Myostatin mRNA Research Articles

Beneficial impact of dietary methyl methionine sulfonium chloride and/or L-carnitine supplementation on growth performance, feed efficiency, and serum biochemical parameters in broiler chicken: role of IGF-1 and MSTN genes.

The purpose of this study was to examine the effect of dietary supplementation with methyl methionine sulfonium chloride (MMSC), and L-carnitine (L-CAR) alone or in combination on the growth performance of broilers through their impact on the expression of IGF-1 and MSTN genes associated with growth in broilers. One-day-old female Ross 308 broiler chicks were allocated into four groups, each of which received a broiler starter diet and water daily ad libitum. The control group (group 1) was given drinking water without any additives. Group 2 received 0.25g L-carnitine per liter of drinking water, group 3 received 0.25g MMSC per liter of drinking water, and group 4 received 0.25g of both L-carnitine and MMSC per liter of drinking water. Birds were given a starter diet to 21days after which they received a broiler grower diet to 35days when the experiment ended. There were five replicate groups of 12 birds per treatment. Body weights and feed intake were recorded weekly. Compared to the control group of birds, supplementation with MMSC either alone or in combination with L-carnitine resulted in an increase in growth rate or feed utilization efficiency; L-carnitine by itself had no effect. MMSC supplementation, again either alone or in combination with L-carnitine, increased jejunal and ileal villi height, increased serum total proteins and globulins, downregulated myostatin (MSTN) mRNA, and upregulated insulin growth factor-1 (IGF-1) mRNA expression. Supplementation with L-carnitine alone showed none of these effects. We conclude that MMSC supplementation improved growth performance through the upregulation of IGF-1 mRNA expression and downregulation of MSTN mRNA expression.

Renal failure suppresses muscle irisin expression, and irisin blunts cortical bone loss in mice.

BackgroundChronic renal failure induces bone mineral disorders and sarcopenia. Skeletal muscle affects other tissues, including bone, by releasing myokines. However, the effects of chronic renal failure on the interactions between muscle and bone remain unclear.MethodsWe investigated the effects of renal failure on bone, muscle, and myokines linking muscle to bone using a mouse 5/6 nephrectomy (Nx) model. Muscle mass and bone mineral density (BMD) were analysed by quantitative computed tomography 8 weeks after Nx.ResultsNephrectomy significantly reduced muscle mass in the whole body (12.1% reduction, P < 0.05), grip strength (10.1% reduction, P < 0.05), and cortical BMD at the femurs of mice (9.5% reduction, P < 0.01) 8 weeks after surgery, but did not affect trabecular BMD at the femurs. Among the myokines linking muscle to bone, Nx reduced the expression of irisin, a proteolytic product of fibronectin type III domain‐containing 5 (Fndc5), in the gastrocnemius muscles of mice (38% reduction, P < 0.01). Nx increased myostatin mRNA levels in the gastrocnemius muscles of mice (54% increase, P < 0.01). In simple regression analyses, cortical BMD, but not trabecular BMD, at the femurs was positively related to Fndc5 mRNA levels in the gastrocnemius muscles of mice (r = 0.651, P < 0.05). The weekly administration of recombinant irisin to mice ameliorated the decrease in cortical BMD, but not muscle mass or grip strength, induced by Nx (6.2% reduction in mice with Nx vs. 3.3% reduction in mice with Nx and irisin treatment, P < 0.05).ConclusionsThe present results demonstrated that renal failure decreases the expression of irisin in the gastrocnemius muscles of mice. Irisin may contribute to cortical bone loss induced by renal failure in mice as a myokine linking muscle to bone.

Skeletal muscle myostatin gene expression and sarcopenia in overweight and obese middle‐aged and older adults

BackgroundMyostatin (MSTN) is a key negative regulator of muscle mass in humans and animals, having direct and indirect influences on molecular regulators of atrophy and hypertrophy, thus potentially impacting fitness and physical function. We have shown that myostatin is elevated in conditions of chronic disability (e.g. paretic limb of stroke). Our hypothesis is that myostatin would be elevated in older adults with sarcopenia. The purpose of this study was to examine the role of skeletal muscle myostatin in sarcopenia.MethodsSixty-four overweight to obese aged 45–81 years underwent a maximal aerobic capacity (VO2max) test, dual-energy X-ray absorptiometry (DXA) scan to determine appendicular lean tissue (ALM), and vastus lateralis muscle biopsy to determine myostatin mRNA expression by quantitative real time PCR (Q-RT-PCR). Rates of sarcopenia were determined using (ALM/BMI), and sarcopenia was defined as <0.789 in men and <0.512 in women. Subjects had low fitness (VO2max: 22.7 ± 0.7 mL/kg/min) and on average 40.9 ± 1% body fat.ResultsThe prevalence of sarcopenia in this cohort was 16%. BMI, % body fat, and fat mass were higher in adults with sarcopenia than those without sarcopenia (all P < 0.001). Myostatin mRNA expression was lower in those without sarcopenia than those with sarcopenia (P < 0.05) and higher in men than women (P < 0.001). Myostatin expression was associated with BMI (r = 0.36, P < 0.01) and mid-thigh intramuscular fat (r = 0.29, P < 0.05).ConclusionGiven that myostatin is important in muscle atrophy, fat accumulation, and sarcopenia, further work could address its implication in other aging cohorts of disability and chronic disease.

