Skeletal Muscle Gastrocnemius Research Articles

Endurance Exercise Training Alters Lipidomic Profiles of Plasma and Eight Tissues in Rats: a MoTrPAC study.

Endurance exercise training (ExT) induces metabolic, structural, and functional adaptations via lipidomic modifications, yet the systematic elucidation of lipidome alterations in response to ExT remains incomplete. As a part of the Molecular Transducers of Physical Activity Consortium (MoTrPAC), we leveraged non-targeted and targeted lipidomics for the systematic discovery of lipid alterations in the brown adipose tissue, heart, hippocampus, kidney, liver, lung, skeletal muscle gastrocnemius, subcutaneous white adipose tissue, and plasma in response to 1, 2, 4 or 8 weeks of ExT in 6-month-old male and female Fischer-344 rats. This study demonstrates that these tissues, each with distinct lipidomic features, underwent dynamic, sexually dimorphic lipid remodeling. Exercise trained animals showed reduced whole-body adiposity and improved cardiorespiratory fitness, along with enhanced utilization of lipid stores and dynamic triacylglycerol remodeling compared to sedentary controls in all tissues except hippocampus. They also showed modifications in phospholipids, lysophospholipids, oxylipins, and ceramides in several tissues. Coordinated changes across tissues reflect systemic tissue communication, with liver-plasma-heart connection potentially playing a key role in systemic lipid metabolism during ExT. These data will improve our understanding of lipid-associated biological processes underlying the health-promoting benefits of ExT.

Skeletal muscle proteome analysis underpins multifaceted mitochondrial dysfunction in Friedreich's ataxia.

Friedreich's ataxia (FRDA) is a severe multisystemic disorder caused by a deficiency of the mitochondrial protein frataxin. While some aspects of FRDA pathology are developmental, the causes underlying the steady progression are unclear. The inaccessibility of key affected tissues to sampling is a main hurdle. Skeletal muscle displays a disease phenotype and may be sampled in vivo to address open questions on FRDA pathophysiology. Thus, we performed a quantitative mass spectrometry-based proteomics analysis in gastrocnemius skeletal muscle biopsies from genetically confirmed FRDA patients (n = 5) and controls. Obtained data files were processed using Proteome Discoverer and searched by Sequest HT engine against a UniProt human reference proteome database. Comparing skeletal muscle proteomics profiles between FRDA and controls, we identified 228 significant differentially expressed (DE) proteins, of which 227 were downregulated in FRDA. Principal component analysis showed a clear separation between FRDA and control samples. Interactome analysis revealed clustering of DE proteins in oxidative phosphorylation, ribosomal elements, mitochondrial architecture control, and fission/fusion pathways. DE findings in the muscle-specific proteomics suggested a shift toward fast-twitching glycolytic fibers. Notably, most DE proteins (169/228, 74%) are target of the transcription factor nuclear factor-erythroid 2. Our data corroborate a mitochondrial biosignature of FRDA, which extends beyond a mere oxidative phosphorylation failure. Skeletal muscle proteomics highlighted a derangement of mitochondrial architecture and maintenance pathways and a likely adaptive metabolic shift of contractile proteins. The present findings are relevant for the design of future therapeutic strategies and highlight the value of skeletal muscle-omics as disease state readout in FRDA.

Transthyretin knockdown recapitulates the insulin-sensitizing effects of exercise and promotes skeletal muscle adaptation to exercise endurance.

Liver transthyretin (TTR) synthesis and release are exacerbated in insulin-resistant states but are decreased by exercise training, in relation to the insulin-sensitizing effects of exercise. We hypothesized that TTR knockdown (TTR-KD) may mimic this exercise-induced metabolic improvement and skeletal muscle remodeling. Adeno-associated virus-mediated TTR-KD and control mice were trained for 8 weeks on treadmills. Their metabolism status and exercise capacity were investigated and then compared with sedentary controls. After treadmill training, the mice showed improved glucose and insulin tolerance, hepatic steatosis, and exercise endurance. Sedentary TTR-KD mice displayed metabolic improvements comparable to the improvements in trained mice. Both exercise training and TTR-KD promoted the oxidative myofiber compositions of MyHC I and MyHC IIa in the quadriceps and gastrocnemius skeletal muscles. Furthermore, training and TTR-KD had an additive effect on running performance, accompanied by substantial increases in oxidative myofiber composition, Ca2+-dependent Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, and the downstream expression of PGC1α as well as the unfolded protein response (UPR) segment of PERK-p-eIF2a pathway activity. Consistent with these findings, electrical pulse stimulation of an in vitro model of chronic exercise (with differentiated C2C12 myoblasts) showed that exogenous TTR protein was internalized and localized in the endoplasmic reticulum, where it disrupted Ca2+ dynamics; this led to decreases in intracellular Ca2+ concentration and downstream pathway activity. TTR-KD may function as an exercise/Ca2+-dependent CaMKII-PGC1α-UPR regulator that upregulates the oxidative myofiber composition of fast-type muscles; it appears to mimic the effect of exercise training on insulin sensitivity-related metabolic improvement and endurance capacity.

