Role of Myostatin in Rheumatoid Arthritis: A Review of the Clinical Impact.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that affects synovial joints and that frequently involves extra-articular organs. A multiplicity of interleukins (IL) participates in the pathogenesis of RA, including IL-6, IL-1ß, transforming growth factor-beta (TGF-ß), and tumor necrosis factor (TNF)-α; immune cells such as monocytes, T and B lymphocytes, and macrophages; and auto-antibodies, mainly rheumatoid factor and anti-citrullinated protein antibodies (ACPAs). Skeletal muscle is also involved in RA, with many patients developing muscle wasting and sarcopenia. Several mechanisms are involved in the myopenia observed in RA, and one of them includes the effects of some interleukins and myokines on myocytes. Myostatin is a myokine member of the TGF-ß superfamily; the overproduction of myostatin acts as a negative regulator of growth and differentiates the muscle fibers, limiting their number and size. Recent studies have identified abnormalities in the serum myostatin levels of RA patients, and these have been found to be associated with muscle wasting and other manifestations of severe RA. This review analyzes recent information regarding the relationship between myostatin levels and clinical manifestations of RA and the relevance of myostatin as a therapeutic target for future research.

Serum myostatin as a candidate disease severity and progression biomarker of spinal muscular atrophy.

The identification of biomarkers for spinal muscular atrophy is crucial for predicting disease progression, severity, and response to new disease-modifying therapies. This study aimed to investigate the role of serum levels of myostatin and follistatin as biomarkers for spinal muscular atrophy, considering muscle atrophy secondary to denervation as the main clinical manifestation of the disease. The study evaluated the differential gene expression of myostatin and follistatin in a lesional model of gastrocnemius denervation in mice, as well as in a meta-analysis of three datasets in transgenic mice models of spinal muscular atrophy, and in two studies involving humans with spinal muscular atrophy. Subsequently, a case-control study involving 27 spinal muscular atrophy patients and 27 controls was conducted, followed by a 12-month cohort study with 25 spinal muscular atrophy cases. Serum levels of myostatin and follistatin were analysed using enzyme-linked immunosorbent assay at a single centre in southern Brazil. Skeletal muscle gene expression of myostatin decreased and of follistatin increased following lesional muscle denervation in mice, consistent with findings in the spinal muscular atrophy transgenic mice meta-analysis and in the iliopsoas muscle of five patients with spinal muscular atrophy type 1. Median serum myostatin levels were significantly lower in spinal muscular atrophy patients (98 pg/mL; 5-157) compared to controls (412 pg/mL; 299-730) (P < 0.001). Lower myostatin levels were associated with greater disease severity based on clinician-rated outcomes (Rho = 0.493-0.812; P < 0.05). After 12 months, there was a further reduction in myostatin levels among spinal muscular atrophy cases (P = 0.021). Follistatin levels did not differ between cases and controls, and no significant changes were observed over time. The follistatin:myostatin ratio was significantly increased in spinal muscular atrophy subjects and inversely correlated with motor severity. Serum myostatin levels show promise as a novel biomarker for evaluating the severity and progression of spinal muscular atrophy. The decrease in myostatin levels and the subsequent favourable environment for muscle growth may be attributed to denervation caused by motor neuron dysfunction.

ß2-Adrenoceptors activation regulates muscle trophic-related genes following acute resistance exercise in mice.

Resistance exercise (RE) training and pharmacological stimulation of ß2-Adrenoceptors (ß2-ARs) alone can promote muscle hypertrophy and prevent muscle atrophy. Although the activation of the sympathetic nervous system (SNS) is a well-established response during RE, the physiological contribution of the endogenous catecholamines and ß2-ARs to the RE-induced changes on skeletal muscle protein metabolism remains unclear. This study investigated the effects of the ß2-ARs blockade on the acute molecular responses induced by a single bout of RE in rodent skeletal muscles. Male C57BL6/J mice were subjected to a single bout of progressive RE (until exhaustion) on a vertical ladder under ß2-AR blockade with ICI 118,551 (ICI; 10 mg kg-1, i. p.), or vehicle (sterile saline; 0.9%, i. p.), and the gene expression was analyzed in gastrocnemius (GAS) muscles by qPCR. We demonstrated that a single bout of RE acutely increased the circulating levels of stress-associated hormones norepinephrine (NE) and corticosterone (CORT), as well as the muscle phosphorylation levels of AMPK, p38 MAPK and CREB, immediately after the session. The acute increase in the phosphorylation levels of CREB was followed by the upregulation of CREB-target genes Sik1, Ppargc1a and Nr4a3 (a central regulator of the acute RE response), 3 h after the RE session. Conversely, ß2-AR blockade reduced significantly the Sik1 and Nr4a3 mRNA levels in muscles of exercised mice. Furthermore, a single bout of RE stimulated the mRNA levels of the atrophic genes Map1lc3b and Gabarapl1 (autophagy-related genes) and Mstn (a well-known negative regulator of muscle growth). Unexpectedly, the gene expression of Igf-1 or Il-6 were not affected by RE, while the atrophic genes Murf1/Trim63 and Atrogin-1/Mafbx32 (ubiquitin-ligases) were increased only in muscles of exercised mice under ß2-AR blockade. Interestingly, performing a single bout of RE under ß2-AR blockade increased the mRNA levels of Mstn in muscles of exercised mice. These data suggest that ß2-ARs stimulation during acute RE stimulates the hypertrophic gene Nr4a3 and prevents the overexpression of atrophic genes such as Mstn, Murf1/Trim63, and Atrogin-1/Mafbx32 in the first hours of postexercise recovery, indicating that he SNS may be physiologically important to muscle adaptations in response to resistance training.

