Skeletal Muscle Myocytes Research Articles

Mitochondrial Turnover Regulates Ischemic Endothelial Angiogenic Capacity in Peripheral Artery Disease

Background: Atherosclerotic occlusions in lower limbs cause ischemia that results in Peripheral artery disease. Despite functional mitochondria, normal endothelial cells use Glycolysis as the primary metabolic pathway for cellular maintenance. While mitochondrial anomalies are extensively studied in skeletal muscle myocytes in PAD, the role of mitochondria in regulating ischemic endothelial (EC) function is largely unknown. Rationale: Based on a recent study that showed Mitochondrial OxPhos is required for EC migration, we aimed to understand the role of EC Mitochondrial OxPhos in regulating ischemic EC angiogenic capacity in PAD. Methods: To study the role of EC-Mitochondrial OxPhos, we developed an EC-specific (using Cdh5-Cre) mitochondrial Transcription factor-A gene (TFAM, shown to be a critical regulator of mitochondrial OxPhos) deficient mice. Since complete deletion of TFAM in ECs resulted in embryonic lethality, male and female EC-TFAM+/− mice were used to study the role of mitochondrial metabolism in regulating EC angiogenic capacity and perfusion recovery in preclinical PAD model (hind limb ischemia (HLI) by femoral artery ligation and resection). Results: HLI significantly decreased perfusion recovery and significantly increased necrosis in both male and female EC-TFAM+/− mice compared to WT controls. Consistent with impaired perfusion recovery, microvascular density was also significantly decreased in EC-TFAM+/− mice vs. WT controls. To understand the mechanism by which EC-TFAM deficiency results in impaired perfusion recovery, we isolated ECs from EC-TFAM+/− and WT mice ischemic muscle at day-7 post HLI, and performed bulk RNA-Seq. GeneOntology Pathway analysis of RNA Seq data showed that Autophagy/mitophagy and OxPhos as the top 2 pathways that were significantly inhibited in EC-TFAM+/− ischemic ECs vs. WT ischemic ECs. Hypothesizing 'accumulation of non-functional mitochondria due to impaired mitophagy inhibits the angiogenic capacity of ischemic ECs in PAD' we systematically examined the expression of key mitophagy gene expression in EC-TFAM+/− ischemic ECs vs. WT ischemic ECs. This mitophagy gene screening showed a significant decrease in Parkin expression in EC-TFAM+/− ischemic ECs vs. WT ischemic ECs. Despite higher mitochondrial DNA content in hypoxia serum starved (HSS, in vitro model for PAD), TFAM+/− ECs vs. WT, seahorse Mitostress assays showed a significant decrease in the OxPhos of HSS-TFAM+/− ECs vs. WT. Accordingly, flow cytometry analysis showed a significant decrease in mitochondrial membrane potential (using Mito-ID) in HSS-TFAM+/− ECs vs. WT. Loss of mitochondrial membrane potential and OxPhos in HSS-TFAM+/− ECs resulted in a significant loss of HSS-TFAM+/− EC angiogenic capacity on growth factor reduced matrigel compared to HSS-WT ECs. Conclusion: These data presented that 'impaired mitophagy results in the accumulation of non-functional mitochondria that inhibits the angiogenic capacity of ischemic ECs resulting in impaired perfusion recovery in PAD. Ongoing experiments are aimed at understanding the causal role of Parkin-Mitophagy in regulating ischemic EC angiogenic capacity and perfusion recovery in PAD. R01HL146673-02. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

In vivo dynamics of hydrogen peroxide and regulation of antioxidant gene expression in mouse skeletal muscle under cooling stimulus

