Skeletal Muscle Type Research Articles

Evidence of Blood and Muscle Redox Status Imbalance in Experimentally Induced Renal Insufficiency in a Rabbit Model.

Chronic kidney disease (CKD) is accompanied by a disturbed redox homeostasis, especially in end-stage patients, which is associated with pathological complications such as anemia, atherosclerosis, and muscle atrophy. However, limited evidence exists about redox disturbances before the end stage of CKD. Moreover, the available redox literature has not yet provided clear associations between circulating and tissue-specific (muscle) oxidative stress levels. The aim of the study was to evaluate commonly used redox status indices in the blood and in two different types of skeletal muscle (psoas, soleus) in the predialysis stages of CKD, using an animal model of renal insufficiency, and to investigate whether blood redox status indices could be reflecting the skeletal muscle redox status. Indices evaluated included reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase (GR), catalase (CAT), total antioxidant capacity (TAC), protein carbonyls (PC), and thiobarbituric acid reactive substances (TBARS). Results showed that blood GSH was higher in the uremic group compared to the control (17.50 ± 1.73 vs. 12.43 ± 1.01, p = 0.033). In both muscle types, PC levels were higher in the uremic group compared to the control (psoas: 1.086 ± 0.294 vs. 0.596 ± 0.372, soleus: 2.52 ± 0.29 vs. 0.929 ± 0.41, p < 0.05). The soleus had higher levels of TBARS, PC, GSH, CAT, and GR and lower TAC compared to the psoas in both groups. No significant correlations in redox status indices between the blood and skeletal muscles were found. However, in the uremic group, significant correlations between the psoas and soleus muscles in PC, GSSG, and CAT levels emerged, not present in the control. Even in the early stages of CKD, a disturbance in redox homeostasis was observed, which seemed to be muscle type-specific, while blood levels of redox indices did not seem to reflect the intramuscular condition. The above results highlight the need for further research in order to identify the key mechanisms driving the onset and progression of oxidative stress and its detrimental effects on CKD patients.

Effects of Two Creatine Monohydrate Feeding Protocols on Doxorubicin‐Induced Skeletal Muscle Dysfunction

The chemotherapy drug doxorubicin (Dox) can negatively impact skeletal muscle function, and interventions to address this side effect are important to improving cancer patient quality of life. The purpose of this study was to investigate the effects of creatine monohydrate (Cr) feeding prior to Dox treatment on skeletal muscle function. Male Sprague‐Dawley rats were randomly assigned to one of four groups: standard chow feeding for four weeks followed by a bolus i.p. 15 mg/kg Dox injection (Dox group); 2% Cr chow feeding for four weeks followed by a bolus i.p. 15 mg/kg Dox injection (Cr1+Dox group); 4% Cr chow feeding for one week then 2% Cr chow feeding for three weeks followed by a bolus i.p. 15 mg/kg Dox injection (Cr2+Dox group); or standard chow feeding for four weeks followed by a bolus i.p. 0.9% NaCl injection to act as a control (Con group). Grip force measurements were obtained prior to the start of nutritional interventions (baseline), prior to injection (after four‐week nutritional intervention), and three days post‐injection. Animals were anesthetized three days post‐injection, the soleus (SOL) and extensor digitorum longus (EDL) muscles were excised, and function was assessed ex vivo using a 100 second fatigue protocol. No grip force differences were observed at baseline or following the four‐week nutritional interventions. Three days after injections, however, the Dox and Cr1+Dox groups had lower grip forces than Con (p&lt;0.05), but this difference was not observed in Cr2+Dox (p&gt;0.05). In the SOL, no between group force differences were observed at baseline or at any of the 10 second time points throughout the fatigue protocol (p&gt;0.05). EDLs from the Dox group generated significantly less force than Con throughout the protocol except the 50 s and 60 s time points (p&lt;0.05). These differences in EDL force production throughout the protocol, however, were not observed in Cr1+Dox and Cr2+Dox (p&gt;0.05). In conclusion, Cr1 and Cr2 feeding protected against Dox‐induced declines in force production in the type II, EDL muscle, and grip force was preserved only with Cr2 feeding. These results suggest that Cr's protection against Dox myotoxicity may be dependent upon Cr dose and skeletal muscle type.Support or Funding InformationDavid S. Hydock, PhD was supported by a Pilot and Exploratory Projects in Palliative Care of Cancer Patients and Their Families, PEP‐17‐193‐01 ‐ PCSM, from the American Cancer Society.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

