Myostatin Protein Expression Research Articles

Context-dependent regulation of pectoralis myostatin and lipid transporters by temperature and photoperiod in dark-eyed juncos

A prominent example of seasonal phenotypic flexibility is the winter increase in thermogenic capacity (=summit metabolism, ) in small birds, which is often accompanied by increases in pectoralis muscle mass and lipid catabolic capacity. Temperature or photoperiod may be drivers of the winter phenotype, but their relative impacts on muscle remodeling or lipid transport pathways are little known. We examined photoperiod and temperature effects on pectoralis muscle expression of myostatin, a muscle growth inhibitor, and its tolloid-like protein activators (TLL-1 and TLL-2), and sarcolemmal and intracellular lipid transporters in dark-eyed juncos Junco hyemalis. We acclimated winter juncos to four temperature (3 °C or 24 °C) and photoperiod [short-day (SD) = 8L:16D; long-day (LD) = 16L:8D] treatments. We found that myostatin, TLL-1, TLL-2, and lipid transporter mRNA expression and myostatin protein expression did not differ among treatments, but treatments interacted to influence lipid transporter protein expression. Fatty acid translocase (FAT/CD36) levels were higher for cold SD than for other treatments. Membrane-bound fatty acid binding protein (FABPpm) levels, however, were higher for the cold LD treatment than for cold SD and warm LD treatments. Cytosolic fatty acid binding protein (FABPc) levels were higher on LD than on SD at 3 °C, but higher on SD than on LD at 24 °C. Cold temperature groups showed upregulation of these lipid transporters, which could contribute to elevated Msum compared to warm groups on the same photoperiod. However, interactions of temperature or photoperiod effects on muscle remodeling and lipid transport pathways suggest that these effects are context-dependent.

Within-Winter Flexibility in Muscle Masses, Myostatin, and Cellular Aerobic Metabolic Intensity in Passerine Birds.

Metabolic rates of passerine birds are flexible traits that vary both seasonally and among and within winters. Seasonal variation in summit metabolic rates (Msum = maximum thermoregulatory metabolism) in birds is consistently correlated with changes in pectoralis muscle and heart masses and sometimes with variation in cellular aerobic metabolic intensity, so these traits might also be associated with shorter-term, within-winter variation in metabolic rates. To determine whether these mechanisms are associated with within-winter variation in Msum, we examined the effects of short-term (ST; 0-7 d), medium-term (MT; 14-30 d), and long-term (LT; 30-yr means) temperature variables on pectoralis muscle and heart masses, pectoralis expression of the muscle-growth inhibitor myostatin and its metalloproteinase activators TLL-1 and TLL-2, and pectoralis and heart citrate synthase (CS; an indicator of cellular aerobic metabolic intensity) activities for two temperate-zone resident passerines, house sparrows (Passer domesticus) and dark-eyed juncos (Junco hyemalis). For both species, pectoralis mass residuals were positively correlated with ST temperature variables, suggesting that cold temperatures resulted in increased turnover of pectoralis muscle, but heart mass showed little within-winter variation for either species. Pectoralis mRNA and protein expression of myostatin and the TLLs were only weakly correlated with ST and MT temperature variables, which is largely consistent with trends in muscle masses for both species. Pectoralis and heart CS activities showed weak and variable trends with ST temperature variables in both species, suggesting only minor effects of temperature variation on cellular aerobic metabolic intensity. Thus, neither muscle or heart masses, regulation by the myostatin system, nor cellular aerobic metabolic intensity varied consistently with winter temperature, suggesting that other factors regulate within-winter metabolic variation in these birds.

Testosterone and trenbolone enanthate increase mature myostatin protein expression despite increasing skeletal muscle hypertrophy and satellite cell number in rodent muscle.