Selenomethionine promotes ANXA2 phosphorylation for proliferation and protein synthesis of myoblasts and skeletal muscle growth

Selenomethionine (Se-Met) has many beneficial effects on higher animals and human, and can regulate cellular physiology through distinct signaling pathways. However, the role and molecular mechanism of Se-Met in skeletal muscle growth remains unclear. In this study, we observed the effects of Se-Met on C2C12 myoblasts and skeletal muscle growth of mice, and explored the corresponding molecular mechanism. Se-Met affected proliferation and protein synthesis of C2C12 myoblasts in a hormesis type of relationship, and had an optimal stimulatory effect at 50 µM concentration. Se-Met also affected mTOR, ANXA2, and PKCα phosphorylation in the same manner. ANXA2 knockdown blocked the stimulation of Se-Met on cell proliferation and protein synthesis and inhibition of Se-Met on autophagy of C2C12 myoblasts. Western blotting analysis showed that PI3K inhibition blocked the stimulation of Se-Met on mTOR phosphorylation. ANXA2 knockdown further blocked the stimulation of Se-Met on PI3K and mTOR phosphorylation. Point mutation experiment showed that ANXA2 mediated the stimulation of Se-Met on the PI3K-mTOR signaling through phosphorylation at Ser26. PKCα interacted with ANXA2, and PKCα knockdown blocked the stimulation of Se-Met on ANXA2 phosphorylation at Ser26. Se-Met addition (7.5mg/kg diet, 4 weeks) increased mouse carcass weight, promoted gastrocnemius skeletal muscle growth and ANXA2 and mTOR phosphorylation in this tissue. Collectively, our findings reveal that Se-Met can promote proliferation and protein synthesis of myoblasts and skeletal muscle growth through ANXA2 phosphorylation.

Opposite Regulation of Homer Signal at the NMJ Postsynaptic Micro Domain between Slow- and Fast-Twitch Muscles in an Experimentally Induced Autoimmune Myasthenia Gravis (EAMG) Mouse Model

Accelerated postsynaptic remodelling and disturbance of neuromuscular transmission are common features of autoimmune neurodegenerative diseases. Homer protein isoform expression, crosslinking activity and neuromuscular subcellular localisation are studied in mouse hind limb muscles of an experimentally induced autoimmune model of Myasthenia Gravis (EAMG) and correlated to motor end plate integrity. Soleus (SOL), extensor digitorum longus (EDL) and gastrocnemius (GAS) skeletal muscles are investigated. nAChR membrane clusters were studied to monitor neuromuscular junction (NMJ) integrity. Fibre-type cross-sectional area (CSA) analysis is carried out in order to determine the extent of muscle atrophy. Our findings clearly showed that crosslinking activity of Homer long forms (Homer 1b/c and Homer2a/b) are decreased in slow-twitch and increased in fast-twitch muscle of EAMG whereas the short form of Homer that disrupts Homer crosslinking (Homer1a) is upregulated in slow-twitch muscle only. Densitometry analysis showed a 125% increase in Homer protein expression in EDL, and a 45% decrease in SOL of EAMG mice. In contrast, nAChR fluorescence pixel intensity decreased in endplates of EAMG mice, more distinct in type-I dominant SOL muscle. Morphometric CSA of EAMG vs. control (CTR) revealed a significant reduction in EDL but not in GAS and SOL. Taken together, these results indicate that postsynaptic Homer signalling is impaired in slow-twitch SOL muscle from EAMG mice and provide compelling evidence suggesting a functional coupling between Homer and nAChR, underscoring the key role of Homer in skeletal muscle neurophysiology.

Reduced Tyrosine and Serine-632 Phosphorylation of Insulin Receptor Substrate-1 in the Gastrocnemius Muscle of Obese Zucker Rat.