Myostatin is associated with the presence and development of acute-on-chronic liver failure.

Background & Aims: Acute-on-chronic liver failure (ACLF) has been linked to different pathophysiological mechanisms, including systemic inflammation and mitochondrial dysfunction. Sarcopenia has also been proposed as a potential mechanism; myostatin is a key factor inducing sarcopenia. Therefore, this study aimed to evaluate the association of myostatin levels with the development of ACLF and mortality in patients with cirrhosis. Methods: We performed a prospective cohort study, including both outpatient and hospitalized patients with cirrhosis. Clinical, biochemical, and nutritional parameters were evaluated, and the development of acute decompensation (AD) or ACLF during follow-up was recorded. ACLF was defined according to the EASL-CLIF criteria. Receiver-operating characteristic, Kaplan-Meier and Cox regression analyses were performed. Results: A total of 186 patients with the whole spectrum of cirrhosis were included; mean age was 53.4 ± 14 years, mean Child-Pugh score was 8 ± 2.5 and mean MELD score was 15 ± 8. There was a stepwise decrease in myostatin levels from a compensated stage to AD and ACLF. Myostatin correlated positively with nutritional markers and negatively with severity scores. The prevalence of sarcopenia was 73.6%. During follow-up, 27.9% of patients developed AD and 25.8% developed ACLF. Most episodes were grade 2-3, mainly (62.5%) precipitated by infections. The most common organ failures observed were in the liver (63.3%) and the kidney (64.6%). Receiver-operating characteristic analysis yielded <1,280 pg/ml as the best serum myostatin cut-off for the prediction of ACLF. In Kaplan-Meier curves and multivariate analysis, myostatin levels remained independently associated with the incidence of ACLF and survival. Conclusions: There is a progressive decrease in myostatin levels as cirrhosis progresses, demonstrating an association of sarcopenia with the development of ACLF and increased mortality. Impact and implications: Myostatin is a muscle hormone, it is decreased in patients with muscle loss and is a marker of impaired muscle function. In this study we show that myostatin levels are decreased in patients with cirrhosis, with lower levels in patients with acute decompensation and acute-on chronic liver failure (ACLF). Low myostatin levels in cirrhosis predict the development of ACLF and mortality independently of liver disease severity and sex.

Effects of Creatine Supplementation on the Myostatin Pathway and Myosin Heavy Chain Isoforms in Different Skeletal Muscles of Resistance-Trained Rats.

Creatine has been used to maximize resistance training effects on skeletal muscles, including muscle hypertrophy and fiber type changes. This study aimed to evaluate the impact of creatine supplementation on the myostatin pathway and myosin heavy chain (MyHC) isoforms in the slow- and fast-twitch muscles of resistance-trained rats. Twenty-eight male Wistar rats were divided into four groups: a sedentary control (Cc), sedentary creatine supplementation (Cr), resistance training (Tc), and resistance training combined with creatine supplementation (Tcr). Cc and Tc received standard commercial chow; Cr and Tcr received a 2% creatine-supplemented diet. Tc and Tcr performed a resistance training protocol on a ladder for 12 weeks. Morphology, MyHC isoforms, myostatin, follistatin, and ActRIIB protein expressions were analyzed in soleus and white gastrocnemius portion samples. The results were analyzed using two-way ANOVA and Tukey's test. Tc and Tcr exhibited higher performance than their control counterparts. Resistance training increased the ratio between muscle and body weight, the cross-sectional area, as well as the interstitial collagen fraction. Resistance training alone increased MyHC IIx and follistatin while reducing myostatin (p < 0.001) and ActRIIB (p = 0.040) expressions in the gastrocnemius. Resistance training induced skeletal muscle hypertrophy and interstitial remodeling, which are more evident in the gastrocnemius muscle. The effects were not impacted by creatine supplementation.

The Effects of Dietary Supplements, Nutraceutical Agents, and Physical Exercise on Myostatin Levels: Hope or Hype?