Introduction: The dynamics of reactive oxygen species (ROS) in the cytosol and mitochondria function as signaling factors that regulates diverse cellular adaptations. Hydrogen peroxide (H2O2) is considered one of the most influential ROS in regulating physiological function; being a ubiquitous molecule with a long half-life and high diffusing capacity compared to other ROS (e.g., superoxide and hydroxyl radicals). Temperature changes within skeletal muscle myocytes constitute an environmental factor that modulates the expression of antioxidant-related genes. Although muscle temperature reduction by icing is a typical physical therapy modality, the relationship between cooling stimuli and in vivo H2O2 dynamics remains unclear. The purpose of this investigation was to elucidate the H2O2 dynamics in the cytosol and mitochondria induced by cooling stimuli and to determine the relationship between these dynamics and the expression of antioxidant-related genes. Specifically, we tested the hypothesis that cooling would reduce [H2O2] in intracellular compartments (cytosol and mitochondria) and suppress antioxidant-related gene expression levels. Methods: Experiment 1. in vivo H2O2 dynamics observation: Cytosolic and mitochondrial H2O2-sensitive fluorescent proteins (HyPer7, n = 6 and MLS-HyPer7, n = 9) were expressed by electroporation in the anterior tibialis muscle (TA) of male C57/BL6 mice (9-14 week of age). The TA was exposed under anesthesia, and cytosolic and mitochondrial [H2O2] were measured when the muscle temperature was reduced from 35 °C to 0 °C using in vivo bioimaging techniques. Experiment 2. Mice (9-12 week of age) were divided into an icing (ICE) group that received cooling stimulation (muscle temperature reduced from 35 °C to 13 °C and back to 35°C, total 6 sets for 60 min, n = 6) and a control (CONT) group (muscle temperature 35 °C for 60 min, n = 6). At 3 hours after temperature intervention, the TA was removed and mRNA levels were measured by Real-time PCR. Results: Experiment 1. In vivo [H2O2] in each compartment were significantly lower in the cytosol (over the range 28-6°C) and mitochondria (over the range 29-1°C) compared to 35 °C conditions. A decreasing phase of [H2O2] in both compartments was observed from 35 °C to around 13 °C, but, subsequently increasing [H2O2] were observed in the lower temperature range (i.e., 12 to 0 °C). Experiment 2: The ICE group significantly increased mRNA levels of antioxidant enzyme-related genes Nfe2l2 and HSF1 compared to CONT (1.2 ± 0.1-fold and 1.1 ± 0.1-fold, respectively, P < 0.01). On the other hand, mRNA levels of mitochondria-related genes such as PGC1α and COX4 were unchanged. Conclusions: This investigation is the first to reveal the H2O2 and related gene expression impact of cooling stimuli in an in vivo model. Cooling mouse skeletal muscle produced temperature-dependent decreases in [H2O2] that reversed with more severe cooling. In both cytosolic and mitochondrial compartments, the lowest point for [H2O2] was found to be 13°C. Repeated cooling cycles (between 35 and 13 °C) with their attendant decreasing [H2O2] elevated antioxidant-related mRNA levels. These results support that icing therapy may improve the antioxidant capacity of muscle despite decreasing muscle ROS levels. This study was supported in part by a Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) for Grant-in-Aid for JSPS Fellows (no. JP23KJ0967) and JSPS KAKENHI Grant (No. JP20H04074). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exercise performance is not improved in mice with skeletal muscle deletion of natriuretic peptide clearance receptor.

Natriuretic peptides (NP), including atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP), play essential roles in regulating blood pressure, cardiovascular homeostasis, and systemic metabolism. One of the major metabolic effects of NP is manifested by their capacity to stimulate lipolysis and the thermogenesis gene program in adipocytes, however, in skeletal muscle their effects on metabolism and muscle function are not as well understood. There are three NP receptors (NPR): NPRA, NPRB, and NPRC, and all three NPR genes are expressed in skeletal muscle and C2C12 myocytes. In C2C12 myocytes treatment with either ANP, BNP, or CNP evokes the cGMP signaling pathway. Since NPRC functions as a clearance receptor and the amount of NPRC in a cell type determines the signaling strength of NPs, we generated a genetic model with Nprc gene deletion in skeletal muscle and tested whether enhancing NP signaling by preventing its clearance in skeletal muscle would improve exercise performance in mice. Under sedentary conditions, Nprc skeletal muscle knockout (MKO) mice showed comparable exercise performance to their floxed littermates in terms of maximal running velocity and total endurance running time. Eight weeks of voluntary running-wheel training in a young cohort significantly increased exercise performance, but no significant differences were observed in MKO compared with floxed control mice. Furthermore, 6-weeks of treadmill training in a relatively aged cohort also increased exercise performance compared with their baseline values, but again there were no differences between genotypes. In summary, our study suggests that NP signaling is potentially important in skeletal myocytes but its function in skeletal muscle in vivo needs to be further studied in additional physiological conditions or with new genetic mouse models.

