cardiac disorders is also discussed.

Myostatin serum concentrations are correlated with the severity of knee osteoarthritis.

OBJECTIVE: Myostatin, a member of the transforming growth factor-ß family, contributes to joint deterioration in mice. Thus, we aimed to assess the correlation of myostatin concentrations with the presence and severity of knee osteoarthritis (OA). MATERIAL AND METHODS: We determined serum and synovial fluid (SF) myostatin concentrations in a population of 184 patients with knee OA and 109 healthy controls. RESULTS: The knee OA group presented with higher serum myostatin concentrations than the controls. Knee OA patients with KL grade 4 showed higher serum and SF myostatin concentrations compared with those with KL grade 2 and 3. Knee OA patients with KL grade 3 had higher serum and SF myostatin concentrations compared with those with KL grade 2. Serum and SF myostatin concentrations were significantly correlated with KL grading. CONCLUSION: Serum and SF myostatin concentrations were correlated with the presence and severity of knee OA.

LC-MS/MS multiplexed assay for the quantitation of a therapeutic protein BMS-986089 and the target protein Myostatin.

BACKGROUND: Therapeutic protein discovery study highlights the need for the development of quantitative bioanalytical methods for determining the levels of both the therapeutic protein and the target protein, as well. RESULTS: For the quantitation of BMS-986089, both accuracy (99-103%) and precision (2.4-12%) were obtained for the analysis of the surrogate peptide (ITYGGNSPVQEFTVPGR), in addition to the accuracy (100-108%) and precision (0.7-18%) that were obtained for the analysis of the surrogate peptide (VVSVLTVLHQDWLNGK). For Myostatin, accuracy (94-103%) and precision (2.4-14.9%) were obtained for the analysis of the surrogate peptide (IPAMVVDR). CONCLUSION: The developed method was applied to the analysis of samples following dosing of BMS-986089 to mice. This method highlights the potential of LC-MS/MS-based methods to eventually assess in vivo drug-target engagement.

Expression Levels of Myostatin and Matrix Metalloproteinase 14 mRNAs in Uterine Leiomyoma are Correlated With Dysmenorrhea.

Uterine leiomyoma is the most common benign neoplasm of female reproductive system, found in about 50% of women in reproductive age. The mechanisms of leiomyoma growth include cell proliferation, which is modulated by growth factors, and deposition of extracellular matrix (ECM). Activin A and myostatin are growth factors that play a role in proliferation of leiomyoma cells. Matrix metalloproteinases (MMPs) are known for their ability to remodel the ECM in different biological systems. The aim of this study was to evaluate the expression levels of activin ßA-subunit, myostatin, and MMP14 messenger RNAs (mRNAs) in uterine leiomyomas and the possible correlation of these factors with clinical features of the disease. Matrix metalloproteinase 14 was highly expressed in uterine leiomyoma and correlated with myostatin and activin A mRNA expression. Moreover, MMP14 and myostatin mRNA expression correlated significantly and directly with the intensity of dysmenorrhea. Overall, the present findings showed that MMP14 mRNA is highly expressed in uterine leiomyoma, where it correlates with the molecular expression of growth factors and is further increased in cases of intense dysmenorrhea.

Myostatin, follistatin and activin type II receptors are highly expressed in adenomyosis.

OBJECTIVE: To evaluate the expression pattern of activins and related growth factor messenger RNA (mRNA) levels in adenomyotic nodules and in their endometrium. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Symptomatic premenopausal women scheduled to undergo hysterectomy for adenomyosis. INTERVENTION(S): Samples from adenomyotic nodules and homologous endometria were collected. Endometrial tissue was also obtained from a control group. MAIN OUTCOME MEASURE(S): Quantitative real-time polymerase chain reaction (PCR) analysis and immunohistochemical localization of activin-related growth factors (activin A, activin B, and myostatin), binding protein (follistatin), antagonists (inhibin-α, cripto), and receptors (ActRIIa, ActRIIb) were performed. RESULT(S): Myostatin mRNA levels in adenomyotic nodule were higher than in eutopic endometrium and myostatin, activin A, and follistatin concentrations were higher than in control endometrium. No difference was observed for inhibin-α, activin B, and cripto mRNA levels. Increased mRNA levels of ActRIIa and ActRIIb were observed in adenomyotic nodules compared with eutopic endometrium and control endometrium. Immunofluorescent staining for myostatin and follistatin confirmed higher protein expression in both glands and stroma of patients with adenomyosis than in controls. CONCLUSION(S): The present study showed for the first time that adenomyotic tissues express high levels of myostatin, follistatin, and activin A (growth factors involved in proliferation, apoptosis, and angiogenesis). Increased expression of their receptors supports the hypothesis of a possible local effect of these growth factors in adenomyosis. The augmented expression of ActRIIa, ActRIIb, and follistatin in the endometrium of these patients may play a role in adenomyosis-related infertility.