Obesity has become a serious health problem in the world, with increased morbidity, mortality, and financial burden on patients and health-care providers. The skeletal muscle is the most extensive tissue, severely affected by a sedentary lifestyle, which leads to obesity and type 2 diabetes. Obesity disrupts insulin signaling in the skeletal muscle, resulting in decreased glucose disposal, a condition known as insulin resistance. Although there is a large body of evidence on obesity-induced insulin resistance in various skeletal muscles, the molecular mechanism of insulin resistance due to a disruption in insulin receptor signaling, specifically in the gastrocnemius skeletal muscle of obese Zucker rats (OZRs), is not fully understood. This study subjected OZRs to a glucose tolerance test (GTT) to analyze insulin sensitivity. In addition, immunoprecipitation and immunoblotting techniques were used to determine the expression and tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and insulin receptor-β (IRβ), and the activation of serine-632-IRS-1 phosphorylation in the gastrocnemius muscle of Zucker rats. The results show that the GTT in the OZRs was impaired. There was a significant decrease in IRS-1 levels, but no change was observed in IRβ in the gastrocnemius muscle of OZRs, compared to Zucker leans. Obese rats had a higher ratio of tyrosine phosphorylation of IRS-1 and IRβ than lean rats. In obese rats, however, insulin was unable to induce tyrosine phosphorylation. Moreover, insulin increased the phosphorylation of serine 632-IRS-1 in the gastrocnemius muscle of lean rats. However, obese rats had a low basal level of serine-632-IRS-1 and insulin only mildly increased serine phosphorylation in obese rats, compared to those without insulin. Thus, we addressed the altered steps of the insulin receptor signal transduction in the gastrocnemius muscle of OZRs. These findings may contribute to a better understanding of human obesity and type 2 diabetes.

Gene expression of sex steroid metabolizing enzymes and receptors in the skeletal muscle of migrant and resident subspecies of white-crowned sparrow (Zonotrichia leucophrys).

Circulating sex steroid concentrations vary dramatically across the year in seasonally breeding animals. The ability of circulating sex steroids to effect muscle function can be modulated by changes in intracellular expression of steroid metabolizing enzymes (e.g., 5α-reductase type 2 and aromatase) and receptors. Together, these combined changes in plasma hormones, metabolizing enzymes and receptors allow for seasonally appropriate changes in skeletal muscle function. We tested the hypothesis that gene expression of sex steroid metabolizing enzymes and receptors would vary seasonally in skeletal muscle and these changes would differ between a migrant and resident life history strategy. We quantified annual changes in plasma testosterone and gene expression in pectoralis and gastrocnemius skeletal muscles using quantitative polymerase chain reaction (qPCR) in free-living migrant (Zonotrichia leucophrys gambelii) and resident (Z. l. nuttalli) subspecies of white-crowned sparrow during breeding, pre-basic molt, and wintering life history stages. Pectoralis muscle profile was largest in migrants during breeding, while residents maintained large muscle profiles year-round. Circulating testosterone peaked during breeding in both subspecies. Pectoralis muscle androgen receptor mRNA expression was lower in females of both subspecies during breeding. Estrogen receptor-α expression was higher in the pectoralis muscle, but not gastrocnemius, of residents throughout the annual cycle when compared to migrants. Pectoralis aromatase expression was higher in resident males compared to migrant males. No differences were observed for 5α-reductase 2. Between these two subspecies, patterns of plasma testosterone and androgen receptors appear to be conserved, however estrogen receptor gene expression appears to have diverged.

Diet-Induced Gene Expression Changes of Cachectic Muscle, Adipose, and Liver

ObjectivesCancer cachexia is a systemic disease characterized by muscle and adipose loss that cannot be reversed by increasing caloric intake. Our previous research has shown insulin resistance precedes cancer cachexia in the C26 mouse model of cachexia, and a diet high in linoleic acid, the essential omega-6 polyunsaturated fatty acid, attenuates the C26-induced insulin resistance. Therefore, to better understand how dietary linoleic acid is improving insulin sensitivity, we characterized gene expression changes in three major tissues responsible for controlling insulin sensitivity: skeletal muscle, adipose, and liver. MethodsMale CD2F1 (Charles River, MA) were randomized to semi-purified diet (24% fat by weight) containing fat prominently from lard, or containing fat prominently from safflower oil (a linoleic acid-rich oil). One week after diet randomization, mice were inoculated with colon-26 (C26) adenocarcinoma cells (1.0E6 cells). 13 days after inoculation mice were euthanized and gastrocnemius skeletal muscle, epididymal white adipose tissue, and liver tissue were collected for total transcriptome analysis using poly-A enriched next generation RNA-sequencing. Differentially expressed genes were selected based on p-values < 0.05. ResultsThere were no detectable differences in body weight or food intake between the two diets in mice with C26 tumors. Between the two diets 12 genes were differentially expressed in the muscle, while 57 genes were differentially expressed in the liver, and 314 genes were differentially expressed in adipose. ConclusionsA linoleic acid enriched diet had little effect on the skeletal muscle transcriptome but induced larger transcriptome changes in liver and adipose. This could suggest dietary linoleic acid increases insulin sensitivity through affecting metabolism in adipose and liver, rather than skeletal muscle. Determining these diet-induced transcriptome changes allows us to better target tissue-specific molecular mechanisms of linoleic acid in future research. Funding SourcesThis work was supported by the Pelotonia Fellowship Program, the Ohio State University Comprehensive Cancer Center, and the National Institutes of Health.