IRE1α regulates skeletal muscle regeneration through Myostatin mRNA decay.

Skeletal muscle can undergo a regenerative process from injury or disease to preserve muscle mass and function, which is critically influenced by cellular stress responses. Inositol-requiring enzyme 1 (IRE1) is an ancient endoplasmic reticulum (ER) stress sensor and mediates a key branch of the unfolded protein response (UPR). In mammals, IRE1α is implicated in the homeostatic control of stress responses during tissue injury and regeneration. Here, we show that IRE1α serves as a myogenic regulator in skeletal muscle regeneration in response to injury and muscular dystrophy. We found in mice that IRE1α was activated during injury-induced muscle regeneration, and muscle-specific IRE1α ablation resulted in impaired regeneration upon cardiotoxin-induced injury. Gain- and loss-of-function studies in myocytes demonstrated that IRE1αacts to sustain both differentiation in myoblasts and hypertrophy in myotubes through regulated IRE1-dependent decay (RIDD) of mRNA encoding Myostatin, a key negative regulator of muscle repair and growth. Furthermore, in the mouse model of Duchenne muscular dystrophy (DMD), loss of muscle IRE1α resulted in augmented Myostatin signaling and exacerbated the dystrophic phenotypes. Thus, these results reveal a pivotal role for the RIDD output of IRE1α in muscle regeneration, offering new insight into potential therapeutic strategies for muscle loss diseases.

Coenzyme Q10 in quail nutrition: effects on growth performance, meat quality, and myostatin gene expression

• Diets without supplemental vitamin E did not impair growth performance of quail. • Supplementation of coenzyme Q10 to basal diet improved growth of broiler quail. • Supplementation of the basal diet with coenzyme Q10 improved meat quality traits. • Expression of myostatin mRNA decreased in quail received 210 mg coenzyme Q10/kg feed. The present experiment was conducted to investigate effects of dietary coenzyme Q10 (CQ10) supplementation on growth performance, meat quality, immune responses, and myostatin gene expression in broiler quail. A total of 605 one d-old Japanese broiler quail ( Coturnix japonica ) were randomly allotted to 11 dietary treatments with 5 replicates pens of 11 chicks each in a completely randomized design during a 5-wk period. Experimental treatments consisted of a control (CTL) diet: basal corn-soybean meal diet supplemented with vitamin E in the vitamin premix as recommended by breeder manual; CTL diet without vitamin E supplementation in the vitamin premix (CTL–), and CTL diet supplemented with 100 IU vitamin E in the vitamin premix (CTL+). The other dietary treatments included CTL diet supplemented with inclusion rates of 15, 30, 45, 60, 75, 90, 105, and 210 mg CQ10/kg. Although CTL– or CTL+ diets did not affect growth performance of broiler quail, supplementation of 105 or 210 mg CQ10/kg to CTL diet improved weight gain and feed conversion ratio so that quail possessed better growth performance ( P < 0.05). The breast muscle of quail fed diets supplemented with 30 to 210 mg CQ10/kg contained greater water holding capacity ( P < 0.05). Dietary supplementation of 60 to 210 mg CQ10/kg increased meat pH compared to quail in the other experimental treatments except for those fed with dietary supplemental 45 mg CQ10/kg ( P < 0.05). Concentration of thiobarbituric acid-reactive substance and cooking loss of meat samples were lower in quail received diets supplemented with 45 to 210 mg CQ10/kg diet ( P < 0.05). Quail received dietary supplemental 105 to 210 mg CQ10/kg had lighter meat than those fed the CTL, CTL+, and CTL– diets ( P < 0.05). Red blood cell and white blood cell counts decreased after feeding quail with diets without supplementation of vitamin E in the premix ( P < 0.05) but improved through feeding quail with CTL, CTL+, and 15 to 90 mg supplemental CQ10/kg of diet ( P < 0.05). Antibody titer against sheep red blood cells increased in quail fed diets supplemented with 75 mg CQ10/kg ( P < 0.05). Myostatin gene expression was greater in quail received 100 IU vitamin E in their feed than quail in CTL– or COQ210 treatments ( P < 0.05). In conclusion, supplementation of CQ10 could increase growth performance of quail beyond what observed for vitamin E and also could replace vitamin E in terms of immune responses.