Myostatin, a secreted growth factor belonging to the transforming growth factor ß (TGF-ß) family, performs a role in hindering muscle growth by inhibiting protein kinase B (Akt) phosphorylation and the associated activation of hypertrophy pathways (e.g., IGF-1/PI3K/Akt/mTOR pathway). In addition to pharmacological agents, some supplements and nutraceutical agents have demonstrated modulatory effects on myostatin levels; however, the clinical magnitude must be appraised with skepticism before translating the mechanistic effects into muscle hypertrophy outcomes. Here, we review the effects of dietary supplements, nutraceutical agents, and physical exercise on myostatin levels, addressing the promise and pitfalls of relevant randomized clinical trials (RCTs) to draw clinical conclusions. RCTs involving both clinical and sports populations were considered, along with wasting muscle disorders (e.g., sarcopenia) and resistance training-induced muscle hypertrophy, irrespective of disease status. Animal models were considered only to expand the mechanisms of action, and observational data were consulted to elucidate potential cutoff values. Collectively, the effects of dietary supplements, nutraceutical agents, and physical exercise on myostatin mRNA expression in skeletal muscle and serum myostatin levels are not uniform, and there may be reductions, increases, or neutral effects. Large amounts of research using resistance protocols shows that supplements or functional foods do not clearly outperform placebo for modulating myostatin levels. Thus, despite some biological hope in using supplements or certain functional foods to decrease myostatin levels, caution must be exercised not to propagate the hope of the food supplement market, select health professionals, and laypeople.

Association Between Serum Myostatin Levels, Hospital Mortality, and Muscle Mass and Strength Following ST-Elevation Myocardial Infarction.

AIM: This study aimed to evaluate the association between serum myostatin levels, hospital mortality, and muscle mass and strength following ST-segment elevation myocardial infarction (STEMI). METHODS: This was a prospective observational study. Within 48 hours of admission, bioelectrical impedance and handgrip strength were assessed and blood samples collected for myostatin evaluation. Hospital mortality was recorded. A multiple logistic regression model was also constructed, adjusted by parameters that exhibited significant differences in the univariate analysis, to evaluate the association between myostatin levels and hospital mortality. RESULTS: One hundred and two (102) patients were included: mean age was 60.5±10.6 years, 67.6% were male, and 6.9% died during hospital stay. Univariate analysis showed that patients with lower myostatin levels had higher mortality rates. Serum myostatin levels positively correlated with handgrip strength (r=0.355; p<0.001) and appendicular skeletal muscle mass index (r=0.268; p=0.007). Receiver operating characteristic (ROC) curve analysis revealed that lower myostatin levels were associated with hospital mortality at the <2.20 ng/mL cut-off. Multiple logistic regression showed that higher serum myostatin levels were associated with reduced hospital mortality when adjusted by ß blocker use (OR, 0.228; 95% CI, 0.054-0.974; p=0.046). CONCLUSIONS: Serum myostatin concentrations positively correlated with muscle mass and strength in STEMI patients. Further assessment of serum myostatin association with mortality should be conducted using a larger sample and assessing the additive value to the Global Registry of Acute Coronary Events (GRACE) or thrombolysis in myocardial infarction (TIMI) risk scores.

Myostatin coordinating the proliferation and differentiation of adipose and skeletal muscle cells and energy metabolism balance based on gene chip technology / A miostatina na coordenação da proliferação e diferenciação de células musculares adiposas e esqueléticas e no equilíbrio do metabolismo energético com base na tecnologia de chips genéticos / Msrn, basado en la tecnología de chips genéticos, coordina la fertilización y el metabolismo de las células musculares grasas y óseas

ABSTRACT Myoblasts fuse into multinucleated muscle fibers to form and promote the growth of skeletal muscle. In order to analyze the role of myostatin (MSTN) in body fat, skeletal muscle cell proliferation and differentiation and energy metabolism, this study will use the antisense RNA technology of gene chip technology to study it. The results showed that the MSTN gene regulated the growth and proliferation of myoblasts and affected the development of skeletal muscle by affecting the expression of Cdc42, bnip2, p38 and other genes; knockout or overexpression of the MSTN gene would lead to a trend of fat-related genes from fat synthesis to fat decomposition; after the MSTN gene was knocked down, the expression levels of cpti-b, PPARG and other genes in the cells were corresponding after MSTN overexpression, the relative expression of the PPARG gene decreased. It is suggested that the knockout or overexpression of MSTN may affect lipid accumulation, and cpti-b and PPARG may directly regulate lipid level. It is hoped that this experiment can provide a reference for the study of MSTN effect on fat deposition.

RESUMO Os mioblastos se fundem eM fibras musculares multinucleadas para formar e promover o crescimento do músculo esquelético. A fim de analisar o papel da miostatina (MSTN) na gordura corporal, proliferação de células musculares esqueléticas e diferenciação e metabolismo energético, este estudo utilizará a tecnologia anti-RNA de chips genéticos para estudá-la. Os resultados mostraram que o gene MSTN regulava o crescimento e a proliferação de mioblastos e afetava o desenvolvimento do músculo esquelético, afetando a expressão de Cdc42, bnip2, p38 e outros genes; a eliminação ou sobrexpressão do gene MSTN conduziria a uma tendência de os genes adiposos sintetizarem a gordura até sua decomposição; após a eliminação do gene MSTN, os níveis de expressão de cpti-b, PPARG e outros genes nas células mostraram-se correspondentes após a sobrexpressão do gene MSTN, e a expressão relativa do gene PPARG diminuiu. Sugere-se que a eliminação ou sobrexpressão da MSTN possa afetar a acumulação de lipídeos, e o cpti-b e o PPARG podem regular diretamente o nível lipídico. Espera-se que esta experiência possa fornecer uma referência para o estudo do efeito da MSTN sobre a deposição de gordura.