Deletion of Myostatin Resolves Myosteatosis and Improves Angiogenesis in Obese Mice

Andrew C. Speese, B.S.1, Caleb A. Padgett M.S.1, Cody L. Rosewater M.S.1, Zachary L. Corley B.S.1, James D. Mintz M.B.A.1, David J. Fulton Ph.D.1,2, and David W. Stepp Ph.D.1,31Vascular Biology Center, 2Department of Pharmacology and Toxicology, and 3Department of Physiology, Medical College of Georgia, Augusta University, Augusta, GAObjectiveTo test the hypothesis that increased muscle mass achieved through deletion of myostatin improves ischemic angiogenesis in obese mice.Methodsdb/db mice, a well‐described model of obesity, were crossed with mice lacking myostatin (MSTN KO), a myokine that negatively regulates muscle differentiation and growth, to generate lean and obese mice with and without elevated muscle mass (MSTN KO). We utilized confocal and electron microscopy (EM) and nuclear magnetic resonance (NMR) spectroscopy to assess morphology and histology in the skeletal muscle (SKM) of db +/‐_MSTN +/‐ mice. Muscle lipid contents were assessed via mass spectrometry. Femoral artery ligation was used to stimulate in vivo angiogenesis in response to ischemia. Gene expression was assessed by RNA‐Seq with mRNA and protein expression follow‐up in isolated gastrocnemius skeletal muscle via RT‐qPCR and western blotting.ResultsAs previously described, myostatin deletion results in significantly increased muscle mass without altering whole‐body mass, fat percentage, or activity levels in obese db/db mice. Blood glucose is markedly improved but plasma lipids remain elevated. In parallel with these improvements, we observed that obesity inhibits and MSTN deletion restores vascularization following hind limb ischemia. Despite persistent plasma lipedema, ectopic lipid deposition in skeletal muscle was largely ameliorated in obese mice lacking myostatin, a reduction largely explained by a decrease in tissue triglyceride levels. EM and confocal microscopy revealed accumulation of lipid in obese muscle cells that was lacking in obese mice with MSTN KO. The resolved deposition occurred predominantly inside muscle cells with fat cell infiltration in external spaces largely unaffected. RNA‐Seq analysis of skeletal muscle revealed marked upregulation of lipogenesis pathways, notably the expression of stearoyl Co‐A desaturase 1 (SCD1), the rate limiting enzyme for the oleate synthesis and subsequent triglyceride production. SCD1 was elevated in muscle from normal obese mice but not those lacking myostatin at both the protein and RNA level.ConclusionIn summary, increased muscle mass in obese mice provides potent protection to vascular and metabolic health. A potent correlate of this improvement is the resolution of myosteatosis in obese, hypermuscular mice. These data suggest that resolution of myosteatosis, independent of muscle mass, may afford similar protection, possibly by targeted deletion or blockade of SCD1.

Endurance exercise training suppresses myostatin upregulation and nuclear factor-kappa B activation in a mouse model of Parkinson's disease.

Background and Aim:Muscle atrophy is common in Parkinson’s disease (PD). Although myostatin has been implicated in muscle atrophy, its expression in PD skeletal muscle has not been investigated. Therefore, this study aimed to elucidate the influence of PD induction and exercise training on myostatin expression in the gastrocnemius skeletal muscle.Materials and Methods:Thirty albino mice were randomly selected and separated into three groups of 10 mice each: Sedentary control, sedentary PD (SPD), and exercised PD (EPD). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid were used to induce chronic parkinsonism in the PD groups. Immunohistochemistry was used to investigate the expression of myostatin and nuclear factor kappa B (NF-kB) in gastrocnemius muscles of all three groups.Results:Myostatin expression and NF-kB nuclear localization, indicative of its activation, were significantly (p<0.01) higher in gastrocnemius skeletal muscle in the SPD group than in the control and EPD groups. Concomitantly, the average cross-sectional area of gastrocnemius muscle fibers in the SPD albino mice was significantly smaller (p<0.01) than in the control and EPD groups, indicating muscle atrophy.Conclusion:The present data are the first to indicate a correlation between PD induction and myostatin overexpression and NF-kB activation in the gastrocnemius muscle, potentially promoting the muscle atrophy commonly seen in PD. Additionally, the current data are the first to indicate the beneficial effects of exercise training on PD-associated myostatin overexpression, NF-κB activation, and muscle atrophy. Thus, our data are the first to suggest that myostatin and NF-κB might be regarded as potential therapeutic targets in an attempt to ameliorate skeletal muscle abnormalities commonly observed in PD.