Molecular Differences in Skeletal Muscle After 1 Week of Active vs. Passive Recovery From High-Volume Resistance Training.

Vann, CG, Haun, CT, Osburn, SC, Romero, MA, Roberson, PA, Mumford, PW, Mobley, CB, Holmes, HM, Fox, CD, Young, KC, and Roberts, MD. Molecular differences in skeletal muscle after 1 week of active vs. passive recovery from high-volume resistance training. J Strength Cond Res 35(8): 2102-2113, 2021-Numerous studies have evaluated how deloading after resistance training (RT) affects strength and power outcomes. However, the molecular adaptations that occur after deload periods remain understudied. Trained, college-aged men (n = 30) performed 6 weeks of whole-body RT starting at 10 sets of 10 repetitions per exercise per week and finishing at 32 sets of 10 repetitions per exercise per week. After this period, subjects performed either active (AR; n = 16) or passive recovery (PR; n = 14) for 1 week where AR completed ∼15% of the week 6 training volume and PR ceased training. Variables related to body composition and recovery examined before RT (PRE), after 6 weeks of RT (POST), and after the 1-week recovery period (DL). Vastus lateralis (VL) muscle biopsies and blood samples were collected at each timepoint, and various biochemical and histological assays were performed. Group × time interactions (p < 0.05) existed for skeletal muscle myosin heavy chain (MHC)-IIa mRNA (AR > PR at POST and DL) and 20S proteasome activity (post-hoc tests revealed no significance in groups over time). Time effects (P < 0.05) existed for total mood disturbance and serum creatine kinase and mechano growth factor mRNA (POST > PRE &D L), VL pressure to pain threshold and MHC-IIx mRNA (PRE&DL > POST), Atrogin-1 and MuRF-1 mRNA (PRE < POST < DL), MHC-I mRNA (PRE < POST & DL), myostatin mRNA (PRE & POST < DL), and mechanistic target of rapamycin (PRE > POST & DL). No interactions or time effects were observed for barbell squat velocity, various hormones, histological metrics, polyubiquitinated proteins, or phosphorylated/pan protein levels of 4E-BP1, p70S6k, and AMPK. One week of AR after a high-volume training block instigates marginal molecular differences in skeletal muscle relative to PR. From a practical standpoint, however, both paradigms elicited largely similar responses.

MiR-2014-5p and miR-1231-5p regulate muscle growth of Larimichthys crocea by targeting MSTN gene.

MicroRNAs (miRNAs) play an important role in regulating gene expression, and myostatin (MSTN) has been widely recognized as a key gene for muscle growth and development. Through high-throughput sequencing to study the effects of starvation on miRNA transcriptomes in Larimichthys crocea muscle tissue, we found that the expression of miR-2014, miR-1231 and miR-1470 were significantly different between fasting and normal feeding Larimichthys crocea. Bioinformatics analysis predicted that miR-2014, miR-1231 and miR-1470 target MSTN mRNA 3'UTR. To verify the accuracy of predictions, we constructed double luciferase plasmids containing MSTN 3'UTR and confirmed that miR-2014-5p and miR-1231-5p can inhibit MSTN expression by targeting MSTN 3'UTR using double luciferase experiments, while miR-1470 is not involved in regulation. Subsequent site-directed mutation experiments reflected the specificity of the target sequence. In addition, quantitative PCR experiments revealed that miR-2014-5p and miR-1231-5p may participate in the regulation of MSTN expression in fasting and refeeding period, respectively. These results implied that miRNA may take part in muscle growth regulation during starvation. It provides some insights into the molecular regulation mechanism of MSTN in response to starvation stress in fish.

Expression of titin-linked putative mechanosensing proteins in skeletal muscle after power resistance exercise in resistance-trained men.