The androgen-induced alterations in adult rodent skeletal muscle fibre cross-sectional area (fCSA), satellite cell content and myostatin (Mstn) were examined in 10-month-old Fisher 344 rats (n=41) assigned to Sham surgery, orchiectomy (ORX), ORX+testosterone (TEST; 7.0mgweek-1 ) or ORX+trenbolone (TREN; 1.0mgweek-1 ). After 29days, animals were euthanised and the levator ani/bulbocavernosus (LABC) muscle complex was harvested for analyses. LABC muscle fCSA was 102% and 94% higher in ORX+TEST and ORX+TREN compared to ORX (p<.001). ORX+TEST and ORX+TREN increased satellite cell numbers by 181% and 178% compared to ORX, respectively (p<.01), with no differences between conditions for myonuclear number per muscle fibre (p=.948). Mstn protein was increased 159% and 169% in the ORX+TEST and ORX+TREN compared to ORX (p<.01). pan-SMAD2/3 protein was ~30-50% greater in ORX compared to SHAM (p=.006), ORX+TEST (p=.037) and ORX+TREN (p=.043), although there were no between-treatment effects regarding phosphorylated SMAD2/3. Mstn, ActrIIb and Mighty mRNAs were lower in ORX, ORX+TEST and ORX+TREN compared to SHAM (p<.05). Testosterone and trenbolone administration increased muscle fCSA and satellite cell number without increasing myonuclei number, and increased Mstn protein levels. Several genes and signalling proteins related to myostatin signalling were differentially regulated by ORX or androgen therapy.

Myocardial myostatin in spontaneously hypertensive rats with heart failure

BackgroundMyostatin has been shown to regulate skeletal and cardiac muscle growth. However, its status on long-term hypertrophied myocardium has not been addressed. The purpose of this study was to evaluate the expression of myocardial myostatin and its antagonist follistatin in spontaneously hypertensive rats (SHR) with heart failure. MethodsEighteen-month-old SHR were evaluated to identify clinical features of heart failure such as tachypnea/labored respiration and weight loss. After heart failure was detected, rats were subjected to echocardiogram and euthanized. Age-matched normotensive Wistar-Kyoto (WKY) rats were used as controls. Myostatin and follistatin protein expression was assessed by Western blotting. Statistical analysis was performed by Student's t test. ResultsAll SHR (n=8) presented right ventricular hypertrophy and five had lung congestion. SHR had left chambers hypertrophy and dilation (left atrial diameter: WKY 5.73±0.59; SHR 7.28±1.17mm; p=0.004; left ventricular (LV) diastolic diameter/body weight ratio: WKY 19.6±3.1; SHR 27.7±4.7mm/kg; p=0.001), and LV systolic dysfunction (midwall fractional shortening: WKY 34.9±3.31; SHR 24.8±3.20%; p=0.003). Myocyte diameter (WKY 23.1±1.50, SHR 25.5±1.33μm; p=0.004) and myocardial interstitial collagen fraction (WKY 4.86±0.01; SHR 8.36±0.02%; p<0.001) were increased in the SHR. Myostatin (WKY 1.00±0.16; SHR 0.77±0.23 arbitrary units; p=0.035) and follistatin (WKY 1.00±0.35; SHR 0.49±0.18 arbitrary units; p=0.002) expression was lower in SHR. Myostatin and follistatin expression negatively correlated with LV diastolic diameter-to-body weight ratio and LV systolic diameter, and positively correlated with midwall fractional shortening. ConclusionMyostatin and follistatin protein expression is reduced in the long-term hypertrophied myocardium from spontaneously hypertensive rats with heart failure.

Combination Antisense Treatment for Destructive Exon Skipping of Myostatin and Open Reading Frame Rescue of Dystrophin in Neonatal mdx Mice

The fatal X-linked Duchenne muscular dystrophy (DMD), characterized by progressive muscle wasting and muscle weakness, is caused by mutations within the DMD gene. The use of antisense oligonucleotides (AOs) modulating pre-mRNA splicing to restore the disrupted dystrophin reading frame, subsequently generating a shortened but functional protein has emerged as a potential strategy in DMD treatment. AO therapy has recently been applied to induce out-of-frame exon skipping of myostatin pre-mRNA, knocking-down expression of myostatin protein, and such an approach is suggested to enhance muscle hypertrophy/hyperplasia and to reduce muscle necrosis. Within this study, we investigated dual exon skipping of dystrophin and myostatin pre-mRNAs using phosphorodiamidate morpholino oligomers conjugated with an arginine-rich peptide (B-PMOs). Intraperitoneal administration of B-PMOs was performed in neonatal mdx males on the day of birth, and at weeks 3 and 6. At week 9, we observed in treated mice (as compared to age-matched, saline-injected controls) normalization of muscle mass, a recovery in dystrophin expression, and a decrease in muscle necrosis, particularly in the diaphragm. Our data provide a proof of concept for antisense therapy combining dystrophin restoration and myostatin inhibition for the treatment of DMD.