Dietary Alaska Pollack Protein Induces Acute and Sustainable Skeletal Muscle Hypertrophy in Rats

Our previous studies suggested that Alaska pollack protein (APP) intake increases skeletal muscle mass and that it may cause a slow-to-fast shift in muscle fiber type in rats fed a high-fat diet after 56 days of feeding. In this study, we explored whether dietary APP induces acute and sustainable skeletal muscle hypertrophy in rats fed a normal-fat diet. Male 5-week-old Sprague–Dawley rats were divided into four groups and fed a purified ingredient-based high-fat diet or a purified ingredient-based normal-fat diet with casein or APP, containing the same amount of crude protein. Dietary APP significantly increased gastrocnemius muscle mass (105~110%) after 2, 7 days of feeding, regardless of dietary fat content. Rats were separated into two groups and fed a normal-fat diet with casein or APP. Dietary APP significantly increased gastrocnemius muscle mass (110%) after 56 days of feeding. Dietary APP significantly increased the cross-sectional area of the gastrocnemius skeletal muscle and collagen-rich connective tissue after 7 days of feeding. It decreased the gene expression of Mstn /Myostatin, Trim63/MuRF1, and Fbxo32/atrogin-1, but not other gene expression, such as serum IGF-1 after 7 days of feeding. No differences were observed between casein and APP groups with respect to the percentage of Type I, Type IIA, and Type IIX or IIB fibers, as determined by myosin ATPase staining after 7 days of feeding. In the similar experiment, the puromycin-labeled peptides were not different between dietary casein and APP after 2 days of feeding. These results demonstrate that APP induces acute and sustainable skeletal muscle hypertrophy in rats, regardless of dietary fat content. Dietary APP, as a daily protein source, may be an approach for maintaining or increasing muscle mass.

Effect of Experimentally Induced Chronic Kidney disease on Skeletal, Cardiac and Smooth Muscle fibers of albino rat

Abstract Background Chronic kidney disease (CKD), or chronic renal failure (CRF) as it was historically termed, includes all degrees of decreased renal function, starting from mild, and moderate, to severe chronic kidney failure. Skeletal muscle atrophy frequently complicates the course of CKD and is associated with excess morbidity and mortality. Cardiovascular diseases have been reported to be the leading causes of death in CKD patients. Chronic Kidney Disease was also reported to be associated with an increased incidence of acid-related gastrointestinal disorders. Aim of the work The aim of this study was to investigate the effect of chronic kidney disease experimentally induced by gentamicin intramuscular injection on the histological structure of gastrocnemius skeletal muscle, left ventricular cardiac muscle and smooth muscle fibers of lower esophagus. Materials and methods Twenty male adult Wistar albino rats were randomly and equally divided into two groups. Group I (control group) received physiological saline intramuscular injection, once daily for 28 consecutive days, in a dose equivalent to that taken in group II. Group II (Gentamicin-treated group) were given Gentamicin intramuscular injection for induction of CKD. Gentamicin was given as Gentamycin sulfate, 40 mg/ml (Sandoz, Switzerland), once daily, in a dose of 80 mg/kg/day for 28 days to induce CKD. After 28 days of the first injection of gentamicin, rats were anaesthetized and blood samples were collected to measure the level of serum urea and creatinine. The left kidneys, the middle third of left gastrocnemius muscle, the lateral wall of left ventricle (LV) and the gastroesophageal junction of all rats of both groups (I and II) were processed for light microscopic study. The middle third of left gastrocnemius muscle, the lateral wall of left ventricle (LV) were further processed for transmission electron microscopic study. Histomorphometrical and statistical analysis were also done. Results The LM examination revealed moderate obliteration of glomeruli, dilatation in some renal tubules and collapse in others, mainly in distal convoluted tubules, with significant fibrosis of renal parenchyma. Serum urea and creatinine levels were increased significantly. The skeletal muscle fibers of the rats in group II (CKD) showed focal areas of myofibers degeneration with siginificant fibrosis. The cardiac muscle fibers of the rats in the group II (CKD) showed focal areas of cardiomyocytes degeneration and other areas of significantly hypertrophied fibers. The smooth muscle fibers of the lower esophageal sphincter of the rats in group II (CKD) showed no significant structural changes compared with the control group, however, the myenetric plexus showed multiple pyknotic and karyolitic nuclei with vacuolated cytoplasm. In addition, insignificant increase in the amount of collagen fibers was observed in almost all layers. Conclusion CKD produced moderate atrophy of skeletal muscle fibers, significant increase in the cardiomyocyte size and no significant structural effect of smooth muscle fibers of the lower esophageal sphincter.

Maslinic Acid Attenuates Denervation-Induced Loss of Skeletal Muscle Mass and Strength.

Maslinic acid (MA) is a pentacyclic triterpene abundant in olive peels. MA reportedly increases skeletal muscle mass and strength in older adults; however, the underlying mechanism is unknown. This study aimed to investigate the effects of MA on denervated muscle atrophy and strength and to explore the underlying molecular mechanism. Mice were fed either a control diet or a 0.27% MA diet. One week after intervention, the sciatic nerves of both legs were cut to induce muscle atrophy. Mice were examined 14 days after denervation. MA prevented the denervation-induced reduction in gastrocnemius muscle mass and skeletal muscle strength. Microarray gene expression profiling in gastrocnemius muscle demonstrated several potential mechanisms for muscle maintenance. Gene set enrichment analysis (GSEA) revealed different enriched biological processes, such as myogenesis, PI3/AKT/mTOR signaling, TNFα signaling via NF-κB, and TGF-β signaling in MA-treated mice. In addition, qPCR data showed that MA induced Igf1 expression and suppressed the expressions of Atrogin-1, Murf1 and Tgfb. Altogether, our results suggest the potential of MA as a new therapeutic and preventive dietary ingredient for muscular atrophy and strength.