Little is known about the molecular responses to power resistance exercise that lead to skeletal muscle remodeling and enhanced athletic performance. We assessed the expression of titin-linked putative mechanosensing proteins implicated in muscle remodeling: muscle ankyrin repeat proteins (Ankrd 1, Ankrd 2, and Ankrd 23), muscle-LIM proteins (MLPs), muscle RING-finger protein-1 (MuRF-1), and associated myogenic proteins (MyoD1, myogenin, and myostatin) in skeletal muscle in response to power resistance exercise with or without a postexercise meal, in fed, resistance-trained men. A muscle sample was obtained from the vastus lateralis of seven healthy men on separate days, 3 h after 90 min of rest (Rest) or power resistance exercise with (Ex + Meal) or without (Ex) a postexercise meal to quantify mRNA and protein levels. The levels of phosphorylated HSP27 (pHSP27-Ser15) and cytoskeletal proteins in muscle and creatine kinase activity in serum were also assessed. The exercise increased (P ≤ 0.05) pHSP27-Ser15 (∼6-fold) and creatine kinase (∼50%), whereas cytoskeletal protein levels were unchanged (P > 0.05). Ankrd 1 (∼15-fold) and MLP (∼2-fold) mRNA increased, whereas Ankrd 2, Ankrd 23, MuRF-1, MyoD1, and myostatin mRNA were unchanged. Ankrd 1 (∼3-fold, Ex) and MLPb (∼20-fold, Ex + Meal) protein increased, but MLPa, Ankrd 2, Ankrd 23, and the myogenic proteins were unchanged. The postexercise meal did not affect the responses observed. Power resistance exercise, as performed in practice, induced subtle early responses in the expression of MLP and Ankrd 1 yet had little effect on the other proteins investigated. These findings suggest possible roles for MLP and Ankrd 1 in the remodeling of skeletal muscle in individuals who regularly perform this type of exercise.NEW & NOTEWORTHY This is the first study to assess the early changes in the expression of titin-linked putative mechanosensing proteins and associated myogenic regulatory factors in skeletal muscle after power resistance exercise in fed, resistance-trained men. We report that power resistance exercise induces subtle early responses in the expression of Ankrd 1 and MLP, suggesting these proteins play a role in the remodeling of skeletal muscle in individuals who regularly perform this type of exercise.

Effect of 6 Weeks of Aerobic Training on TGF-Β1, Myostatin and Matrix Metalloproteinase 9 Genes Expression in the Tendon of Fast- and Slow-Twitch Muscles of Male Wistar Rats

Background and Aim: Tendon, like the skeletal muscle, exhibits mechanical and morphological adaptations resulted from exercise training; however, little is known about the basic cellular and molecular mechanisms that regulate these responses. The aim of the present study was therefore to investigate the effect of 6 weeks of aerobic training on the TGF-β1, myostatin and MMP9 mRNAs expression in the tendon of fast- and slow-twitch muscles. Methods &amp; Materials For this purpose, 12 male Wistar rats at 8 weeks of age were randomly divided into two groups: experimental (n=6) and control (n=6). The exercise group performed aerobic training for 6 weeks, 5 sessions per week. Forty-eight hours after the last training session, all rats were sacrificed and the tendons of soleus and Extensor Digitorum Longus (EDL) muscles were extracted. Expression of TGF-β1, myostatin and MMP9 mRNAs were assayed using RealTime-PCR. Independent t-test was also used for statistical analysis. Ethical Considerations: All stages of the study were conducted according to the ethical guidelines and authorization of Research Deputy of Islamic Azad University, Central Tehran Branch No. IR.IAU.PS.REC.1398.296. Results: The results showed that the expression of TGF-β1 mRNA in EDL and soleus tendons significantly increased (P≤0.001), whereas the expression of myostatin in EDL tendon was significantly reduced (P≤ 0.001). Increased mRNA expression of MMP9 in the tendon of EDL and soleus muscles was not statistically significant (P&gt;0.05). Conclusion: It seems that aerobic exercise can modulate the expression of genes involved in the regulation of tendon collagen in a muscle type-dependent manner.

Regular exercise and branched-chain amino acids prevent ischemic acute kidney injury-related muscle wasting in mice.

Acute kidney injury (AKI) causes glucose and protein metabolism abnormalities that result in muscle wasting, thereby affecting the long‐term prognosis of critical illness survivors. Here, we examined whether early intervention with treadmill exercise and branched‐chain amino acids (BCAA) can prevent AKI‐related muscle wasting and reduced physical performance in mice. Unilateral 15 min ischemia‐reperfusion injury was induced in contralateral nephrectomized mice, and muscle histological and physiological changes were assessed and compared with those of pair‐fed control mice, since AKI causes severe anorexia. Mice exercised for 30 min each day and received oral BCAA for 7 days after AKI insult. By day 7, ischemic AKI significantly decreased wet weight, myofiber cross‐sectional area, and central mitochondrial volume density of the anterior tibialis muscle, and significantly reduced maximal exercise time. Regular exercise and BCAA prevented AKI‐related muscle wasting and low physical performance by suppressing myostatin and atrogin‐1 mRNA upregulation, and restoring reduced phosphorylated Akt and PGC‐1α mRNA expression in the muscle. Ischemic AKI induces muscle wasting by accelerating muscle protein degradation and reducing protein synthesis; however, we found that regular exercise and BCAA prevented AKI‐related muscle wasting without worsening kidney damage, suggesting that early rehabilitation with nutritional support could prevent AKI‐related muscle wasting.