RESUMEN Los mioblastos se funden en fibras musculares multinucleadas para formar y promover el crecimiento del músculo esquelético. A fin de analizar el papel de la miostatina (MSTN) en la grasa corporal, proliferación de células musculares esqueléticas y diferenciación y metabolismo energético, este estudio utilizará la tecnología anti-RNA de chips genéticos para estudiarla. Los resultados mostraron que el gen MSTN regulaba el crecimiento y la proliferación de mioblastos y afectaba el desarrollo del músculo esquelético, afectando la expresión de Cdc42, bnip2, p38 y otros genes; la eliminación o sobreexpresión del gen MSTN conduciría a una tendencia de que los genes adiposos sinteticen la grasa hasta su descomposición; después de la eliminación del gen MSTN, los niveles de expresión de cpti-b, PPARG y otros genes en las células se mostraron correspondientes después de la sobreexpresión del gen MSTN, y la expresión relativa del gen PPARG disminuyó. Se sugiere que la eliminación o sobreexpresión de la MSTN pueda afectar la acumulación de lipídos, y el cpti-b y el PPARG pueden regular directamente el nivel lipídico. Se espera que esta experiencia pueda proveer una referencia para el estudio del efecto de la MSTN sobre el depósito de grasa.

Skeletal Muscle Anti-Atrophic Effects of Leucine Involve Myostatin Inhibition.

Lack of mechanical load leads to skeletal muscle atrophy, and one major underlying mechanism involves the myostatin pathway that negatively regulates protein synthesis and also activates Atrogin-1/MAFbx and MuRF1 genes. In hindlimb immobilization, leucine was observed to attenuate the upregulation of the referred atrogenes, thereby shortening the impact on fiber cross-sectional area, nonetheless, the possible connection with myostatin is still elusive. This study sought to verify the impact of leucine supplementation on myostatin expression. Male Wistar rats were supplemented with leucine and hindlimb immobilized for 3 and 7 days, after which soleus muscles were removed for morphometric measurements and analyzed for gene and protein expression by real-time PCR and Western blotting, respectively. Muscle wasting was prominent 7 days after immobilization, as expected, leucine feeding mitigated this effect. Atrogin-1/MAFbx gene expression was upregulated only after 3 days of immobilization, and this effect was attenuated by leucine supplementation. Atrogin-1/MAFbx protein levels were elevated after 7 days of immobilization, which leucine supplementation was not able to lessen. On the other hand, myostatin gene expression was upregulated in immobilization for 3 and 7 days, which returned to normal levels after leucine supplementation. Myostatin protein levels followed gene expression at a 3-day time point only. Follistatin gene expression was upregulated during immobilization and accentuated by leucine after 3 days of supplementation. Concerning protein expression, follistatin was not altered neither by immobilization nor in immobilized animals treated with leucine. In conclusion, leucine protects against skeletal muscle mass loss during disuse, and the underlying molecular mechanisms appear to involve myostatin inhibition and Atrogin-1 normalization independently of follistatin signaling.

The myostatin rs1805086 variant is associated with obesity in Mexican adults, independently of metabolic risk factors.

AIM: To determine whether rs1805086 is associated with obesity and metabolic disturbances in a Mexican adult population. SUBJECTS AND METHODS: We genotyped rs1805086 in 1024 men and women aged 18-58 years. Anthropometric and body fat data were used to estimate obesity. Biochemical parameters were measured and DNA was used to determine the rs1805086 genotype. RESULTS: rs1805086 heterozygous AG frequency was 5.4%, and the homozygous for the risk allele GG was absent. Heterozygous had higher levels of body mass index (BMI) and waist/height ratio (WHtR). Heterozygous subjects showed a greater total and central obesity compared to the homozygous for ancestral allele AA (OR BMI > 30 kg/m2 = 2.35, 95% CI 1.29-4.29; OR WHtR > 0.5 = 2.03, 95% CI 1.19-3.45; OR elevated fat mass (EFM) %= 1.72, 95% CI 1.01-2.92; OR fat mass index (FMI)>p85 = 1.96, 95% CI 1.05-3.68). rs1805086 was not associated with metabolic alterations. CONCLUSION: Heterozygosity for rs1805086 showed a predisposition to having elevated overall and central obesity parameters. This association with adiposity seems to be independent of metabolic risk.