Protective Effects of Clenbuterol against Dexamethasone-Induced Masseter Muscle Atrophy and Myosin Heavy Chain Transition

BackgroundGlucocorticoid has a direct catabolic effect on skeletal muscle, leading to muscle atrophy, but no effective pharmacotherapy is available. We reported that clenbuterol (CB) induced masseter muscle hypertrophy and slow-to-fast myosin heavy chain (MHC) isoform transition through direct muscle β2-adrenergic receptor stimulation. Thus, we hypothesized that CB would antagonize glucocorticoid (dexamethasone; DEX)-induced muscle atrophy and fast-to-slow MHC isoform transition.MethodologyWe examined the effect of CB on DEX-induced masseter muscle atrophy by measuring masseter muscle weight, fiber diameter, cross-sectional area, and myosin heavy chain (MHC) composition. To elucidate the mechanisms involved, we used immunoblotting to study the effects of CB on muscle hypertrophic signaling (insulin growth factor 1 (IGF1) expression, Akt/mammalian target of rapamycin (mTOR) pathway, and calcineurin pathway) and atrophic signaling (Akt/Forkhead box-O (FOXO) pathway and myostatin expression) in masseter muscle of rats treated with DEX and/or CB.Results and ConclusionMasseter muscle weight in the DEX-treated group was significantly lower than that in the Control group, as expected, but co-treatment with CB suppressed the DEX-induced masseter muscle atrophy, concomitantly with inhibition of fast-to-slow MHC isoforms transition. Activation of the Akt/mTOR pathway in masseter muscle of the DEX-treated group was significantly inhibited compared to that of the Control group, and CB suppressed this inhibition. DEX also suppressed expression of IGF1 (positive regulator of muscle growth), and CB attenuated this inhibition. Myostatin protein expression was unchanged. CB had no effect on activation of the Akt/FOXO pathway. These results indicate that CB antagonizes DEX-induced muscle atrophy and fast-to-slow MHC isoform transition via modulation of Akt/mTOR activity and IGF1 expression. CB might be a useful pharmacological agent for treatment of glucocorticoid-induced muscle atrophy.

MicroRNA-128 targets myostatin at coding domain sequence to regulate myoblasts in skeletal muscle development

MicroRNAs (miRNAs or miRs) play a critical role in skeletal muscle development. In a previous study we observed that miR-128 was highly expressed in skeletal muscle. However, its function in regulating skeletal muscle development is not clear. Our hypothesis was that miR-128 is involved in the regulation of the proliferation and differentiation of skeletal myoblasts. In this study, through bioinformatics analyses, we demonstrate that miR-128 specifically targeted mRNA of myostatin (MSTN), a critical inhibitor of skeletal myogenesis, at coding domain sequence (CDS) region, resulting in down-regulating of myostatin post-transcription. Overexpression of miR-128 inhibited proliferation of mouse C2C12 myoblast cells but promoted myotube formation; whereas knockdown of miR-128 had completely opposite effects. In addition, ectopic miR-128 regulated the expression of myogenic factor 5 (Myf5), myogenin (MyoG), paired box (Pax) 3 and 7. Furthermore, an inverse relationship was found between the expression of miR-128 and MSTN protein expression in vivo and in vitro. Taken together, these results reveal that there is a novel pathway in skeletal muscle development in which miR-128 regulates myostatin at CDS region to inhibit proliferation but promote differentiation of myoblast cells.

Exercise alters myostatin protein expression in sedentary and exercised streptozotocin-diabetic rats.