Macrophage Subpopulations and the Acute Inflammatory Response of Elderly Human Skeletal Muscle to Physiological Resistance Exercise.

The current model for repair of damaged tissue includes immune cells, mediating the progression from a pro-inflammatory to an anti-inflammatory environment. How this process changes with aging in human skeletal muscle under conditions of physiological exercise loading remains unclear. To investigate this, 25 elderly males (mean age 70 ± SD 7 years), as well as 12 young (23 ± 3 years) and 12 elderly (74 ± 3 years) females, performed a unilateral bout of heavy resistance leg extension exercise. Biopsies were collected from the vastus lateralis muscle of the rested (control) leg, and post exercise from the exercised leg at 4.5 h, and on days 1, 4, and 7 for the male participants, or on day 5 for the female participants. Total macrophages (CD68+) as well as pro- (CD11b+) and anti-inflammatory (CD163+, CD206+) subpopulations were identified on sections by immunohistochemistry. Gene expression levels of COL1A1, TNF-a, CD68, myostatin, TCF7L2, IL-1B, IL-1R, IL-10, and Ki67 were determined by real-time RT-PCR. At rest, the muscle tissue from the elderly vs. young females was characterized by higher gene expression levels of CD68, IL-10, lower myostatin mRNA, and trends for a greater number of macrophages, while COL1A1 mRNA post exercise values were greater in the elderly vs young. For the male participants, mRNA levels of the inflammatory cytokines IL-1B, IL-1R were elevated in the early phase following exercise, followed by increases in COL1A1 and Ki67 on days 4 and 7. In general, exercise induced increases in all types of macrophages counted in the elderly, but not in young, individuals. Cells expressing CD68, CD11b, and CD206 simultaneously were the most frequently observed cell type, which raises the possibility that pure pro- and anti-inflammatory macrophages populations do not exist in healthy human skeletal muscle within the spectrum of tissue remodeling induced by physiological exercise designed to induce hypertrophy. Together these data provide insight into the time course of macrophage activity and associated molecular targets in human skeletal muscle in the context of aging and exercise.

Effects Of Adipose Mesenchymal Stem Cells Injected Intramuscularly On Myostatin Signaling Skeletal Muscle In Rats After Eccentric Exercise

PURPOSE: To observe the effect of adipose mesenchymal stem cells (ASCs) on myostatin signal of skeletal muscle in rats at different time points after eccentric exercise, and to explore the mechanism of ASCs injection on skeletal muscle injury and repair after eccentric exercise. METHODS: Phosphate buffer saline (PBS) were injected into the gastrocnemius muscle in the left leg, and ASCs were injected into the gastrocnemius muscle of the right leg of SD rats after eccentric exercise. The rats were randomly divided into four groups: one day D1, three days D3, seven days W1 and fourteen days W2 after exercise. Skeletal muscle ultrastructure was observed by electron microscopic. The content of serum creatine kinase (CK), skeletal muscle troponin I (sTnI), myostatin (MSTN), follistatin (FST) were measured by ELISA. Real-time PCR was used to detect the expressions of MSTN, ACVR2B, FST mRNA in skeletal muscle. The expression of MSTN, ACVR2B, FST, p-Smad2/3 were detected by Western Blot. RESULTS: Compared with group PBS, adipose mesenchymal stem cell injection significantly promoted the repair of muscle fibers. Compared with group D1, the level of CK in group W2 was significantly decreased, the content of sTnI level in group D3 and group W1 were remarkably increased, the serum MSTN contents in group W1 were significantly decreased, which in group W2 was remarkably increased. Compared with group PBS, the relative expression quantities of MSTN mRNA were significantly decreased at time point D3 and which were extremely significantly down-regulated at time point W2. Compared with group PBS, the expression of MSTN protein were significantly decreased at time point D3, the expression of ACVR2B protein were remarkably increased at time point D1,D3 and W1, however, which was significantly decreased at time point W2. Compared with group PBS, the phosphorylation of Smad2/3 shown significantly decreased at the time point W2 of group ASCs. CONCLUSIONS: After eccentric exercise, allogeneic adipose mesenchymal stem cells injected intramuscularly can decreased the transcription of MSTN in skeletal muscle. Adipose mesenchymal stem cells injected intramuscularly may improve the regeneration and repair of skeletal muscle after eccentric exercise through affecting the downstream signaling pathway of MSTN.

The Effects of Cold Water Immersion and Active Recovery on Molecular Factors That Regulate Growth and Remodeling of Skeletal Muscle After Resistance Exercise.