Histone Deacetylase 2 Suppresses Skeletal Muscle Atrophy and Senescence via NF-κB Signaling Pathway in Cigarette Smoke-Induced Mice with Emphysema.

BackgroundExposure to cigarette smoke (CS) is the main risk factor for chronic obstructive pulmonary disease (COPD). CS not only causes chronic airway inflammation and lung damage but also is involved in skeletal muscle dysfunction (SMD). Previous studies have shown that histone deacetylase 2 (HDAC2) plays an important role in the progression of COPD. The aim of this study was to determine the role of HDAC2 in CS-induced skeletal muscle atrophy and senescence.MethodsGastrocnemius muscle weight and cross-sectional area (CSA) were measured in mice with CS-induced emphysema, and changes in the expression of atrophy-related markers and senescence-related markers were detected. In addition, the relationship between HDAC2 expression and skeletal muscle atrophy and senescence was also investigated.ResultsMice exposed to CS for 24 weeks developed emphysema and gastrocnemius atrophy and exhibited a decrease in gastrocnemius weight and skeletal muscle cross-sectional area. In addition, the HDAC2 protein levels were significantly decreased while the levels of atrophy-associated markers, including MURF1 and MAFbx, and senescence-associated markers, including P53 and P21, were significantly increased in the gastrocnemius muscle. In vitro, the exposure of C2C12 cells to cigarette smoke extract (CSE) significantly increased the MAFbx and MURF1 protein levels and decreased the HDAC2 protein levels. Moreover, overexpression of HDAC2 significantly ameliorated CSE-induced atrophy and senescence and reversed the increased MURF1, MAFbx, P53, and P21 expression in C2C12 cells. In addition, CSE treatment significantly increased the IKK and NF-κB p65 protein levels, and PTDC (an NF-kB inhibitor) ameliorated atrophy and senescence.ConclusionOur findings suggest that HDAC2 plays an important role in CS-induced skeletal muscle atrophy and senescence, possibly through the NF-κB pathway.

Myosin Heavy Chain Isoform Mrna Expression In Low- And High-capacity Running Rats

Differential expression of Myosin Heavy Chain (MHC) isoforms in skeletal muscle determines its metabolic and functional characteristics. Endurance exercise training is associated with fast-to-slow MHC isoform transition, and which is considered to mediate the beneficial effects of exercise on metabolic health. Rats genetically bred for low-capacity running (LCR) and high-capacity running (HCR) are characterized by the health benefits typically described after endurance exercise training. LCR and HCR rats differed in maximal running capacity 14 fold. PURPOSE: To quantify mRNA expression for slow (MHC-I) and fast (MCH-IIa, MCH-IIb) MHC isoforms in soleus, gastrocnemius and vastus skeletal muscle of LCR and HCR male adult rats, and determine if HCR rats have higher mRNA expression for slow, and lower mRNA expression for fast, MHC isoforms. METHODS: Muscle tissue from 5 HCR and 5 LCR rats was homogenized and mRNA was isolated. The mRNA content of MHC isoforms in muscle was quantified using reverse transcriptase polymerase chain reaction (RT-PCR), and standardized to the 18S mRNA content in muscle. Differences between groups were calculated using the comparative CT method (2-ΔΔCT). LCR were the control group. RESULTS: Expression of mRNA of the MHC isoforms differed between groups and the type of skeletal muscle analyzed. MHC-I mRNA expression was lower in the HCR compared to LCR across all muscle groups, but this difference (2-ΔΔCT) was more pronounced for the gastrocnemius muscle [LCR: 1.0 ± 0.6 (mean±SE); HCR: 0.3 ± 0.2; P = 0.08]. Although MHC-IIa mRNA expression appeared lower in soleus and vastus in the HCR, it was higher in the HCR compared to LCR in the gastrocnemius muscle [LCR: 1.0 ± 1.0; HCR: 4.6 ± 3.6; P = 0.07]. MHC-IIb mRNA expression was lower in the HCR compared to LCR across all muscle groups, but this difference was more pronounced for the soleus muscle [LCR: 1.0 ± 1.1; HCR: 0.2 ± 0.4; P = 0.08]. CONCLUSION: Genetic differences linked to the MHC transcriptome (i.e., decrease in MHC-I mRNA) in muscle do not explain the exercise performance observed in HCR rats. Changes in the muscle MHC transcriptome may not directly be responsible for the health benefits associated with exercise training. It remains to be determined on whether the differences we observed at the MHC mRNA level are translated to the MHC proteome level.