3D mitochondrial network formation during postnatal skeletal muscle development of mice

Mitochondrial connectivity plays an important role for muscle energy distribution. However, it is obscure how their structural formation is developed during postnatal muscle growth. Here, using a high‐resolution electron microscopy (i.e., FIB‐SEM), we have sought to examine three dimensional (3D) mitochondrial network formations in oxidative (soleus) and glycolytic (gastrocnemius) muscles of C57BL/6N mice at postnatal (P) 1, 7, 14, 21, and 42 day, respectively. After semi‐automated machine learning process, 3D mitochondrial reticulum formations were rendered and quantitatively analyzed by ImageJ software. Both types of skeletal muscles showed similarly fiber parallel mitochondrial network formations during early days (P1–P7). After P14, oxidative muscles developed grid‐like mitochondrial reticulum, while glycolytic muscle mitochondria were found as perpendicular formations along the Z‐lines. Unlikely to glycolytic muscles where individual mitochondrial volumes were similarly maintained across the development, oxidative muscles significantly increased those volumes by P14 and then largely decreased to the level comparable to glycolytic muscles. After P14, oxidative muscles maintained notably higher total mitochondrial volumes as compared to glycolytic muscles. Furthermore, we observed that at P14, glycolytic muscles showed remarkably higher ratio of mitochondrial surface area to volume, as well as sphericity, than oxidative muscles. Another interesting point was that during early muscle growth (P1–P7), mitochondrial elongation, aspect ratio, and length were all shown to be higher in glycolytic muscles than in oxidative muscles. In conclusion, this preliminary study provides how 3D mitochondrial networks are changed over the course of postnatal muscle development, and also suggests that P14 may be a critical time point for the mitochondrial transformation. However, further studies are needed to better understand 3D muscle mitochondrial development along with contractile components and other subcellular elements such as lipid droplets and sarcoplasmic reticulum.Support or Funding InformationThis work is supported by the Division of Intramural Research of the National Heart, Lung and Blood Institute and the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of elamipretide on skeletal muscle in dogs with experimentally induced heart failure.

AimsElamipretide (ELAM), an aromatic–cationic tetrapeptide, interacts with cardiolipin and normalizes dysfunctional mitochondria of cardiomyocytes. This study examined the effects of ELAM on skeletal muscle mitochondria function in dogs with chronic heart failure (HF).Methods and resultsStudies were performed in skeletal muscle biopsy specimens obtained from normal dogs (n = 7) and dogs with chronic intracoronary microembolization‐induced HF (n = 14) treated with subcutaneous ELAM 0.5 mg/kg (HF + ELAM, n = 7) or vehicle (normal saline control, HF‐CON, n = 7). After 3 months of therapy, triceps skeletal muscle samples were obtained from all dogs, and the proportion of type 1 and type 2 fibres was assessed. Mitochondria isolated from myofibrils of the vastus lateralis skeletal muscle exposed in vitro to ELAM for 1 h were used to assess mitochondrial function. The proportion of skeletal muscle type 1 fibres was lower in HF‐CON dogs compared with normal dogs (23 ± 4 vs. 32 ± 5%, P < 0.05). Treatment with ELAM restored a near‐normal fibre‐type composition (31 ± 7%, P < 0.05 vs. HF‐CON). Skeletal muscle mitochondria showed significantly lower levels of adenosine diphosphate‐dependent mitochondrial respiration (100 ± 9 vs. 164 ± 15 natom O/min/mg protein, P < 0.05), mitochondrial membrane potential (0.17 ± 0.03 vs. 0.53 ± 0.03 red/green fluorescence ratio, P < 0.05), mitochondrial permeability transition pore (38 ± 3 vs. 62 ± 2 relative light units, P < 0.05), maximum rate of adenosine triphosphate synthesis (3284 ± 418 vs. 8835 ± 423 RLU/μg protein, P < 0.05), and cytochrome c oxidase activity (1390 ± 108 vs. 2459 ± 210 natom O/min/mg protein, P < 0.05) compared with normal dogs. Exposure of skeletal muscle myofibrillar mitochondria from HF dogs to ELAM showed a dose‐dependent improvement/normalization of all measures of mitochondrial function. In mitochondria from skeletal muscle of HF dogs exposed to 0.10 μM ELAM, adenosine diphosphate‐dependent mitochondrial respiration increased to 183 ± 18 natom O/min/mg protein, membrane potential increased to 0.30 ± 0.03 red/green fluorescence ratio, mitochondrial permeability transition pore increased to 54 ± 4 RLU, maximum rate of adenosine triphosphate synthesis increased to 4423 ± 414, and cytochrome c oxidase activity increased to 2033 ± 191 natom O/min/mg protein. Exposure of skeletal muscle myofibrillar mitochondria from normal dogs to ELAM had no effect on mitochondrial function parameters.ConclusionsThe results indicate that ELAM, previously shown to positively influence mitochondrial function of the failing heart, can also positively impact mitochondrial function of skeletal muscle and potentially help restore skeletal muscle function and improve exercise tolerance.