Regular postexercise cooling attenuates muscle hypertrophy, yet its effects on the key molecular factors that regulate muscle growth and remodeling are not well characterized. In the present study, nine men completed two sessions of single-leg resistance exercise on separate days. On 1 day, they sat in cold water (10°C) up to their waist for 10 min after exercise. On the other day, they exercised at a low intensity for 10 min after exercise. Muscle biopsies were collected from the exercised leg before, 2, 24, and 48 h after exercise in both trials. These muscle samples were analyzed to evaluate changes in genes and proteins involved in muscle growth and remodeling. Muscle-specific RING finger 1 mRNA increased at 2 h after both trials (P < 0.05), while insulin-like growth factor (IGF)-1 Ec, IGF-1 receptor, growth arrest and DNA damage-inducible protein 45, collagen type I alpha chain A, collagen type III alpha chain 1, laminin and tissue inhibitor of metallopeptidase 1 mRNA increased 24−48 h after both trials (P < 0.05). By contrast, atrogin-1 mRNA decreased at all time points after both trials (P < 0.05). Protein expression of tenascin C increased 2 h after the active recovery trial (P < 0.05), whereas FoxO3a protein expression decreased after both trials (P < 0.05). Myostatin mRNA and ubiquitin protein expression did not change after either trial. These responses were not significantly different between the trials. The present findings suggest that regular cold water immersion attenuates muscle hypertrophy independently of changes in factors that regulate myogenesis, proteolysis and extracellular matrix remodeling in muscle after exercise.

Enhanced myostatin expression and signalling promote tubulointerstitial inflammation in diabetic nephropathy

Myostatin (MSTN), a family member of the transforming growth factor (TGF)-β super family, has been detected in the tubuli of pig kidney, but its role in the human kidney is not known. In this study we observed upregulation of MSTN mRNA (~8 to 10-fold increase) both in the glomeruli and tubulointerstitium in diabetic nephropathy (DN). In DN, immunoreactive MSTN was mainly localized in the tubuli and interstitium (∼4–8 fold increase), where it colocalized in CD45+ cells. MSTN was also upregulated in the glomeruli and the arterial vessels. Tubulointerstitial MSTN expression was directly related to interstitial fibrosis (r = 0.54, p < 0.01). In HK-2 tubular epithelial cells, both high (30 mmol) glucose and glycated albumin upregulated MSTN mRNA and its protein (p < 0.05–0.01). MSTN-treated HK-2 cells underwent decreased proliferation, together with NF-kB activation and CCL-2 and SMAD 2,3 overexpression. In addition, MSTN induced intracellular ROS release and upregulated NADPH oxidase, effects which were mediated by ERK activation. In conclusion, our data show that MSTN is expressed in the human kidney and overexpressed in DN, mainly in the tubulointerstitial compartment. Our results also show that MSTN is a strong inducer of proximal tubule activation and suggest that MSTN overexpression contributes to kidney interstitial fibrosis in DN.

MOTS‐c inhibits high‐fat diet‐induced muscle wasting by suppressing myostatin expression via the PTEN/AKT/FOXO1 signaling pathway

Insulin resistance is thought to contribute to the development of muscle wasting. MOTS‐c, a novel mitochondrial‐derived peptide, improves insulin resistance in skeletal muscle, but its effects on muscle wasting have yet to be explored. Here, we used a diet‐induced obese mouse model to examine the effect of MOTS‐c administration on high fat diet‐induced muscle wasting. Three weeks of MOTS‐c administration by intraperitoneal (IP) injection in high‐fat diet fed mice resulted in higher total skeletal muscle mass of soleus, gastrocnemius, and plantaris compared to its high‐fat diet fed control group. Myostatin mRNA expression levels were significantly decreased in the MOTS‐c treated group compared to the high‐fat diet fed controls in the gastrocnemius muscle. Furthermore, myostatin mRNA expression negatively correlated with the total skeletal muscle mass of soleus, gastrocnemius, and plantaris. To examine the detailed mechanism of MOTS‐c action, we injected high‐fat diet fed mice with MOTS‐c for eight weeks and detected significantly lower plasma myostatin levels than high‐fat diet fed controls, as well as mRNA expression of myostatin and atrogin‐1 in the skeletal muscle. We further examined the upstream signaling pathways of myostatin regulation in the skeletal muscle and showed that MOTS‐c treatment inhibited PTEN activity, leading to elevated AKT phosphorylation and inhibition of FOXO1 transcription activity, resulting in myostatin and atrogin‐1 suppression. These results suggest that potent MOTS‐c analogues could potentially treat conditions such as high‐fat diet‐induced skeletal muscle wasting, as well as other muscle wasting phenotypes including sarcopenia, and that circulating levels of myostatin may serve as a pharmacodynamic biomarker of MOTS‐c activity.