Undernutrition regulates the expression of a novel splice variant of myostatin and insulin-like growth factor 1 in ovine skeletal muscle.

Undernutrition suppresses the growth of skeletal muscles and alters the expression of insulin-like growth factor 1 (IGF1), a key mitogen, and myostatin, a potent inhibitor of myogenesis. These changes can explain, at least in part, the reduced growth of skeletal muscles in underfed lambs. We have recently identified a myostatin splice variant (MSV) that binds to and antagonizes the canonical signaling of myostatin. In the present study, we hypothesized that the expression of MSV would be reduced in conjunction with myostatin and IGF1 in response to underfeeding in skeletal muscles of sheep. Young growing ewes were fed either ad libitum or an energy-restricted diet (30% of maintenance requirements) for 28 d. This regime of underfeeding resulted in a 24% reduction in body mass (P < 0.001) and a 36% reduction in the mass of the semitendinosus muscles relative to controls (P < 0.001) by day 28. The concentrations of MSV and IGF1 messenger RNA (mRNA) were reduced (both P < 0.001), but myostatin mRNA was not altered in semitendinosus muscles. Unlike the reduced expression of mRNA, the abundance of MSV protein was increased (P < 0.05) and there was no change in the abundance of myostatin protein. Our results suggest that undernutrition for 28 d decreases the signaling of myostatin by increasing the abundance of MSV protein. Although this action may reduce the growth inhibitory activity of myostatin, it cannot prevent the loss of growth of skeletal muscles during undernutrition.

Stable suppression of myostatin gene expression in goat fetal fibroblast cells by lentiviral vector-mediated RNAi.

Myostatin (MSTN) is a secreted growth factor that negatively regulates skeletal muscle mass, and therefore, strategies to block myostatin-signaling pathway have been extensively pursued to increase the muscle mass in livestock. Here, we report a lentiviral vector-based delivery of shRNA to disrupt myostatin expression into goat fetal fibroblasts (GFFs) that were commonly used as karyoplast donors in somatic-cell nuclear transfer (SCNT) studies. Sh-RNA positive cells were screened by puromycin selection. Using real-time polymerase chain reaction (PCR), we demonstrated efficient knockdown of endogenous myostatin mRNA with 64% down-regulation in sh2 shRNA-treated GFF cells compared to GFF cells treated by control lentivirus without shRNA. Moreover, we have also demonstrated both the induction of interferon response and the expression of genes regulating myogenesis in GFF cells. The results indicate that myostatin-targeting siRNA produced endogenously could efficiently down-regulate myostatin expression. Therefore, targeted knockdown of the MSTN gene using lentivirus-mediated shRNA transgenics would facilitate customized cell engineering, allowing potential use in the establishment of stable cell lines to produce genetically engineered animals.

Expression profiles of myostatin, myogenin, and Myosin heavy chain in skeletal muscles of two rabbit breeds differing in growth rate.

The purpose of the present study was to compare mRNA levels of myostatin (MSTN), myogenin (MyoG), and fiber type compositions in terms of myosin heavy chain (MyHC) in skeletal muscles of two rabbit breeds with different body sizes and growth rates. Longissimus dorsi and biceps femoris muscles of 16 Californian rabbits (CW) and 16 Germany great line of ZIKA rabbits (GZ) were collected at the ages of 35d and 84d (slaughter age). The results showed that the live weights of GZ rabbits of 35d and 84d old were approximately 36% and 26% greater than those of CW rabbits, respectively. Quantitative real-time PCR analysis revealed that at the age of 84d GZ rabbits contained significantly lower MSTN mRNA level and higher MyoG mRNA level in both longissimus dorsi and biceps femoris muscles than CW rabbits, and mRNA levels of MSTN and MyoG exhibited opposite changes from the age of 35d to 84d, suggesting that GZ rabbits were subjected to less growth inhibition from MSTN at slaughter age, which occurred most possibly in skeletal muscles. Four types of fiber were identified by real-time PCR in rabbit muscles, with MyHC-1 and MyHC-2D, MyHC-2B were the major types in biceps femoris and longissimus dorsi muscles, respectively. At the age of 84d, GZ rabbits contained greater proportion of MyHC-1 and decreased proportion of MyHC-2D and decreased lactate dehydrogenase activity in biceps femoris than CW rabbits, and the results were exactly opposite in longissimus dorsi, suggesting that GZ rabbits show higher oxidative capacity in biceps femoris muscle than CW rabbits. In conclusion, the trends of mRNA levels of MSTN and fiber types in GZ rabbits' skeletal muscles might be consistent with the putative fast growth characteristic of GZ rabbits compared to CW rabbits.