Efficacy of Multi-exon Skipping Treatment in Duchenne Muscular Dystrophy Dog Model Neonates

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, which codes for dystrophin. Because the progressive and irreversible degeneration of muscle occurs from childhood, earlier therapy is required to prevent dystrophic progression. Exon skipping by antisense oligonucleotides called phosphorodiamidate morpholino oligomers (PMOs), which restores the DMD reading frame and dystrophin expression, is a promising candidate for use in neonatal patients, yet the potential remains unclear. Here, we investigate the systemic efficacy and safety of early exon skipping in dystrophic dog neonates. Intravenous treatment of canine X-linked muscular dystrophy in Japan dogs with a 4-PMO cocktail resulted in ∼3%-27% in-frame exon 6-9 skipping and dystrophin restoration across skeletal muscles up to 14% of healthy levels. Histopathology was ameliorated with the reduction of fibrosis and/or necrosis area and centrally nucleated fibers, significantly in the diaphragm. Treatment induced cardiac multi-exon skipping, though dystrophin rescue was not detected. Functionally, treatment led to significant improvement in the standing test. Toxicity was not observed from blood tests. This is the first study to demonstrate successful multi-exon skipping treatment and significant functional improvement in dystrophic dogs. Early treatment was most beneficial for respiratory muscles, with implications for addressing pulmonary malfunction in patients.

Discrepant effects of copper (II) stress on different types of skeletal muscles in chicken: Elements and amino acids

Different distributions of 28 elements and 17 amino acids in pectoralis, wing biceps brachii and leg gastrocnemius of chicken upon CuSO4 (300 mg/kg) exposure for 90 days were investigated. Accompanied by copper accumulation, pathological injuries were observed in those three kinds of skeletal muscles using histological and ultrastructural methods. Based on data obtained, we analyzed leg gastrocnemius displayed the most increases (P < 0.000) in all three kinds of elements detected, including macroelements (131%), essential microelements (129%) and toxic microelements (179%) than the other two skeletal muscles. Furthermore, decreased total amino acids (P = 0.006), a susceptibility of lipid peroxidation/oxidative stress and a disequilibrium of nutrition and taste were analyzed in the leg gastrocnemius, indicating an unsuitability for human consumption. Intriguingly, these anomalies were scarce in pectoralis and wing biceps brachii. Combined with multivariate analysis we may conclude that leg gastrocnemius are more vulnerable to copper stress than pectoralis and wing biceps brachii in chicken.

Body temperature elevation during exercise is essential for activating the Akt signaling pathway in the skeletal muscle of type 2 diabetic rats.

This study examined the effect of changes in body temperature during exercise on signal transduction-related glucose uptake in the skeletal muscle of type 2 diabetic rats. Otsuka Long-Evans Tokushima Fatty rats (25 weeks of age), which have type 2 diabetes, were divided into the following four weight-matched groups; control (CON, n = 6), exercised under warm temperature (WEx, n = 8), exercised under cold temperature (CEx, n = 8), and heat treatment (HT, n = 6). WEx and CEx animals were subjected to running on a treadmill at 20 m/min for 30 min under warm (25°C) or cold (4°C) temperature. HT animals were exposed to single heat treatment (40–41°C for 30 min) in a heat chamber. Rectal and muscle temperatures were measured immediately after exercise and heat treatment, and the gastrocnemius muscle was sampled under anesthesia. Rectal and muscle temperatures increased significantly in rats in the WEx and HT, but not the CEx, groups. The phosphorylation levels of Akt, AS160, and TBC1D1 (Thr590) were significantly higher in the WEx and HT groups than the CON group (p < 0.05). In contrast, the phosphorylation levels of AMP-activated protein kinase, ACC, and TBC1D1 (Ser660) were significantly higher in rats in the WEx and CEx groups than the CON group (p < 0.05) but did not differ significantly between rats in the WEx and CEx groups. Body temperature elevation by heat treatment did not activate the AMPK signaling. Our data suggest that body temperature elevation during exercise is essential for activating the Akt signaling pathway in the skeletal muscle of rats with type 2 diabetic rats.