The aim of this study was to analyze the effect of exercise on the pattern of muscle myostatin (MSTN) protein expression in two important metabolic disorders, i.e., obesity and diabetes mellitus. MSTN, is a negative regulator of skeletal muscle mass. We evaluated the effect of exercise on MSTN protein expression in diabetes mellitus and high fat diet-induced obesity. MSTN protein expression in gastrocnemius muscle was analyzed by Western Blot. P < 0.05 was assumed. Exercise induced a significant decrease in glycemia in both diabetic and obese animals. The expression of precursor and processed protein forms of MSTN and the weight of gastrocnemius muscle did not vary in sedentary or exercised obese animals. Diabetes reduced gastrocnemius muscle weight in sedentary animals. However, gastrocnemius muscle weight increased in diabetic exercised animals. Both the precursor and processed forms of muscle MSTN protein were significantly higher in sedentary diabetic rats than in control rats. The precursor form was significantly lower in diabetic exercised animals than in diabetic sedentary animals. However, the processed form did not change. These results demonstrate that exercise can modulate the muscle expression of MSTN protein in diabetic rats and suggest that MSTN may be involved in energy homeostasis.

The Green Tea Polyphenol Epigallocatechin-3-Gallate (EGCg) Attenuates Skeletal Muscle Atrophy in a Rat Model of Sarcopenia.

Sarcopenia-the loss of muscle mass and functionality occurring with age-is a pervasive problem with few effective treatments beyond exercise. We examined the ability of the green tea catechin, epigallocatechin-3-gallate (EGCg), to impact muscle mass and the molecular pathway involved in muscle atrophy in a rat model of sarcopenia. 20-month-old Sprague-Dawley rats were treated for 8 weeks with control diet or control plus 200mg/kg body weight of EGCg diet. EGCg-supplemented animals had significantly greater gastrocnemius muscle mass than the aged controls, and showed a trend for increased muscle fiber cross-sectional areas (CSA) (p=0.06). These changes were associated with significantly lower protein expressions of the intramuscular 19S and 20S proteasome subunits and the MuRF1 and MAFbx ubiquitin ligases in the EGCg-treated animals. Proteasome activity as determined by 'Chymotrypsin-like' enzyme activity was also significantly reduced by EGCg. Muscle mRNA expression of IL-15 and IGF-1 were significantly increased in the EGCg group vs. the aged controls. In comparison to younger adult animals (6 month), the protein expression of 19S, 20S, MuRF1, MAFbx, and myostatin were increased between approximately 4- and 12-fold in the aged controls, but only up to ~2-fold in the aged EGCg animals. EGCg supplementation was able to preserve muscle in sarcopenic rats, partly through attenuating protein degradation via the ubiquitin-proteasome pathway, together with increased expression of anabolic factors.

Myostatin in the placentae of pregnancies complicated with gestational diabetes mellitus

IntroductionGestational diabetes mellitus (GDM) is characterised by maternal glucose intolerance and insulin resistance during pregnancy. Myostatin, initially identified as a negative regulator of muscle development may also function in the regulation of placental development and glucose uptake. Myostatin expression in placentae of GDM complicated pregnancies is unknown. However, higher myostatin levels occur in placentae of pregnancies complicated with preeclampsia. We hypothesise that myostatin will be differentially expressed in GDM complicated pregnancies. MethodsMyostatin concentrations (ELISA) were evaluated in plasma of presymptomatic women who later developed GDM and compared to plasma of normal glucose tolerant (NGT) women. Furthermore, myostatin protein expression (Western blot) was studied in placentae of pregnant women with GDM (treated with diet or insulin) compared to placentae of NGT women. ResultsNo significant difference in myostatin concentration was seen in plasma of pre-symptomatic GDM women compared to NGT women. In placenta significant differences in myostatin protein expressions (higher precursor; p < 0.05and lower dimer: p < 0.005) were observed in GDM complicated compared to NGT pregnancies. Furthermore, placentae of GDM women treated with insulin compared to diet have higher dimer (p < 0.005) and lower precursor (p < 0.05). Compared to lean women, placentae of obese NGT women were lower in myostatin dimer expression (p < 0.05). DiscussionMyostatin expression in placental tissue is altered under stress conditions (e.g. obesity and abnormal glucose metabolism) found in pregnancies complicated with GDM. We hypothesise that myostatin is active in these placentae and could affect glucose homoeostasis and/or cytokine production thereby altering the function of the placenta.

Preliminary investigation into a potential role for myostatin and its receptor (ActRIIB) in lean and obese horses and ponies.