Muscle wasting in collagen-induced arthritis and disuse atrophy.

The mechanisms of muscle wasting and decreased mobility have a major functional effect in rheumatoid arthritis, but they have been poorly studied. The objective of our study is to describe muscular involvement and the pathways in an experimental model of arthritis compared to the pathways in disuse atrophy. Female Wistar rats were separated into three groups: control (CO), collagen-induced arthritis (CIA), and immobilized (IM). Spontaneous locomotion and weight were evaluated weekly. The gastrocnemius muscle was evaluated by histology and immunoblotting to measure the expression of myostatin (a negative regulator), LC3 (autophagy), MuRF-1 (proteasome-mediated proteolysis), MyoD, and myogenin (satellite-cell activation). The significance level was set at P < 0.05, and histological analysis of joints confirmed the severity of the arthropathy. There was a significant difference in spontaneous locomotion in the CIA group. Animal body weight, gastrocnemius muscle weight, and relative muscle weight decreased 20%, 30%, and 20%, respectively, in the CIA rats. Inflammatory infiltration and swelling were present in the gastrocnemius muscles of the CIA rats. The mean cross-sectional area was reduced by 30% in the CIA group and by 60% in the IM group. The expressions of myostatin and LC3 between the groups were similar. There was increased expression of MuRF-1 in the IM (1.9-fold) and CIA (3.1-fold) groups and of myogenin in the muscles of the CIA animals (1.7-fold), while MyoD expression was decreased in the IM (20%) rats. This study demonstrated that the development of experimental arthritis is associated with decreased mobility, body weight, and muscle loss. Both IM and CIA animal models presented muscle atrophy, but while proteolysis and the regeneration pathways were activated in the CIA model, there was no activation of regeneration in the IM model. We can assume that muscle atrophy in experimental arthritis is associated with the disease itself and not simply with decreased mobility.

Associação entre proteínas do plasma seminal, motilidade e viabilidade espermática em coelhos submetidos a doping genético / Association among seminal plasma proteins, sperm motility and sperm viability in rabbits submitted to gene doping

Neste trabalho foi estudada a correlação entre o perfil proteico do plasma seminal e a motilidade e viabilidade espermática em coelhos submetidos ao tratamento com vetores de expressão contendo o gene da eritropoetina (EPO) e com EPO recombinante humana. Foram identificadas, em coelhos submetidos ao tratamento com vetor de DNA contendo o gene da EPO, duas bandas proteicas associadas a alterações na motilidade espermática - 48kDa à baixa motilidade (P<0,05) e 18kDa à alta motilidade (P<0,05) - e esse fator foi associado a maior viabilidade espermática (P<0,05). Em coelhos submetidos ao tratamento com EPO recombinante, um fator proteico, 63kDa, associou-se à alta motilidade espermática (P<0,05), enquanto dois, 26 e 40kDa, foram associados à alta viabilidade espermática (P<0,05). Esses resultados sugerem que o doping genético pode ocasionar mudanças no perfil proteico do plasma seminal, provocando alterações na motilidade e viabilidade espermática.

In this study the correlation between seminal plasma protein profile and the sperm motility and sperm viability in rabbits submitted to treatment with an expression vector containing EPO gene and with human recombinant EPO was evaluated. In rabbits submitted to treatment with EPO expression vector, two protein bands were associated to sperm motility - 48kDa associated to low motility (P<0.05) and 18kDa to high motility (P<0.05) - and this protein band was also associated to high sperm viability (P<0.05). In rabbits submitted to treatment with human recombinant EPO, a protein factor, 63kDa, was associated to high sperm motility (P<0.05) while two protein factors, 26 and 40kDa, were associated to high sperm viability (P<0.05). These results suggest that gene doping leads to changes in rabbit seminal plasma protein, altering sperm motility and sperm viability.

Human masseter muscle fiber type properties, skeletal malocclusions, and muscle growth factor expression.