The evaluation of RS1805086 and RS1805065 polymorphisms in Mstn gene and anthropometric properties of national and amateur arm wrestlers / Evaluación de los polimorfismos RS1805086 y RS1805065 en el gen Mstn y las propiedades antropométricas de los luchadores de brazo nacionales y amateurs

The aim of this study is to investigate rs1805086 and rs1805065 polymorphisms of MSTN gene of national and amateur Turkish arm wrestlers and people leading a sedentary lifestyle, and the anthropometric properties such as hand, wrist, and forearm circumferences of national and amateur Turkish arm wrestlers are aimed to be explored. In this study, a total of 79 volunteers who were 24 national (7 females, 17 males) Turkish arm wrestlers, 21 amateur (7 females, 14 males) Turkish arm wrestlers and 34 sedentary people (12 females, 22 males) participated. To analyse the data, Statistical Package for the Social Sciences, SPSS 22 (SPSS Inc., Chicago, IL, USA) was used. As a result of the study, when data on rs1805086 and rs1805065 polymorphisms of MSTN gene were examined respectively, it was found out that MSTN 153KK genotype was 100.0% dominant in both national (n=24) and amateur (n=21) arm wrestlers, and it was 94.12 % dominant in sedentary people. KR genotype was observed in 5.88 % of the sedentary people. The data from the other rs1805065 polymorphism of MSTN gene showed that all participants (n = 45, 100.0 %) were carriers of normal homozygous genotype. Furthermore, for both female group and male group, there found to be statistically significant difference in terms of anthropometric properties. It can be concluded that though there was no significant difference between national and amateur Turkish arm wrestlers in terms of their MSTN gene characteristics; in terms of anthropometric properties, significant differences were discovered. It was found out that on these athletes, not MSTN gene polymorphisms but anthropometric properties were effective.

El objetivo de este estudio fue investigar los polimorfismos rs1805086 y rs1805065 del gen MSTN de luchadores de brazos turcos, nacionales y aficionados, y personas que llevan un estilo de vida sedentario, y las propiedades antropométricas además de las circunferencias de manos, muñecas y antebrazos de los luchadores de brazos turcos nacionales y aficionados. En este estudio, participaron un total de 79 voluntarios: 24 luchadores de brazos turcos nacionales (7 mujeres, 17 hombres), 21 luchadores de brazos turcos aficionados (7 mujeres, 14 hombres) y 34 personas sedentarias (12 mujeres, 22 hombres). Para analizar los datos, se utilizó el Paquete Estadístico para las Ciencias Sociales, SPSS 22 (SPSS Inc., Chicago, IL, EE. UU.). Como resultado del estudio, cuando se examinaron los datos sobre los polimorfismos rs1805086 y rs1805065 del gen MSTN respectivamente, se descubrió que el genotipo MSTN 153KK era 100,0 % dominante en luchadores de brazos nacionales (n = 24) y aficionados (n = 21) , y era 94,12 % dominante en personas sedentarias. El genotipo KR se observó en el 5,88 % de las personas sedentarias. Los datos del otro polimorfismo rs1805065 del gen MSTN mostraron que todos los participantes (n = 45; 100,0 %) eran portadores del genotipo homocigoto normal. Además, tanto para el grupo femenino como para el masculino, se encontró una diferencia estadísticamente significativa en términos de propiedades antropométricas. Se puede concluir que, aunque no hubo una diferencia significativa entre los luchadores de brazos turcos nacionales y aficionados en términos de sus características genéticas MSTN; en términos de propiedades antropométricas, se descubrieron diferencias significativas. Se descubrió que, en estos atletas, no fueron los polimorfismos del gen MSTN sino las propiedades antropométricas las efectivas.

Muscle proteolysis via ubiquitin-proteasome system (UPS) is activated by BthTx-I Lys49 PLA2 but not by BthTx-II Asp49 PLA2 and Bothrops jararacussu venom.

Bites by viperid snakes belonging to Bothrops genus produce fast and intense local edema, inflammation, bleeding and myonecrosis. In this study, we investigated the role of Myogenic Regulatory Factors (MRFs: MyoD; Myog), negatively regulated by GDF-8 (Myostatin), and ubiquitin-proteasome system pathway (UPS: MuRF-1; Fbx-32) in gastrocnemius muscle regeneration after Bothrops jararacussu snake venom (Bjussu) or its isolated phospholipase A2 myotoxins, BthTx-I (Lys-49 PLA2) and BthTx-II (Asp-49 PLA2) injection. Male Swiss mice received a single intra-gastrocnemius injection of crude Bjussu, at a dose/volume of 0.83 mg/kg/20 µl, and BthTx-I or BthTx-II, at a dose/volume of 2.5 mg/kg/20 µl. Control mice (Sham) received an injection of sterile saline solution (NaCl 0.9%; 20 µl). At 24, 48, 72 and 96 h post injection, right gastrocnemius was collected for protein expression analyses. Based on the temporal expressional dynamics of MyoD, Myog and GDF-8/Myostatin, it was possible to propose that the myogenesis pathway was impacted most badly by BthTx-II followed by BthTx-I and lastly by B. jararacussu venom, thus suggesting that catalytic activity has likely inhibitory role on the satellite cells-mediated reparative myogenesis pathway. Inversely, the catalytic activity seems to be not a determinant for the activation of proteins ubiquitination by MuRF-1 and Fbx-32/Atrogin-1 E3 proteasome ligases, given proteolysis pathway through UPS was activated neither after Bjussu, nor after BthTx-II, but just after the catalytically-inactive BthTx-I Lys-49 PLA2-homologue exposure. The findings of this study disclose interesting perspective for further mechanistic studies about pathways that take part in the atrophy and repair after permanent damage induced by bothropic snakebites.