Obesity is a widespread problem across the leisure population of horses and ponies in industrialised nations. Skeletal muscle is a major contributor to whole body resting energy requirements and communicates with other tissues through the secretion of myokines into the circulation. Myostatin, a myokine and negative regulator of skeletal muscle mass, has been implicated in obesity development in other species. This study evaluated gene and protein expression of myostatin and its receptor, ActRIIB in adipose tissues and skeletal muscles and serum myostatin concentrations in six lean and six obese animals to explore putative associations between these factors and obesity in horses and ponies. Myostatin mRNA expression was increased while ActRIIB mRNA was decreased in skeletal muscles of obese animals but these differences were absent at the protein level. Myostatin mRNA was increased in crest fat of obese animals but neither myostatin nor ActRIIB proteins were detected in this tissue. Mean circulating myostatin concentrations were significantly higher in obese than in lean groups; 4.98 ng/ml (±2.71) and 9.00 ng/ml (±2.04) for the lean and obese groups, respectively. In addition, there was a significant positive association between these levels and myostatin gene expression in skeletal muscles (average R2 = 0.58; p<0.05). Together, these results provide further basis for the speculation that myostatin and its receptor may play a role in obesity in horses and ponies.

Poor maternal nutrition inhibits muscle development in ovine offspring.

BackgroundMaternal over and restricted nutrition has negative consequences on the muscle of offspring by reducing muscle fiber number and altering regulators of muscle growth. To determine if over and restricted maternal nutrition affected muscle growth and gene and protein expression in offspring, 36 pregnant ewes were fed 60%, 100% or 140% of National Research Council requirements from d 31 ± 1.3 of gestation until parturition. Lambs from control-fed (CON), restricted-fed (RES) or over-fed (OVER) ewes were necropsied within 1 d of birth (n = 18) or maintained on a control diet for 3 mo (n = 15). Semitendinosus muscle was collected for immunohistochemistry, and protein and gene expression analysis.ResultsCompared with CON, muscle fiber cross-sectional area (CSA) increased in RES (58%) and OVER (47%) lambs at 1 d of age (P < 0.01); however at 3 mo, CSA decreased 15% and 17% compared with CON, respectively (P < 0.01). Compared with CON, muscle lipid content was increased in OVER (212.4%) and RES (92.5%) at d 1 (P < 0.0001). Muscle lipid content was increased 36.1% in OVER and decreased 23.6% in RES compared with CON at 3 mo (P < 0.0001). At d 1, myostatin mRNA abundance in whole muscle tended to be greater in OVER (P = 0.07) than CON. Follistatin mRNA abundance increased in OVER (P = 0.04) and tended to increase in RES (P = 0.06) compared with CON at d 1. However, there was no difference in myostatin or follistatin protein expression (P > 0.3). Phosphorylated Akt (ser473) was increased in RES at 3 mo compared with CON (P = 0.006).ConclusionsIn conclusion, maternal over and restricted nutrient intake alters muscle lipid content and growth of offspring, possibly through altered gene and protein expression.

Growth hormone influences atrophy pathways in skeletal muscle of heart failure rats (1163.3)

Skeletal muscle atrophy is usually observed in heart failure. Growth hormone (GH) increases muscular mass in several clinical settings. Objective: To evaluate GH administration effects on myogenin, MyoD, MRF4, myostatin, follistatin, PI3K, Akt, atrogin‐1 and MuRF‐1 protein expression, in the gastrocnemius muscle of rats with aortic stenosis‐induced heart failure. Methods: Male Wistar rats were subjected to thoracotomy for ascending aortic stenosis induction. After developing tachypnea, rats were evaluated by echocardiogram and subdivided into three groups (n=8): Sham, aortic stenosis (AS) and AS treated with GH (AS‐GH, 2mg/kg/day for 14 days). At the end of treatment, animals were subjected to echocardiogram and euthanized. Protein expression was analized by Western‐Blot. Statistical analysis: Anova and Tukey. Results: Before treatment, cardiac variables did not differ between AS and AS‐GH groups. GH administration preserved body weight (BW, Sham 492±32; AS 420±32; AS‐GH 486±69 g; p&lt;0.05 AS vs Sham and AS‐GH). Left ventricular posterior wall thickness (Sham 3.22±0.20; AS 3.80±0.36; AS‐GH 4.37±0.67 mm) and right ventricle weight/BW ratio (Sham 0.55±0.03; AS 1.15±0.25; AS‐GH 1.18±0.15 mg/g) were higher in AS and AS‐GH than Sham. MyoD expression was higher in AS group than Sham and unchanged in AS‐GH and Sham (Sham 1.00±1.28; AS 1.71±1.37; AS‐GH 1.21±1.21 arbitrary units). MRF4 levels were higher in AS‐GH than Sham and similar in AS and Sham (Sham 1.00±0.73; AS 1.58±0.42; AS‐GH 3.14±1.61 arbitrary units). Atrogin‐1 was lower decreased in AS‐GH than Sham and AS (Sham 1.00±0.58; AS 1.14±0.87; AS‐GH 0.31±0.16 arbitrary units). Protein expression of myostatin, follistatin, myogenin, PI3K, Akt and MuRF‐1 did not differ between groups. Conclusion: Growth hormone administration modulates myogenic regulatory factors and atrogin‐1 expression in the gastrocnemius muscle of heart failure rats.Grant Funding Source: Supported by Fapesp