PURPOSE: We identified masseter muscle fiber type property differences in subjects with dentofacial deformities. PATIENTS AND METHODS: Samples of masseter muscle were collected from 139 young adults during mandibular osteotomy procedures to assess mean fiber areas and percent tissue occupancies for the 4 fiber types that comprise the muscle. Subjects were classified into 1 of 6 malocclusion groups based on the presence of a skeletal Class II or III sagittal dimension malocclusion and either a skeletal open, deep, or normal bite vertical dimension malocclusion. In a subpopulation, relative quantities of the muscle growth factors IGF-I and GDF-8 gene expression were quantified by real-time polymerase chain reaction. RESULTS: Fiber properties were not different in the sagittal malocclusion groups, but were very different in the vertical malocclusion groups (P ≤ .0004). There were significant mean fiber area differences for type II (P ≤ .0004) and type neonatal-atrial (P = .001) fiber types and for fiber percent occupancy differences for both type I-II hybrid fibers and type II fibers (P ≤ .0004). Growth factor expression differed by gender for IGF-I (P = .02) and GDF-8 (P < .01). The ratio of IGF-I:GDF-8 expression associates with type I and II mean fiber areas. CONCLUSION: Fiber type properties are very closely associated with variations in vertical growth of the face, with statistical significance for overall comparisons at P ≤ .0004. An increase in masseter muscle type II fiber mean fiber areas and percent tissue occupancies is inversely related to increases in vertical facial dimension.

Myostatin: genetic variants, therapy and gene doping

Since its discovery, myostatin (MSTN) has been at the forefront of muscle therapy research because intrinsic mutations or inhibition of this protein, by either pharmacological or genetic means, result in muscle hypertrophy and hyperplasia. In addition to muscle growth, MSTN inhibition potentially disturbs connective tissue, leads to strength modulation, facilitates myoblast transplantation, promotes tissue regeneration, induces adipose tissue thermogenesis and increases muscle oxidative phenotype. It is also known that current advances in gene therapy have an impact on sports because of the illicit use of such methods. However, the adverse effects of these methods, their impact on athletic performance in humans and the means of detecting gene doping are as yet unknown. The aim of the present review is to discuss biosynthesis, genetic variants, pharmacological/genetic manipulation, doping and athletic performance in relation to the MSTN pathway. As will be concluded from the manuscript, MSTN emerges as a promising molecule for combating muscle wasting diseases and for triggering wide-ranging discussion in view of its possible use in gene doping.

Desde sua descoberta, a miostatina (MSTN) entrou na linha de frente em pesquisas relacionadas às terapias musculares porque mutações intrínsecas ou inibição desta proteína tanto por abordagens farmacológicas como genéticas resultam em hipertrofia muscular e hiperplasia. Além do aumento da massa muscular, a inibição de MSTN potencialmente prejudica o tecido conectivo, modula a força muscular, facilita o transplante de mioblastos, promove regeneração tecidual, induz termogênese no tecido adiposo e aumenta a oxidação na musculatura esquelética. É também sabido que os atuais avanços em terapia gênica têm uma relação com o esporte devido ao uso ilícito de tal método. Os efeitos adversos de tal abordagem, seus efeitos no desempenho de atletas e métodos para detectar doping genético são, contudo, desconhecidos. O objetivo da presente revisão de literatura foi discutir biossíntese, variantes genéticas, manipulação genética e farmacológica, e doping relacionado à via da MSTN. Como será concluído do manuscrito, a MSTN emerge como uma molécula promissora para combater doenças atróficas musculares e para gerar muitas discussões devido à sua possível utilização em doping genético.

[Reduction of muscle mass mediated by myostatin in an experimental model of pulmonary emphysema]. / Reducción de la masa muscular mediada por miostatina en un modelo experimental de enfisema pulmonar.

INTRODUCTION: Among the extrapulmonary manifestations of COPD, dysfunction and loss of muscle mass/weight are those that have the greatest impact on the quality of life of patients. Our objective was to evaluate the molecular mechanisms that are potentially implicated in the limited development of muscle mass in the diaphragm and gastrocnemius of mice with experimentally-induced emphysema. METHODS: An experimental model in mice, in which emphysema was induced by means of the local instillation of elastase (n=6), while saline was administered to the controls (n=7). We determined the levels of oxidative stress, proteolytic systems, signaling pathways, growth factors and cell differentiation (western-blot) in the diaphragm and gastrocnemius of all the mice after 34 weeks. RESULTS: Upon comparing the mice with emphysema with the controls, the following findings were observed: (1) lower total body weight and lower weight of the diaphragm and gastrocnemius; (2) in the diaphragm, the levels of protein oxidation were increased, the mitochondrial antioxidant systems reduced, the levels of myostatin and of the ERK1/2 and FoxO1 signaling pathways were higher, and the myosin content was lower (67%); and (3) in the gastrocnemius of the emphysematous mice, the cytosolic antioxidants were decreased and the levels of myostatin and of the JNK and NF-kB signaling pathways were increased. CONCLUSIONS: The reduction of the myosin content observed in the diaphragm of mice with emphysema could explain their smaller size. Oxidative stress, myostatin and FoxO could be implicated in the loss of this structural protein.