Attenuation of metabolic dysfunctions in the skeletal muscles of type 1 diabetic rats by Stevia rebaudiana extracts, via AMPK upregulation and antioxidant activities.

Stevia rebaudiana Bertoni leaves are well-known for their sweetness and have been used as a non-caloric sweetener in several countries. It has numerous therapeutic properties which have been proven safe and effective over hundreds of years. In the present study, we aimed to evaluate the possible antioxidant effects of stevia extracts and their role in regulating AMPK in type-1 diabetic rats. Fifty male Sprague Dawely rats were divided into: (1) normal control (NC) group; normal rats receiving 0.5 ml normal saline, (2) DM group; diabetic rats receiving 0.5 ml normal saline, (3) DM + MSE group; DM rats receiving 200 mg/kg of methanolic extract of stevia, (4) DM + S group; DM rats receiving 2 mg/kg of pure stevioside, and (5) DM + CGA group; DM rats receiving 10 mg/kg of pure chlorogenic acid. Four weeks after treatment, AMPK activity, GLUT4 mRNA and oxidative stress markers were measured in frozen muscles. Also, fasting blood glucose in serum, insulin and HbA1c were measured at the end of experiment. DM caused a significant increase in serum fasting glucose, HbA1c and muscle MDA with significant reduction in serum insulin, muscle SOD, catalase, GPx, AMPK activity and GLUT4 expression (p < 0.05). Treatment with stevia extract, pure stevioside and chlorogenic acid caused significant improvements in the studied parameters (p < 0.05). We concluded that stevia extracts and derivatives may improve metabolic dysfunction in skel- etal muscles via upregulation of AMPK and GLUT4 and suppression of oxidative stress.

Attenuation of metabolic dysfunctions in the skeletal muscles of type 1 diabetic rats by Stevia rebaudiana extracts, via AMPK upregulation and antioxidant activities [pdf

Attenuation of metabolic dysfunctions in the skeletal muscles of type 1 diabetic rats by Stevia rebaudiana extracts, via AMPK upregulation and antioxidant activities [pdf

Exercise and Mitochondrial Remodeling in Skeletal Muscle in Type 2 Diabetes.

Exercise is regarded as a potent stimulus in modulation of glucose utility and mitochondrial adaptations in skeletal muscle, leading to enhanced metabolic health. As mitochondria play a crucial role in sustaining metabolic homeostasis, and disturbances in mitochondrial function are highly linked with development of metabolic diseases, a comprehensive understanding of exercise-mediated mitochondrial remodeling under the pathophysiological condition of type 2 diabetes is warranted to develop an efficient therapeutic strategy. Although it is evident that the primary etiology of type 2 diabetes is insulin resistance, there is accumulating evidence linking abnormal mitochondrial functional and morphological properties to development of type 2 diabetes. Despite this, the precise molecular and cellular events that underline these phenomena remain uncertain. Mitochondria are highly dynamic subcellular organelles that can change mass and shape as necessary via coordinated processes such as mitochondrial fusion, fission, and biogenesis. Mitochondrial fusion is controlled by proteins, including mitofusin-1, mitofusin-2, and optic atrophy protein 1, while the fission process is mainly modulated by control of fission protein 1 and dynamin-related protein 1. Peroxisome proliferator-activated receptor gamma coactivator-1α acts as a master controller of mitochondrial biogenesis. The present review’s primary aims were to briefly discuss the cellular mechanisms of muscle fiber type-dependent glucose uptake and to highlight emerging evidence linking disturbances in mitochondrial dynamics to development of insulin resistance and type 2 diabetes. The potential for exercise to normalize type 2 diabetes-induced aberrant mitochondrial integrity is also addressed.

Lactate administration activates the ERK1/2, mTORC1, and AMPK pathways differentially according to skeletal muscle type in mouse.