Acute Dietary Protein Intake Restriction Is Associated with Changes in Myostatin Expression after a Single Bout of Resistance Exercise in Healthy Young Men1,2

Skeletal muscle satellite cells (SCs) play an important role in the myogenic adaptive response to exercise. It remains to be established whether nutrition plays a role in SC activation in response to exercise. In the present study, we assessed whether dietary protein alters the SC response to a single bout of resistance exercise. Twenty healthy young (aged 21 ± 2 y) males were randomly assigned to consume a 4-d controlled diet that provided either 1.2 g protein ⋅ kg body weight−1 ⋅ d−1 [normal protein diet (NPD)] or 0.1 g protein ⋅ kg body weight−1 ⋅ d−1 [low protein diet (LPD)]. On the second day of the controlled diet, participants performed a single bout of resistance exercise. Muscle biopsies from the vastus lateralis were collected before and after 12, 24, 48, and 72 h of post-exercise recovery. SC content and activation status were determined using immunohistochemistry. Protein and mRNA expression were determined using Western blotting and reverse transcription polymerase chain reaction. The number of myostatin + SCs decreased significantly at 12, 24, and 48 h (range, −14 to −49%; P < 0.05) after exercise cessation, with no differences between groups. Although the number of myostatin + SCs returned to baseline in the type II fibers on the NPD after 72 h of recovery, the number remained low on the LPD. At the 48 and 72 h time points, myostatin protein expression was elevated (86 ± 26% and 88 ± 29%, respectively) on the NPD (P < 0.05), whereas it was reduced at 72 h (−36 ± 12% compared with baseline) in the LPD group (P < 0.05). This study demonstrates that dietary protein intake does not modulate the post-exercise increase in SC content but modifies myostatin expression in skeletal muscle tissue. This trial was registered at clinicaltrials.gov> as NCT01220037.

Androgenic and estrogenic regulation of Atrogin-1, MuRF1 and myostatin expression in different muscle types of male mice

The molecular factors targeted by androgens and estrogens on muscle mass are not fully understood. The current study aimed to explore gene and protein expression of Atrogin-1, MuRF1, and myostatin in an androgen deprivation-induced muscle atrophy model. We examined the effects of Orx either with or without testosterone (T) or estradiol (E2) administration on Atrogin-1 gene expression, and MuRF1 and myostatin gene and protein expression. Measurements were made in soleus (SOL), extensor digitorum longus (EDL) and levator ani/bulbocavernosus (LA/BC) of male C57BL/6 mice. Thirty days of Orx resulted in a reduction in weight gain and muscle mass. These effects were prevented by T. In LA/BC, Atrogin-1 and MuRF1 mRNA was increased throughout 30 days of Orx, which was fully reversed by T and partially by E2 administration. In EDL and SOL, a less pronounced upregulation of both genes was only detectable at the early stages of Orx. Myostatin mRNA levels were downregulated in LA/BC and upregulated in EDL following Orx. T, but not E2, reversed these effects. No changes in protein levels of MuRF1 and myostatin were found in EDL at any time point following Orx. The atrophy in SOL and EDL in response to androgen deprivation, and its restoration by T, is accompanied by only minimal changes in atrogenes and myostatin gene expression. The marked differences in muscle atrophy and atrogene and myostatin mRNA between LA/BC and the locomotor muscles suggest that the murine LA/BC is not an optimal model to study Orx-induced muscle atrophy.