Ameliorative Effect of Carica papaya Seed Extract on Diabetic Rat Model with Muscle Atrophy

This study aims to prove that papaya seed extract has the potential to improve muscle cell atrophy in diabetic conditions. Type 2 diabetes mellitus (DM) animal model were made with conditions similar to type 2 DM in humans i.e. with long-term (56 days) high sugar (fructose) consumption. Then, the next 14 days were treated with papaya seed extract at the dose of 100, 200, and 300 mg/kg BW orally. Body weight and blood glucose levels were monitored throughout the study period. At the end of the study, histopathological examination of gastrocnemius skeletal muscle tissue was carried out using hematoxylin-eosin staining and measuring myocyte cell area. Results showed that there is a correlation between blood glucose levels with the area of muscle cells which explains that the decrease in blood glucose levels is in line with the increase in the area of muscle fiber cells. In the diabetic group and the treatment group the dose of 100 mg/kg BW has a smaller area, whereas in the treatment group the dose of 200 and 300 mg/kg BW has an area close to the area of the muscle cell in the normal group. This is supported by the results of measurements of the area of muscle fiber cells observed through cross sections and measured at the end of this study. The treatment group at the dose of 200 and 300 mg/kg BW did not have significant difference (P&gt;0.05) compared to the normal group. It can be concluded that papaya seed extract therapy at the dose of 200 and 300 mg/kg BW on diabetic rats can reduce the fasting blood glucose levels so that it can ameliorate cell atrophy in the diabetic conditions.

Oral glutamine supplementation attenuates inflammation and oxidative stress-mediated skeletal muscle protein content degradation in immobilized rats: Role of 70 kDa heat shock protein

Skeletal muscle disuse results in myofibrillar atrophy and protein degradation, via inflammatory and oxidative stress-mediated NF-kB signaling pathway activation. Nutritional interventions, such as l-glutamine (GLN) supplementation have shown antioxidant properties and cytoprotective effects through the modulation on the 70-kDa heat shock protein (HSP70) expression. However, these GLN-mediated effects on cell signaling pathways and biochemical mechanisms that control the myofibrillar protein content degradation in muscle disuse situations are poorly known yet. This study investigated the effects of oral GLN plus l-alanine (ALA; GLN ​+ ​ALA-solution) supplementation, either in their free or dipeptide (L-alanyl-l-glutamine-DIP) form, on GLN-glutathione (GSH) axis and cytoprotection mediated by HSP70 protein expression in the slow-twitch soleus and fast-twitch gastrocnemius skeletal muscle of rats submitted to 14-days of hindlimb immobilization-induced disuse muscle atrophy. Forty-eight Wistar rats were distributed into 6 groups: hindlimb immobilized (IMOB group) and hindlimb immobilized orally supplemented with either GLN (1 g kg−1) plus ALA (0.61 g kg−1) ​(GLN ​+ ​ALA-IMOB group) or 1.49 ​g ​kg−1 of DIP (DIP-IMOB group) and; no-immobilized (CTRL) and no-immobilized supplemented GLN ​+ ​ALA and DIP baselines groups. All animals, including CTRL and IMOB rats (water), were supplemented via intragastric gavage for 14 days, concomitantly to immobilization period. Plasma and muscle GLN levels, lipid (thiobarbituric acid reactive substances-TBARS) and protein (carbonyl) peroxidation, erythrocyte concentration of reduced GSH and GSH disulfide (GSSG), plasma and muscle pro-inflammatory TNF-α levels, muscle IKKα/β-NF-kB signaling pathway and, the myofibrillar protein content (MPC) were measured. The MPC was significantly lower in IMOB rats, compared to CTRL, GLN ​+ ​ALA, and DIP animals (p ​< ​0.05). This finding was associated with reduced plasma and muscle GLN concentration, equally in IMOB animals. Conversely, both GLN ​+ ​ALA and DIP supplementation restored plasma and muscle GLN levels, which equilibrated GSH and intracellular redox status (GSSG/GSH ratio) in erythrocytes and skeletal muscle even as, increased muscle HSP70 protein expression; attenuating oxidative stress and TNF-α-mediated NF-kB pathway activation, fact that reverberated on reduction of MPC degradation in GLN ​+ ​ALA-IMOB and DIP-IMOB animals (p ​< ​0.05). In conclusion, the findings shown herein support the oral GLN ​+ ​ALA and DIP supplementations as a therapeutic and effective nutritional alternative to attenuate the deleterious effects of the skeletal muscle protein degradation induced by muscle disuse.