Skeletal muscle is described as an endocrine organ, constitutively or intermittently secreting bioactive molecules. The signaling pathways by which these molecules mediate changes in skeletal muscle and regulate interorgan crosstalk are only partly understood. Lactate is widely described as a signaling molecule in different cells, but the role of lactate as a signaling molecule in mature skeletal muscle has not been fully unveiled. The aim of this study was to determine the role of lactate on activation of signaling pathways in adult mouse skeletal muscle. Male mice were injected intraperitoneally with lactate or saline, and tissues were dissected after 40 min. Phosphorylation levels of relevant proteins in muscle were assessed by Western blotting. After lactate administration, we found an increase in p‐ERK1/2Thr202/Tyr204 (3.5‐fold; P = 0.004) and p‐p70S6KT hr389 (1.9‐fold; P = 0.01) in quadriceps; and an increase in p‐rpS6Ser235/236 in both quadriceps (6.3‐fold; P = 0.01) and EDL (2.3‐fold; P = 0.01), without changes in soleus. There was a tendency toward an increase in p‐AMPKT hr172 (1.7‐fold; P = 0.08), with a significant increase in p‐ACCS er79 (1.5‐fold; P = 0.04) in soleus, without changes in quadriceps and EDL. These results support the hypothesis that lactate plays a role in the molecular signaling related to hypertrophy and to oxidative metabolism on adult skeletal muscle and suggest that this activation depends on the skeletal muscle type. The mechanisms that underlie the effect of lactate in mature skeletal muscles remain to be established.

Critical power is positively related to skeletal muscle capillarity and type I muscle fibers in endurance-trained individuals.

The asymptote [critical power (CP)] and curvature constant ( W') of the hyperbolic power-duration relationship can predict performance within the severe-intensity exercise domain. However, the extent to which these parameters relate to skeletal muscle morphology is less clear, particularly in endurance-trained individuals, who, relative to their lesser-trained counterparts, possess skeletal muscles that can support high levels of oxygen transport and oxidative capacity, i.e., elevated type I fiber proportion and cross-sectional area (CSA) and capillarity. Fourteen endurance-trained men performed a maximal incremental test to determine peak oxygen uptake (V̇o2peak; 63.2 ± 4.1 ml·min-1·kg-1, mean ± SD) and maximal aerobic power (406 ± 63 W) and three to five constant-load tests to task failure for the determination of CP (303 ± 52 W) and W' (17.0 ± 3.0 kJ). Skeletal muscle biopsies were obtained from the vastus lateralis and analyzed for percent proportion of fiber types, CSA, and indexes of capillarity. CP was positively correlated with the percent proportion ( r = 0.79; P = 0.001) and CSA ( r = 0.73; P = 0.003) of type I fibers, capillary-to-fiber ratio ( r = 0.88; P < 0.001), and capillary contacts around type I fibers ( r = 0.94; P < 0.001) and type II fibers ( r = 0.68; P = 0.008). W' was not correlated with any morphological variables. These data reveal a strong positive association between CP and skeletal muscle capillarity. Our findings support the assertion that CP is an important parameter of aerobic function and offer novel insights into the physiological bases of CP. NEW & NOTEWORTHY This investigation demonstrated very strong positive correlations between critical power and skeletal muscle capillarity, particularly around type I fibers, and type I fiber composition. These correlations were demonstrated in endurance-trained individuals expected to possess well-adapted skeletal muscles, such as high levels of oxygen transport structures and high oxidative capacities, supporting the view that critical power is an important parameter of aerobic function. In contrast, the curvature constant W' was not associated with fiber type composition or capillarity.

Increased Ratio of Non-mercaptalbumin-1 Among Total Plasma Albumin Demonstrates Potential Protein Undernutrition in Adult Rats.