Aerobic exercise affects myostatin expression in aged rat skeletal muscles: a possibility of antiaging effects of aerobic exercise related with pelvic floor muscle and urethral rhabdosphincter.

PurposeAging-induced loss of muscle mass and subsequent reduction of strength is a fundamental cause of frailty, functional decline, and disability. And this may lead to muscular dysfunction, voiding dysfunction, or urinary incontinence due to pelvic muscle weakness induced by aging. Physical exercise has been recommended for the prevention and the treatment of these age-related frail states. We investigated the effects of treadmill exercise on muscle strength, myostatin mRNA and protein expression, and gastrocnemius myocytes proliferation in aged rats to investigate the possible antiaging effects of aerobic exercise on skeletal muscles such as pelvic floor muscles and urethral rhabdosphincter muscle.MethodsIn this study, 5-month-old male Sprague-Dawley rats were used as the young-age group (n=20) and 24-month-old rats were used as the old-age group (n=20). Each group was randomly divided into two groups (n=10 in each group): the sedentary and the treadmill exercise group. The rats in the exercise groups were forced to run on a motorized treadmill for 30 minutes, once a day, for 6 weeks. For this study, a weight load test, hematoxylin and eosin staining, real-time and reverse transcription polymerase chain reaction for myostatin mRNA, myostatin western blot, and 5-bromo-2'-deoxyuridine immunohistochemistry were performed in the gastrocnemius muscle.ResultsThe age-induced reduction of muscle mass and strength was associated with a decrease in myocyte proliferation and an increase in myostatin mRNA and protein expression in the gastrocnemius. However, treadmill exercise improved muscle mass and strength through suppression of myostatin mRNA and protein expression, and myocyte proliferation increase in the gastrocnemius against the aging process.ConclusionsAerobic exercise is a useful strategy for enhancing muscle function against aging-induced loss of skeletal muscle mass and functions.

Attenuation of Conducted Vasodilation in Skeletal Muscle Arterioles during Hyperhomocysteinemia

Background: Vasomotor responses conducted from terminal arterioles to proximal vessels may contribute to match tissue demands and blood supply during skeletal muscle contraction. Conduction of vasodilatation (CVD) from distal resistance arterioles to the proximal arterioles and feeding arteries during metabolic demand is mediated by intercellular gap junctions in the vascular endothelium. The role of hyperhomocysteinemia (HHcy) in the musculoskeletal system during CVD is unclear. We hypothesize that during HHcy, there is impaired CVD due to decreased expression of endothelial-associated connexins and thus decreased tissue perfusion to the contracting skeletal muscles. Methods: CVD studies were performed in a gluteus maximus muscle preparation of wild-type (C57BL6/J) and CBS-/+ (HHcy) mice using intravital microscopy. Expression of connexins and myostatin protein (an antiskeletal muscle statin) was studied by Western blot and immunohistochemistry methods. Tissue perfusion to acetylcholine was assessed by the laser Doppler technique. Results: There was decreased CVD and tissue perfusion in response to acetylcholine in CBS-/+ mice compared to wild-type controls. There was decreased expression of connexins 37, 40 and 43 and increased expression of myostatin in CBS-/+ mice compared to wild-type controls. Conclusion: Our findings suggest that CVD in skeletal muscle is decreased during HHcy due to decreased expression of gap junction connexins.