Myostatin levels in skeletal muscle of hibernating ground squirrels.

Myostatin, a negative regulator of muscle mass, is elevated during disuse and starvation. Mammalian hibernation presents a unique scenario, where animals are hypocaloric and in torpor, but the extent of muscle protein loss is minimized. We hypothesized that myostatin expression, which is usually increased early in disuse and under hypocaloric conditions, could be suppressed in this unique model. Skeletal muscle was collected from thirteen-lined ground squirrels, Spermophilus tridecemlineatus, at six time points during hibernation: control euthermic (CON); entrance into hibernation (ENT), body temperature (T(b)) falling; early hibernation (EHib), stable T(b) in torpor for 24 h; late hibernation (LHib), stable T(b) in torpor for 3 days; early arousal (EAr), T(b) rising; and arousal (AR), T(b) restored to 34-37°C for about 18 h. There was no significant increase of myostatin during ENT, EHib or LHib. Unexpectedly, there were approximately sixfold increases in myostatin protein levels as squirrels arose from torpor. The elevation during EAr remained high in AR, which represented an interbout time period. Mechanisms that could release the suppression or promote increased levels of myostatin were assessed. SMAD2 and phosphorylated SMAD2 were increased during EHib, but only the phosphorylated SMAD2 during AR mirrored increases in myostatin. Follistatin, a negative regulator of myostatin, did not follow the same time course as myostatin or its signaling pathway, indicating more control of myostatin at the signaling level. However, SMAD7, an inhibitory SMAD, did not appear to play a significant role during deep hibernation. Hibernation is an excellent natural model to study factors involved in the endogenous intracellular mechanisms controlling myostatin.

Influence of different feeding systems on the growth performance and muscle development of Japanese Black steers.

This study investigated the growth performance and gene expression for muscle development between grass hay-fed (GH) and concentrate-fed (CT) steers. Daily gain and energy intake during the fattening period of the GH group were lower than those of the CT group. Analysis of C/EBPα, PPARγ2, myosin heavy chain (MHC), and myostatin gene expressions was performed by real-time PCR. Expressions of C/EBPα and myostatin in semitendinosus and longissimus lumborum (LL) muscles were higher in the CT group than in the GH group at the end of fattening. In LL muscle, MHC expression at the end of fattening was greater in the GH group than in the CT group. These results suggest that regulation of adipogenesis and myogenesis by the expression of genes involved in muscle development might have occurred in the skeletal muscle of the GH group by the feeding of grass hay and/or because of the low energy intakes.

Angiotensin II activates myostatin expression in cultured rat neonatal cardiomyocytes via p38 MAP kinase and myocyte enhance factor 2 pathway.

Angiotensin II (AngII) plays a critical role in cardiac remodeling and promotes cardiac myocyte hypertrophy. Myostatin, a negative regulator of muscle growth, is increased in hypertrophied and infarcted heart. The direct effect of AngII on cardiac myocyte myostatin expression has not been previously investigated. We hypothesized that myostatin may act as a cardiac endocrine inhibitor for AngII. AngII-induced myostatin protein expression in cultured rat neonatal cardiomyocytes was dose-dependent. AngII significantly increased myostatin protein and mRNA expression in a time-dependent manner. Addition of losartan, SB203580, or p38 siRNA 30 min before AngII stimulation significantly blocked the increase of myostatin protein by AngII. AngII significantly increased phosphorylation of p38 while SB205380 and losartan attenuated the phosphorylation of p38 induced by AngII. AngII increased, while myostatin-Mut plasmid, SB203580, losartan, and myocyte enhance factor 2 (MEF-2) antibody abolished the myostatin promoter activity. Co-stimulation with myostatin and AngII significantly inhibited the protein synthesis induced by AngII. In conclusion, AngII enhances myostatin expression in cultured rat neonatal cardiomyocytes. The AngII-induced myostatin is mediated through p38 MAP kinase and MEF-2 pathway.