The redox state of plasma albumin shifts in response to dietary protein intake in growing rats, and the shift is more sensitive than that of plasma albumin level, a classical marker of protein nutritional status. While it has been suggested that plasma albumin redox state could be useful as a novel marker of protein nutritional status, the above animal model is highly sensitive to dietary protein intake and the observation may not be extrapolated widely to humans. This study aimed to investigate whether albumin redox state also reflects protein nutritional status in adult rats, which have a lower dietary protein requirement and are less responsive to protein intake. Male adult rats were placed on AIN-93M diet (14% casein), or AIN-93M-based low protein diets (10 or 5% casein) ad libitum for 24 weeks. Whereas there was no significant difference in body weight between the groups at the end of the experimental period, the 5% casein diet group had the smallest gastrocnemius muscle weight among the groups, which was significantly lower than that of the 10% casein diet group. Plasma albumin level was also lower in the 5% casein diet group compared with the other groups, but the differences were limited and inconsistent during the experimental period. Among the albumin redox isoforms such as mercaptalbumin, non-mercaptalbumin-1, and non-mercaptalbumin-2, the ratio of non-mercaptalbumin-1 among total albumin was significantly higher in the 5% casein diet group, and the increase remained constant throughout the experimental period. Increased non-mercaptalbumin-1 ratio would thus demonstrate the presence of potential protein undernutrition in adult rats, as manifested only by a decreased gain in a specific type of skeletal muscle; non-mercaptalbumin-1 among total albumin ratio could be useful as a robust marker of protein nutritional status, contributing to prevention of protein undernutrition-related diseases such as frailty and sarcopenia.

High-intensity interval, but not endurance, training induces muscle fiber type-specific subsarcolemmal lipid droplet size reduction in type 2 diabetic patients.

This study compared the effects of moderate-intensity endurance training and high-intensity interval training on fiber type-specific subcellular volumetric content and morphology of lipid droplets and mitochondria in skeletal muscles of type 2 diabetic patients. Sixteen sedentary type 2 diabetic patients (57 ± 7 yr old) were randomized to complete 11 wk of either 40-min cycling at 50% peak workload (Endurance, n = 8) or 10 1-min cycling intervals at 95% peak workload separated by 1 min of recovery (High-Intensity Interval, n = 8), three times per week. Assessments for cardiorespiratory fitness, body composition, glycemic control, together with muscle biopsies were performed before and after the intervention. Morphometric analyses of lipid droplets and mitochondria were conducted in the subcellular fractions of biopsied muscle fibers using quantitative electron microscopy. The training intervention increased cardiorespiratory fitness, lowered fat mass, and improved nonfasting glycemic control ( P < 0.05), with no difference between training modalities. In the subsarcolemmal space, training decreased lipid droplet volume ( P = 0.003), and high-intensity interval, but not endurance, training reduced the size of lipid droplets, specifically in type 2 fibers ( P < 0.001). No training-induced change in intermyofibrillar lipid droplets was observed in both fiber types. Subsarcolemmal mitochondrial volume was increased by high-intensity interval ( P = 0.02), but not endurance, training ( P = 0.79). Along with improvement in glycemic control, low-volume high-intensity interval training is an alternative time-saving training modality that affects subcellular morphology and volumetric content of lipid droplets in skeletal muscle of type 2 diabetic patients.

Long-Term Insulin Treatment Alters Muscle-Related Protein in OLETF Rat Skeletal Muscle

Purpose: The objective of this study was to investigate molecular and physiological changes in response to long-term insulin glargine treatment in the skeletal muscle of Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Methods: Protein changes in the skeletal muscle of type 2 diabetic rats was detected by Western blot analysis, and muscle fiber type composition was assessed by real-time PCR. Results: Insulin glargine (1 U/kg) treatment in OLETF rats (OLETF-G) for 24 weeks were changed in blood glucose levels in intraperitoneal glucose tolerance tests compared with age-matched, untreated OLETF rats (OLETF-C), and the area under the curve was significantly decreased for OLETF-G rats compared with OLETF-C rats (P &amp;lt; 0.05). The protein levels of myosin heavy chain (MHC) isoforms were altered in gastrocnemius muscle of OLETF rats, and the proportions of MHC type I and II fibers were lower and higher, respectively, in OLETF-G compared with OLETF-C rats. Activation of myokines (interleukin [IL]-6, IL-15, fibronectin type III domain containing 5 [FNDC5]/irisin, and myostatin) in gastrocnemius muscle was significantly inhibited in OLETF-G compared with OLETF-C rats (P &amp;lt; 0.05). Moreover, myogenin determination gene D and myogenin levels were decreased, while insulin-like growth factor-I levels were increased, in the skeletal muscle of OLETF-G rats (P &amp;lt; 0.05). Insulin glargine treatment significantly increased the phosphorylation levels of 5’-AMP-activated protein kinase, sirtuin 1, and peroxisome proliferator-activated receptor γ coactivator 1α. Conclusions: Our results suggest that long-term insulin glargine treatment can effectively alter protein expression in the gastrocnemius muscle of OLETF rats. Disclosure H. Kwon: None. O. Hong: None. H. Kwon: None. Y. Kang: None. S. Lee: None. S. Kim: None. S. Yoo: None.