The effect of myostatin silencing by lentiviral-mediated RNA interference on goat fetal fibroblasts

Myostatin is a transforming growth factor-β family member that acts as a negative regulator of skeletal muscle mass. To identify possible myostatin inhibitors that may promote muscle growth, we used RNA interference mediated by a lentiviral vector to knockdown myostatin in goat fetal fibroblast cells. We also investigated the expression changes in relevant myogenic regulatory factors (MRFs) and adipogenic regulatory factors in the absence of myostatin in goat fetal fibroblasts. Quantitative RT-PCR revealed that myostatin transcripts were significantly reduced by 75 % (P < 0.01). Western blot showed that myostatin protein expression was reduced by 95 % (P < 0.01). We also found that the mRNA expression of activin receptor IIB (ACVR2B) significantly increased by 350 % (P < 0.01), and p21 increased 172 % (P < 0.01). Furthermore, myostatin inhibition decreased Myf5 and increased MEF2C mRNA expression in goat fetal fibroblasts, suggesting that myostatin regulates MRFs differently in fibroblasts compared to muscle. In addition, the expression of adipocyte marker genes peroxisome proliferator-activated receptor (PPAR) γ and leptin, but not CCAAT/enhance-binding protein (C/EBP) α and C/EBPβ, were upregulated at the transcript level after myostatin silencing. These results suggest that we have generated a novel way to block myostatin in vitro, which could be used to improve livestock meat production and gene therapy of musculoskeletal diseases. This also suggests that myostatin plays a negative role in regulating the expression of adipogenesis related genes in goat fetal fibroblasts.

GROWTH HORMONE ADMINISTRATION ALTERS PROTEIN EXPRESSION IN SKELETAL MUSCLE OF RATS WITH AORTIC STENOSIS‐INDUCED HEART FAILURE

In this study we evaluated the effects of growth hormone (GH) administration on protein expression of myostatin, follistatin, and the myogenic regulatory factors in skeletal muscle of rats with ascending aortic stenosis‐induced heart failure.MethodsMale Wistar rats were subjected to thoracotomy to induce aortic stenosis (AS). After developing tachypnea/labored respiration, rats were subjected to echocardiogram and assigned to three groups (n=8): Sham, AS, and AS treated with GH (AS‐GH, 2 mg/kg/day) for 14 days. After treatment, rats were subjected to echocardiogram and euthanatized. Protein expression was evaluated by Western‐blot.Statistical analysisAnova and Tukey.ResultsBefore GH treatment, cardiac parameters did not differ between AS and AS‐GH groups. GH preserved body weight. Right ventricular weight/BW ratio and left ventricular posterior wall thickness were higher in both AS groups than Sham. MyoD was higher in AS than Sham and similar between AS‐GH and Sham (Sham 1.0±1.3; AS 1.7±1.4; AS‐GH 1.2±1.2 arbitrary units). MRF4 expression was higher in AS‐GH than Sham (Sham 1.0±0.7; AS 1.6±0.4; AS‐GH 3.1±1.6 arbitrary units). Myostatin, follistatin, and myogenin expression did not differ between groups.ConclusionGrowth hormone increases MRF4 and normalizes MyoD expression in skeletal muscle of heart failure rats. Myostatin/follistatin pathway is not changed in this experimental model.

Expression of Active and Latent Myostatin in Overload‐ Induced Hypertrophied Rat Skeletal Muscle

Myostatin (GDF‐8) is the chief chalone in skeletal muscle and negatively controls adult skeletal muscle growth. The role of myostatin during overload‐induced hypertrophy of adult muscle is unclear. We tested the hypothesis that overloaded adult rodent skeletal muscle would reduce expression of myostatin. Overload‐induced hypertrophy was induced by unilateral tenotomy of the gastrocnemius tendon in 11 male adult Sprague‐Dawley rats followed by a 2‐week period of compensatory overload of the plantaris and soleus muscles. Western blot analysis was performed to evaluate changes in active and latent complex myostatin protein expression. Significant hypertrophy was noted in the soleus (494 ± 29 vs 405 ± 15 mg, p&lt;0.05) and plantaris (289 ± 12 vs 179 ± 37 mg, p&lt;0.05) muscles following overload. Overloaded plantaris muscle decreased expression of the active myostatin protein by 28.0 ± 6.6 % (p&lt;0.01) while the myostatin precursor decreased slightly (p=0.04). Overloaded soleus muscle decreased expression of the active myostatin protein by 22.5 ± 5.4% (p&lt;0.01) while myostatin precursor was unchanged. Myostatin latent complex expression decreased in the overloaded soleus by 11.4 ± 4.2% (p = 0.03) but was unchanged in the plantaris. These data support the hypothesis that the myostatin‐signaling pathway in overloaded muscles is generally reduced and contributes to muscle hypertrophy.