Expression profiles of growth-related genes in two Nile tilapia strains and their crossbred provide insights into introgressive breeding effects.

The Nile tilapia (Oreochromis niloticus) is a prominent farmed fish in aquaculture worldwide. Crossbreeding has recently been carried out between the Red-Stirling and the wt Chitralada strains of Nile tilapia, producing a heterotic hybrid (7/8 Chitralada and 1/8 Red-Stirling) that combines the superior growth performance of the Chitralada with the reddish coloration of the Red-Stirling strain. While classical selective breeding and crossbreeding strategies are well known, the molecular mechanisms underlying the phenotypic expression of economically advantageous traits in tilapia remain largely unknown. Molecular investigations have shown that variable expression of growth hormone (gh), insulin-like growth factors (igf1 and 2) and somatolactin (smtla) - components of the growth hormone/insulin-like growth factor (GH/IGF) axis - and myostatin (mstn) genes can affect traits of economic relevance in farmed animals. The aim of this study was to assess and compare the gene expression signature among Chitralada, Red-Stirling and their backcross hybrid in order to gain insights into the effects of introgressive breeding in modulation of the GH/IGF axis. Gene expression analyses in distinct tissues showed that most genes of the GH/IGF axis were up-regulated and mstn was down-regulated in backcross animals in comparison with Red-Stirling and Chitralada animals. These gene expression profiles revealed that backcross animals displayed a distinctive expression signature, which attests to the effectiveness of the introgressive breeding technique. Our findings also suggest that the GH/IGF axis and mstn genes might be candidate markers for fish performance and prove useful within genetic improvement programs aimed at the production of superior-quality tilapia strains using introgressive breeding.

Post-transcriptional silencing of myostatin-1 in the spotted rose snapper (Lutjanus guttatus) promotes muscle hypertrophy.

Muscle growth is regulated by several factors including the growth differentiation factor 8, known as myostatin, an inhibitor of myocyte differentiation and proliferation. Research on myostatin regulation was already conducted to improve growth rates in farmed animals, including aquatic species. To explore the effects of myostatin inactivation in a commercial marine fish (spotted rose snapper, Lutjanus guttatus) in vivo, we induced post-transcriptional silencing (knockdown) of myostatin-1 (mstn-1) by injecting dsiRNA directly into the muscle of juvenile fish (87 days post-hatch) using a commercial polymer as vehicle. Results show a significant decrease in mstn-1 expression starting at 2 days after injection and for up to 5 days. Knockdown of mstn-1 caused muscle fiber hypertrophy (but not hyperplasia); however, there were no significant changes in weight or length. Although still experimental, this study provides evidence that temporary knockdown of mstn-1 in a commercial marine fish in vivo promotes fiber hypertrophy and therefore could potentially help grow-out programmes in fish aquaculture.

Non-synonymous polymorphisms in candidate gene associated with growth traits in Channel catfish (Ictalurus punctatus, Rafinesque, 1818).

Resequencing of Myostatin, Growth Hormone, Follistatin-A-like, Insulin-like Growth Factor I (IGF-I) and Myogenin (MYOG) genes was completed to discover novel genetic variations and assess non synonymous (ns) polymorphisms (SNPs) effect on growth related traits of channel catfish. Wild and farmed animals were used as a discovering population. Resequencing lead to the identification of 59 new variants in the five analyzed genes; 66% found in introns and 34% in coding regions. From coding regions, 14 variants were synonyms and six were ns variations. A mutation rate of one in 129 bp was estimated. Four ns variations were selected for validation and association analysis. In IGF-I two ns polymorphisms, at IGF-I19 the G wild type allele was fixed in population and for IGF-I63 the C allele had a frequency of 0.972 and for mutate allele G of 0.027. In MYOG two ns SNPs were assessed. MYOG131 presented a frequency of alleles T and A, of 0.754 and 0.246, respectively and MYOG233, with a frequency of G and C of 0.775 and 0.225, respectively. Only MYOG131 (g.529T>A) was significantly associated (P < 0.04) to some growth traits. Results suggest MYOG131 g.529T>A as candidate locus for genetic enhancement of growth traits in channel catfish.

G protein-coupled receptor kinase 2 regulates mitochondrial bioenergetics and impairs myostatin-mediated autophagy in muscle cells.