Increased AQP7 abundance in skeletal muscle from obese men with type 2 diabetes.

Aquaglyceroporin 7 (AQP7) facilitates the transport of glycerol across cell membranes. In mice, fasting and refeeding regulate adipose tissue AQP7 abundance, and a role in controlling triglyceride accumulation in adipose tissue has been proposed. AQP7 is also expressed in skeletal muscle, where its function remains to be determined. Here, the abundance of AQP7 in abdominal subcutaneous adipose tissue (SAT) and skeletal muscle was evaluated in the overnight fasted and postprandial state in eight lean and eight obese men with type 2 diabetes (T2D). A biopsy from SAT and muscle was collected after an overnight fast and 2 h after ingestion of a low-fat test meal. Palmitate turnover was evaluated using a [9,10-3H] palmitate dilution technique. Tissue samples were analyzed by immunoblotting. Meal intake did not affect AQP7 expression in SAT or skeletal muscle. No association between the SAT AQP7 abundance and palmitate turnover was found. SAT AQP7 abundance was similar in lean and obese T2D men, whereas muscle AQP7 abundance was more than fourfold higher in obese T2D men. In conclusion, meal intake did not affect AQP7 protein abundance in SAT or skeletal muscle. In addition, SAT AQP7 expression does not appear to be involved in the regulation of adipose tissue lipolysis. However, in contrast to SAT AQP7, skeletal muscle AQP7 protein abundance is markedly increased in obese T2D men, potentially contributing to the excess lipid accumulation in skeletal muscle in type 2 diabetes.

Expression of Pannexin 1 and Pannexin 3 during skeletal muscle development, regeneration, and Duchenne muscular dystrophy.

Pannexin 1 (Panx1) and Pannexin 3 (Panx3) are single membrane channels recently implicated in myogenic commitment, as well as myoblast proliferation and differentiation in vitro. However, their expression patterns during skeletal muscle development and regeneration had yet to be investigated. Here, we show that Panx1 levels increase during skeletal muscle development becoming highly expressed together with Panx3 in adult skeletal muscle. In adult mice, Panx1 and Panx3 were differentially expressed in fast- and slow-twitch muscles. We also report that Panx1/PANX1 and Panx3/PANX3 are co-expressed in mouse and human satellite cells, which play crucial roles in skeletal muscle regeneration. Interestingly, Panx1 and Panx3 levels were modulated in muscle degeneration/regeneration, similar to the pattern seen during skeletal muscle development. As Duchenne muscular dystrophy is characterized by skeletal muscle degeneration and impaired regeneration, we next used mild and severe mouse models of this disease and found a significant dysregulation of Panx1 and Panx3 levels in dystrophic skeletal muscles. Together, our results are the first demonstration that Panx1 and Panx3 are differentially expressed amongst skeletal muscle types with their levels being highly modulated during skeletal muscle development, regeneration, and dystrophy. These findings suggest that Panx1 and Panx3 channels may play important and distinct roles in healthy and diseased skeletal muscles.

MicroRNA‑29a is involved lipid metabolism dysfunction and insulin resistance in C2C12 myotubes by targeting PPARδ.

MicroRNA‑29a (miR‑29a) expression has been reported to be closely associated with skeletal muscle insulin resistance and type 2 diabetes. The present study investigated the effect of miR‑29a on palmitic acid (PA)‑induced lipid metabolism dysfunction and insulin resistance in C2C12 myotubes via overexpressing or silencing of miR‑29a expression. Mouse C2C12 myoblasts were cultured, differentiated and transfected with miR‑29a or miR‑29a inhibitor lentiviral with or without subsequent palmitic acid (PA) treatment. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis were performed to assess the mRNA and protein levels of related genes, respectively. PA treatment increased the expression of miR‑29a in a time‑ and dose‑ dependent manner. miR‑29a silencing improved insulin‑induced glucose uptake and increased glucose transporter‑4 (GLUT4) transportation to the plasma membrane by upregulating its target peroxisome proliferator‑activated receptor δ (PPARδ). Furthermore, it was observed that miR‑29a regulated the expression of genes associated with lipid metabolism, including pyruvate dehydrogenase kinase isoform, mitochondrial uncoupling protein (UCP)2, UCP3, long chain specific acyl‑CoA dehydrogenase, mitochondrial and fatty acid transport protein 2. The results confirmed that silencing miR‑29a induced a decrease in glucose transport and affected lipid metabolism in PA‑treated C2C12 cells, and therefore may be involved in insulin resistance by targeting PPARδ in skeletal muscle. Therefore, the inhibition of miR‑29a may be a potential novel strategy for treating insulin resistance and type 2 diabetes.