Myostatin in the Arterial Wall of Patients with End-Stage Renal Disease.

Aim: Myostatin (Mstn) has been described as a trigger for the progression of atherosclerosis. In this study, we evaluated the role of Mstn in arterial remodeling in patients with end-stage renal disease (ESRD).Methods: Vascular specimens were collected from 16 ESRD patients (56.4 ± 7.9 years) undergoing renal transplant (recipients) and 15 deceased kidney non-uremic donors (55.4 ± 12.1 years). We studied gene and protein expression of Mstn, ubiquitin ligases, Atrogin-1, and muscle ring finger protein-1 (MuRF-1), inflammatory marker CCL2, cytoskeleton components, and Klotho by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Moreover, we assessed vascular calcification and collagen deposition. Finally, we studied the effects of recombinant Mstn on rat vascular smooth muscle cells (VSMCs, A7r5) and evaluated the effects of uremic serum (US) on primary human VSMCs.Results: Myostatin mRNA was upregulated in the arterial vascular wall of recipients compared with donors (∼15-folds, p < 0.05). This response was accompanied by the upregulation of gene expression of Atrogin-1 and MuRF-1 (+2.5- and +10-fold) and CCL2 (+3-fold). Conversely, we found downregulation of protein expression of Smoothelin, α-smooth muscle actin (α-SMA), vimentin, and Klotho (−85%, −50%, −70%, and −80%, respectively; p < 0.05) and gene expression of vimentin and Klotho. Exposition of A7r5 to Mstn induced a time-dependent SMAD 2/SMAD 3 phosphorylation and expression of collagen-1 and transforming growth factor β (TGFβ) mRNA, while US induced overexpression of Mstn and Atrogin-1 and downregulation of Smoothelin and Klotho.Conclusions: Our data suggest that uremia might induce vascular Mstn gene expression together with a complex pathway of molecular and structural changes in the vascular wall. Myostatin, in turn, can translate the metabolic alterations of uremia into profibrotic and stiffness inducing signals.

Molecular characterization of the myostatin gene and its regulation on muscle growth in Yesso scallop Patinopecten yessoensis

Myostatin (MSTN) belongs to the transforming growth factor β (TGF-β) family, which plays a critical role in negatively regulating muscle growth in vertebrates. However, little is known about its function in muscle growth of scallops that possess a large adductor muscle composed of striated and smooth muscle fibers. In this study, we first indentify and characterize the complete cDNA sequence of the myostatin gene from Yesso scallop P. yessoensis, an economically important species in Asian countries. The C-terminal mature TGF-β peptides are highly conserved in scallops, with sequence identity of >97% among these species. Gene expression at developmental stages revealed by whole-mount in situ hybridization indicates that myostatin is mainly expressed on the profusely ciliated rim of velum in veligers. The significant increase of muscle cellularity induced by RNA interference of myostatin can be explained as a result of a combination of hyperplasia and hypertrophy of MHC II striated myofibers, rather than myofiber growth of the paramyosin-rich smooth muscle. This is also evidenced by the quantification results of muscle gene expression, which indicate the significant increase of MHC expression after the inhibition of myostatin mRNA in the striated muscle, but not in the smooth muscle. For both of the striated and smooth muscles, however, no significant difference between the treatment and control groups is detected by real-time quantitative PCR analysis in other muscle genes. These findings suggest that the cellular targets of myostatin in scallop muscles are probably regulated by signaling directly to striated MHC. The new additional nuclei are potentially supplied by a population of satellite cells or muscle stem cell-like adult muscle precursors that may fuse with existing muscle fibers during muscle hypertrophy. These evidences found in this study highlight that myostatin may not only be involved in the development of larval musculature, but also play an important role in regulating scallop muscle growth.

Ammonia Induces a Myostatin-Mediated Atrophy in Mammalian Myotubes, but Induces Hypertrophy in Avian Myotubes