G protein-coupled receptor kinase 2 (GRK2) is an important protein involved in ß-adrenergic receptor desensitization. In addition, studies have shown GRK2 can modulate different metabolic processes in the cell. For instance, GRK2 has been recently shown to promote mitochondrial biogenesis and increase ATP production. However, the role of GRK2 in skeletal muscle and the signaling mechanisms that regulate GRK2 remain poorly understood. Myostatin is a well-known myokine that has been shown to impair mitochondria function. Here, we have assessed the role of myostatin in regulating GRK2 and the subsequent downstream effect of myostatin regulation of GRK2 on mitochondrial respiration in skeletal muscle. Myostatin treatment promoted the loss of GRK2 protein in myoblasts and myotubes in a time- and dose-dependent manner, which we suggest was through enhanced ubiquitin-mediated protein loss, as treatment with proteasome inhibitors partially rescued myostatin-mediated loss of GRK2 protein. To evaluate the effects of GRK2 on mitochondrial respiration, we generated stable myoblast lines that overexpress GRK2. Stable overexpression of GRK2 resulted in increased mitochondrial content and enhanced mitochondrial/oxidative respiration. Interestingly, although overexpression of GRK2 was unable to prevent myostatin-mediated impairment of mitochondrial respiratory function, elevated levels of GRK2 blocked the increased autophagic flux observed following treatment with myostatin. Overall, our data suggest a novel role for GRK2 in regulating mitochondria mass and mitochondrial respiration in skeletal muscle.

CRISPR/Cas9-mediated MSTN gene editing induced mitochondrial alterations in C2C12 myoblast cells

Background: Myostatin (MSTN) negatively regulates muscle mass and is a potent regulator of energy metabolism. However, MSTN knockout have affect mitochondrial function. This research assessed the mitochondrial energy metabolism of Mstn−/+ KO cells, and wondered whether the mitochondria biogenesis are affected. Results: In this study, we successfully achieved Mstn knockout in skeletal muscle C2C12 cells using a CRISPR/Cas9 system and measured proliferation and differentiation using the Cell-Counting Kit-8 assay and qPCR, respectively. We found that MSTN dysfunction could promote proliferation and differentiation compared with the behaviour of wild-type cells. Moreover, Mstn KO induced an increase in KIF5B expression. The mitochondrial content was significantly increased in Mstn KO C2C12 cells, apparently associated with the increases in PGC-1α, Cox1, Cox2, ND1 and ND2 expression. However, no differences were observed in glucose consumption and lactate production. Interestingly, Mstn KO C2C12 cells showed an increase in IL6 and a decrease in TNF-1α levels. Conclusion: These findings indicate that MSTN regulates mitochondrial biogenesis and metabolism. This gene-editing cells provided favourable evidence for animal breeding and metabolic diseases.

Skeletal Muscle Response to Deflazacort, Dexamethasone and Methylprednisolone.

Glucocorticoids represent some of the most prescribed drugs that are widely used in the treatment of neuromuscular diseases, but their usage leads to side effects such as muscle atrophy. However, different synthetic glucocorticoids can lead to different muscle effects, depending upon its chemical formulation. Here, we intended to demonstrate the muscle histologic and molecular effects of administering different glucocorticoids in equivalency and different dosages. Methods: Seventy male Wistar rats distributed into seven groups received different glucocorticoids in equivalency for ten days or saline solution. The study groups were: Control group (CT) saline solution; dexamethasone (DX) 1.25 or 2.5 mg/kg/day; methylprednisolone (MP) 6.7 or 13.3mg/kg/day; and deflazacort (DC) 10 or 20 mg/kg/day. At the end of the study, the animals were euthanized, and the tibialis anterior and gastrocnemius muscles were collected for metachromatic ATPase (Cross-sectional area (CSA) measurement), Western blotting (protein expression of IGF-1 and Ras/Raf/MEK/ERK pathways) and RT-PCR (MYOSTATIN, MuRF-1, Atrogin-1, REDD-1, REDD-2, MYOD, MYOG and IRS1/2 genes expression) experiments. Results: Muscle atrophy occurred preferentially in type 2B fibers in all glucocorticoid treated groups. DC on 10 mg/kg/day was less harmful to type 2B fibers CSA than other doses and types of synthetic glucocorticoids. In type 1 fibers CSA, lower doses of DC and DX were more harmful than high doses. DX had a greater effect on the IGF-1 pathway than other glucocorticoids. MP more significantly affected P-ERK1/2 expression, muscle fiber switching (fast-to-slow), and expression of REDD1 and MyoD genes than other glucocorticoids. Compared to DX and MP, DC had less of an effect on the expression of atrogenes (MURF-1 and Atrogin-1) despite increased MYOSTATIN and decreased IRS-2 genes expression. Conclusions: Different glucocorticoids appears to cause muscle atrophy affecting secondarily different signaling mechanisms. MP is more likely to affect body/muscles mass, MEK/ERK pathway and fiber type transition, DX the IGF-1 pathway and IRS1/2 expression. DC had the smallest effect on muscle atrophic response possibly due a delayed timing on atrogenes response.

Omega-3 multiple effects increasing glucocorticoid-induced muscle atrophy: autophagic, AMPK and UPS mechanisms.