Efficient High-Throughput Screening by Endoplasmic Reticulum Ca2+ Measurement to Identify Inhibitors of Ryanodine Receptor Ca2+-Release Channels.

Genetic mutations in ryanodine receptors (RyRs), Ca2+-release channels in the sarcoplasmic reticulum essential for muscle contractions, cause various skeletal muscle and cardiac diseases. Because the main underlying mechanism of the pathogenesis is overactive Ca2+ release by gain-of-function of the RyR channel, inhibition of RyRs is expected to be a promising treatment of these diseases. Here, to identify inhibitors specific to skeletal muscle type 1 RyR (RyR1), we developed a novel high-throughput screening (HTS) platform using time-lapse fluorescence measurement of Ca2+ concentrations in the endoplasmic reticulum (ER) ([Ca2+]ER). Because expression of RyR1 carrying disease-associated mutation reduces [Ca2+]ER in HEK293 cells through Ca2+ leakage from RyR1 channels, specific drugs that inhibit RyR1 will increase [Ca2+]ER by preventing such Ca2+ leakage. RyR1 carrying the R2163C mutation and R-CEPIA1er, a genetically encoded ER Ca2+ indicator, were stably expressed in HEK293 cells, and time-lapse fluorescence was measured using a fluorometer. False positives were effectively excluded by using cells expressing wild-type (WT) RyR1. By screening 1535 compounds in a library of well characterized drugs, we successfully identified four compounds that significantly increased [Ca2+]ER They include dantrolene, a known RyR1 inhibitor, and three structurally different compounds: oxolinic acid, 9-aminoacridine, and alexidine. All the hit compounds, except for oxolinic acid, inhibited [3H]ryanodine binding of WT and mutant RyR1. Interestingly, they showed different dose dependencies and isoform specificities. The highly quantitative nature and good correlation with the channel activity validated this HTS platform by [Ca2+]ER measurement to explore drugs for RyR-related diseases.

Patterns of Suppressed Mitochondrial Respiration in Isolated Muscle Fibers from Type 2 Diabetics

The quality of skeletal muscle is markedly influenced by mitochondrial function. Aberrant mitochondrial function has been associated with reduced contractile and metabolic dysfunction in skeletal muscle, potentially leading to insulin resistance and type 2 diabetes. However, the direct relationship between mitochondrial dysfunction and type 2 diabetes remains to be fully understood. In the present study, vastus lateralis muscle biopsies were obtained from individuals with type 2 diabetes (N = 7) and non‐diabetic, age, gender and weight‐matched controls (N = 7). Maximal mitochondrial oxygen consumption was assessed in isolated muscle fibers and normalized to wet tissue weight. Preliminary findings illustrate that reductions in carbohydratedependent ADP‐stimulated respiration (~30%) were sustained in muscle fibers from type 2 diabetics, even with the addition of the Krebs Cycle anaplerotic substrates, glutamate (~30%) and succinate (~32%). Interestingly, trends of similar magnitudes persisted for lipid substrate metabolism, as fatty acid‐dependent ADP‐stimulated respiration also tended to be lower (~35%) compared to controls. Furthermore, rotenone‐treated respiration in diabetic skeletal muscle tended to be reduced (~37%). To conclude, these initial findings provide support for a systematic impairment in the oxidative metabolism of both carbohydrates and lipids in skeletal muscle of type 2 diabetic individuals. In addition, findings from rotenone‐treated conditions indicate that investigating functional deficits of Complex I of the Electron Transport Chain (ETC) may provide a more insightful understanding of the relationship between mitochondrial respiratory control and type 2 diabetes.Support or Funding Information*This study is supported by funds from the American Diabetic Association, ADA‐1‐16‐ICIS‐015, (D.P.C.) and start‐up funds from the Department of Health and Human Physiology at the University of Iowa (V.A.L.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.