Ammonia, a byproduct of protein catabolism that is generally regarded as toxic, is processed by the liver for excretion. In diseases resulting in hepatic insufficiency, circulating ammonia levels increase dramatically, ensuing secondary disorders. Sarcopenia, or loss of muscle mass, is commonly associated with hyperammonemia. In mammalian models of cirrhosis, increased myostatin is consistent, contributes to muscle autophagy, and reduces satellite cell activation and differentiation, whereas, avian species show a positive myogenic response to ammonia. The objective of the study was to elucidate the effect of ammonia in chicken, mouse, and rat derived myotubes. Primary myoblasts were isolated from the pectoralis major (breast) and biceps femoris (thigh) of embryonic day 17 chicken embryos, and from the hindlimbs of 3-day-old rat pups. C2C12 cells were used for mouse myoblasts. Myotubes were exposed to 10mM ammonium acetate (AA) or 10mM sodium acetate (SA) for 24 hours to determine myogenic response to ammonia. Relative expression of myostatin mRNA, determined by quantitative real-time PCR, was significantly higher in mammalian myotubes compared to chicken myotubes (P < 0.001). Western blot analysis of myostatin protein confirmed a significant increase in ammonia treated rat myotubes, while chicken breast myotubes showed a significant decrease in myostatin (P < 0.05). Myotube diameter significantly increased in chicken breast and thigh cultures treated with ammonia, while diameter was significantly reduced in mouse and rat myotubes (P < 0.05). Intracellular glutamine is significantly higher in chicken thigh, but not breast, myotubes treated with AA compared to SA treated myotubes (P < 0.05). To investigate fiber type differences in ammonia metabolism, Western blot analysis of protein from AA and SA treated myotubes was examined for fast and slow myosin heavy chain isoforms. AA treatment resulted in a higher ratio of fast to slow isoforms of myosin heavy chain in both types of chicken myotubes, while fast isoforms were decreased in AA treated mouse and rat myotubes. These data demonstrate that chicken myotubes respond positively to ammonia while rodent myotubes respond negatively. Further, there is evidence that ammonia induces a fast fiber type shift in avian muscle, but a slow phenotype shift in mammalian muscle.

Effects of equine myostatin (MSTN) genotype variation on transcriptional responses in Thoroughbred skeletal muscle

Myostatin gene (MSTN) variation influences distance aptitude in Thoroughbreds as a consequence of functional physiological effects including skeletal muscle fibre type and muscle hypertrophy variation. A promotor region short interspersed nuclear element (SINE) insertion, tagged by SNP g.66493737-C, alters MSTN mRNA expression. We tested the hypothesis that skeletal muscle gene expression varies among MSTN genotypes due to differential up- or down-stream gene signalling pathways that may be influenced by exercise and training and consequently contribute to variation in exercise phenotypes. Skeletal muscle biopsies were collected from Thoroughbreds previously genotyped for MSTN (n=35 CC, n=50 CT, n=9 TT) at three different time-points: untrained at rest (UR), untrained after exercise (UE) and trained at rest (TR). Gene differential expression (DE) was determined from RNAseq data using DESeq2 (Benjamini-Hochberg P-value &lt;0.05). Functional over-representation analysis was performed in DAVID. In UR samples, one, nine and 47 genes were DE between CC vs CT, CT vs TT and C:C vs TT, respectively. The OSGEPL1 gene, located &lt;250 Kb proximal to MSTN, was DE among all cohorts. Six genes were DE in UE between CC vs TT including OSGEPL1, FGF10 and COQ8A. There was significant enrichment for GO categories related to mitochondria in TR. Comparison of the exercise response (UR vs UE) revealed patterns of expression that were opposing; i.e. CHRNG was 0.857 log2FC in the TT cohort but 2.055 log2FC in the CC cohort. Genes located in proximity to MSTN and involved in mitochondrial function were most significantly different among genotype cohorts. Patterns of DE among genotypes suggests gene-regulated influence on the phenotype. Understanding these patterns may assist genotype-guided training strategies.

Expression of MSTN gene and its correlation with pectoralis muscle fiber traits in the domestic pigeons (Columba livia)

Myostatin (MSTN) is a negative regulator of skeletal muscle growth and plays an important role in muscle development. In this research, we constructed a tissue expression profile of the pigeon MSTN gene in eight tissues and a spatio-temporal expression profile in the pectoral muscle using qRT-PCR method. And the pectoralis muscle fiber traits during pigeon post-hatching stages at 1, 7, 14, 21, and 28 D were analyzed through the paraffin sections. Then the correlations between the muscle fiber diameter, cross-sectional area, density, and the expression of MSTN in the pectoral muscle were analyzed. Results showed that MSTN mRNA was mainly expressed in breast muscle, heart, spleen, and kidney and it was almost unexpressed in the liver and lungs. Moreover, the MSTN mRNA expression level in breast muscle was significantly higher than that in other tissues (P < 0.05), and showed an interesting trend that it decreased in the first week and then increased with age. Meanwhile, decrease of myostatin transcripts was accompanied by the down-regulation of Myf5 and the up-regulation of MyoG during the first week post-hatching. In addition, the paraffin sections analysis results revealed that the diameter and cross-sectional area of pectoralis muscle fiber significantly increased with age (P < 0.05), and a significant positive correlation was shown between the MSTN gene expression level and muscle fiber diameter (P < 0.05). These fundamental researches might contribute to further understanding of the roles MSTN played in the post-hatching muscle fiber development in pigeon.