Muscle atrophy occurs in many conditions, including use of glucocorticoids. N-3 (omega-3) is widely consumed due its healthy properties; however, concomitant use with glucocorticoids can increase its side effects. We evaluated the influences of N-3 on glucocorticoid atrophy considering IGF-1, Myostatin, MEK/ERK, AMPK pathways besides the ubiquitin-proteasome system (UPS) and autophagic/lysosomal systems. Sixty animals constituted six groups: CT, N-3 (EPA 100 mg/kg/day for 40 days), DEXA 1.25 (DEXA 1.25 mg/kg/day for 10 days), DEXA 1.25 + N3 (EPA for 40 days + DEXA 1.25 mg/kg/day for the last 10 days), DEXA 2.5 (DEXA 2.5 mg/kg/day for 10 days), and DEXA 2.5 + N3 (EPA for 40 days + DEXA 2.5 mg/kg/day for 10 days). Results: N-3 associated with DEXA increases atrophy (fibers 1 and 2A), FOXO3a, P-SMAD2/3, Atrogin-1/MAFbx (mRNA) expression, and autophagic protein markers (LC3II, LC3II/LC3I, LAMP-1 and acid phosphatase). Additionally, N-3 supplementation alone decreased P-FOXO3a, PGC1-alpha, and type 1 muscle fiber area. Conclusion: N-3 supplementation increases muscle atrophy caused by DEXA in an autophagic, AMPK and UPS process.

Rheumatoid arthritis-increased gene expressions in muscle atrophy are restored back to control as a response to acute resistance exercise / Estímulo agudo de musculação promove regulação de genes alterados pela atrofi a muscular

This study had as objective to analyze the acute eff ects of resistance exercise (RE) on the mRNA levels of the following genes (MyoD, myogenin, IGF-1, atrogin-1, MuRF-1, and myostatin) in rheumatoid arthritis (experimental arthritis). Therefore, 26 females rats were randomly allocated into four groups, control (CT, n=7), exercise (Ex, n=6), rheumatoid arthritis (RA, n=6) and RA with exercise (RAEx, n=7). Met-BSA was injected into the tibiotarsal joint in the RA and RAEx groups. After 15 days from injection, the animals were submitted to an acute bout of RE and six hours post protocol the animals were euthanized. We evaluated the joint thickness, infl ammation score, cross-sectional area (CSA) of gastrocnemius muscle fi bers and mRNA expression of the IGF-1, MyoD, myogenin, myostatin, MuRF-1, atrogin-1 and GAPDH. It was observed that the joint thickness and score strongly increased in arthritic rats (p <0.001) while the CSA decreased (p ≤ 0.05). Increased mRNA levels of IGF-1 (2.0 fold), myostatin (4.5 fold), atrogin-1 (2.5 fold), MyoD (3.7-fold) and myogenin (5 fold) were observed in muscle of arthritic rats. The mRNA expression of myostatin, atrogin-1, MyoD and myogenin decreased in the RAEx group. In this way, we can conclude that experimental arthritis-increased gene expressions in muscle atrophy myostatin, atrogin-1, MyoD and myogenin) are restored back to control as a response to acute RE....(AU)

O presente estudo teve como objetivo analisar o efeito agudo do Exercício com pesos sobre os níves de mRNA de genes envolvidos no anabolismo ou catabolismo muscular em um modelo experimental de Artrite Reumatóide. Para tanto, 26 ratas fêmeas foram randomicamente alocadas em quatro grupos, controle (CT, n=7), Exercício (Ex, n=6), Artrite Reumatóide (AR, n=6) e Artrite Reumatóide com exercício (AREx, n=7). Uma substância contendo Albumina bovina metilada foi injetada na articulação tíbio-tarsal nos grupos AR e AREx para indução da Artrite Reumatóide. Após 15 dias da injeção, os animais foram submetidos a um estímulo agudo de treinamento com pesos e 6 horas após o exercício os animais foram eutanasiados. Nós avaliamos a espessura da articulação, escore de infl amação, a área de secção transversa (AST) das fi bras do músculo Gastrocnêmio e a mRNA de IGF-1, MyoD, Myogenina (genes envolvidos no anabolismo muscular), e MuRF-1, atrogina-1 (genes envolvidos no catabolismo muscular), além do gene controle , GAPDH. Foi observado que a espessura articular e o escore de infl amação aumentaram fortemente nas ratas induzidas a Artrite Reumatóide (p <0,001), enquanto a AST reduziu (p ≤ 0,05). Um aumento nos níveis de mRNA de IGF-1 (2,0 vezes), miostatina (4,5 vezes), atrogina-1 (2,5 vezes), MyoD (3,7 vezes) e miogenina (5 vezes) foi observado no músculo das ratas induzidas a Artrite Reumatóide. mRNA de miostatina, atrogina-1, MyoD e miogenina reduziu no grupo RAEx. Desta forma, podemos concluir, que o modelo experimental de Artrite Reumatóide induziu um aumento da expressão de genes durante a atrofi a muscular (myostatin, atrogin-1, MyoD and myogenin) e que estas alterações foram reguladas pelo Exercício com peso....(AU)