LIPOTOXICIDAD EN MÚSCULO ESQUELÉTICO Y SU RELACIÓN CON LA RESISTENCIA INSULÍNICA. ESTUDIOS EN UN MODELO EXPERIMENTAL DE SÍNDROME METABÓLICO

Introducción: el acúmulo de lípidos en el músculo esquelético se encuentra estrechamente vinculado con el desarrollo de la resistencia insulínica. Esta última cumple un rol patogénico central en el desarrollo de numerosos desórdenes metabólicos incluidos en el síndrome metabólico.Objetivos: analizar algunas vías metabólicas implicadas en el acúmulo de lípidos en el músculo esquelético y su asociación con la resistencia insulínica en un modelo experimental que mimetiza el fenotipo del síndrome metabólico humano.Materiales y métodos: ratas macho Wistar recibieron una dieta control (DC) o una dieta rica en sacarosa (DRS) durante seis meses. Al final del período experimental se analizó en músculo esquelético gastrocnemio: contenido de triglicéridos (TG), acil-CoA de cadena larga y diacilglicerol, actividad enzimática carnitina palmitoil transferasa muscular (M-CPT1, M-CPT2 y M-CPT total) y masa proteica del PPARα, AMPK y AMPKp. Se determinaron los niveles séricos de TG, AGNE, glucosa, insulina, TNFα y adiponectina. La sensibilidad insulínica se midió por la técnica clamp euglucémica-hiperinsulinémica.

Identification of Metabolic Changes in Ileum, Jejunum, Skeletal Muscle, Liver, and Lung in a Continuous I.V. Pseudomonas aeruginosa Model of Sepsis Using Nontargeted Metabolomics Analysis

Sepsis is a multiorgan disease affecting the ileum and jejunum (small intestine), liver, skeletal muscle, and lung clinically. The specific metabolic changes in the ileum, jejunum, liver, skeletal muscle, and lung have not previously been investigated. Live Pseudomonas aeruginosa, isolated from a patient, was given via i.v. catheter to pigs to induce severe sepsis. Eighteen hours later, ileum, jejunum, medial gastrocnemius skeletal muscle, liver, and lung were analyzed by nontargeted metabolomics analysis using gas chromatography/mass spectrometry. The ileum and the liver demonstrated significant changes in metabolites involved in linoleic acid metabolism: the ileum and lung had significant changes in the metabolism of valine/leucine/isoleucine; the jejunum, skeletal muscle, and liver had significant changes in arginine/proline metabolism; and the skeletal muscle and lung had significant changes in aminoacyl-tRNA biosynthesis, as analyzed by pathway analysis. Pathway analysis also identified changes in metabolic pathways unique for different tissues, including changes in the citric acid cycle (jejunum), β-alanine metabolism (skeletal muscle), and purine metabolism (liver). These findings demonstrate both overlapping metabolic pathways affected in different tissues and those that are unique to others and provide insight into the metabolic changes in sepsis leading to organ dysfunction. This may allow therapeutic interventions that focus on multiple tissues or single tissues once the relationship of the altered metabolites/metabolism to the underlying pathogenesis of sepsis is determined.

Differential activation of the calpain system involved in individualized adaptation of different fast-twitch muscles in hibernating Daurian ground squirrels.

We examined the lateral gastrocnemius (LG), plantaris (PL), and extensor digitorum longus (EDL) muscles to determine whether differential activation of the calpain system is related to the degree of atrophy in these fast-twitch skeletal muscles during hibernation in Daurian ground squirrels (Spermophilus dauricus). Results from morphological indices showed various degrees of atrophy in the order LG > PL > EDL. Furthermore, all three muscles underwent fast-to-slow fiber-type conversion in hibernation. In regard to the calpain system in the LG muscle, cytosolic Ca2+ increased significantly in hibernation, followed by recovery in posthibernation. Furthermore, calpastatin expression significantly decreased, and calpain 1 and 2 expression significantly increased, which may be responsible for the increased degradation of desmin during hibernation compared with that during summer activity. In the EDL muscle, Ca2+ overload was observed during interbout arousal, and calpastatin showed an increase during hibernation and interbout arousal, which could explain the increased levels of troponin T during both periods compared with levels during summer activity. These findings suggest that cytosolic Ca2+ overload and subsequent calpain 1 and 2 activation may be an important mechanism of LG muscle atrophy during hibernation. Cytosolic Ca2+ homeostasis and high expression of calpain inhibitor calpastatin during hibernation may also be an important mechanism for the EDL muscle to maintain muscle mass. Thus, the differential activation of the calpain system and selective degradation of downstream substrates may be involved in muscle atrophy of different fast-twitch muscles during hibernation.NEW & NOTEWORTHY We found that the extent of both muscle atrophy and calpain system activation differed in fast-twitch lateral gastrocnemius (LG), plantaris (PL), and extensor digitorum longus (EDL) skeletal muscles in hibernating Daurian ground squirrels, but similar hierarchies in the order of LG > PL > EDL. The differential activation of the calpain system and selective degradation of downstream substrates may be involved in muscle atrophy in different fast-twitch muscles during hibernation.