Impaired Cardiac and Skeletal Muscle Energetics Following Anthracycline Therapy for Breast Cancer.

Anthracycline-related cardiac toxicity is a recognized consequence of cancer therapies. We assess resting cardiac and skeletal muscle energetics and myocyte, sarcomere, and mitochondrial integrity in patients with breast cancer receiving epirubicin. In a prospective, mechanistic, observational, longitudinal study, we investigated chemotherapy-naive patients with breast cancer receiving epirubicin versus sex- and age-matched healthy controls. Resting energetic status of cardiac and skeletal muscle (phosphocreatine/gamma ATP and inorganic phosphate [Pi]/phosphocreatine, respectively) was assessed with 31P-magnetic resonance spectroscopy. Cardiac function and tissue characterization (magnetic resonance imaging and 2D-echocardiography), cardiac biomarkers (serum NT-pro-BNP and high-sensitivity troponin I), and structural assessments of skeletal muscle biopsies were obtained. All study assessments were performed before and after chemotherapy. Twenty-five female patients with breast cancer (median age, 53 years) received a mean epirubicin dose of 304 mg/m2, and 25 age/sex-matched controls were recruited. Despite comparable baseline cardiac and skeletal muscle energetics with the healthy controls, after chemotherapy, patients with breast cancer showed a reduction in cardiac phosphocreatine/gamma ATP ratio (2.0±0.7 versus 1.1±0.5; P=0.001) and an increase in skeletal muscle Pi/phosphocreatine ratio (0.1±0.1 versus 0.2±0.1; P=0.022). This occurred in the context of increases in left ventricular end-systolic and end-diastolic volumes (P=0.009 and P=0.008, respectively), T1 and T2 mapping (P=0.001 and P=0.028, respectively) but with preserved left ventricular ejection fraction, mass and global longitudinal strain, and no change in cardiac biomarkers. There was preservation of the mitochondrial copy number in skeletal muscle biopsies but a significant increase in areas of skeletal muscle degradation (P=0.001) in patients with breast cancer following chemotherapy. Patients with breast cancer demonstrated a reduction in skeletal muscle sarcomere number from the prechemotherapy stage compared with healthy controls (P=0.013). Contemporary doses of epirubicin for breast cancer treatment result in a significant reduction of cardiac and skeletal muscle high-energy 31P-metabolism alongside structural skeletal muscle changes. URL: https://www. gov; Unique identifier: NCT04467411.

Plasma proteins as potential biomarkers of aging of single tissue and cell type.

Plasma proteins serve as biomarkers of aging and various age-related diseases. While a number of plasma proteins have been identified that increase or decrease with age, the interpretation of each protein is challenging. This is due to the nature of plasma, which is a mixture of factors secreted by many different tissues and cells. Therefore, the catalog of age-related proteins secreted by a single cell type in a single tissue would be useful for understanding tissue-specific aging patterns. In this study, the author addressed this challenge by integrative data mining of the Human Protein Atlas and the recently published result of large-scale aging proteomics research. Finally, we identified the 17 age-related proteins produced by a single tissue and a single cell type: MBL2 and HP in the liver (hepatocytes), SFTPC in the lung (type II alveolar cells), PRL and POMC in the pituitary (anterior cells), GCG, CUZD1 and CPA2 in the pancreas (pancreatic cells), MYBPC1 in skeletal muscle (myocytes), PTH in the parathyroid gland (glandular cells), LPO and AMY1A in the salivary gland (glandular cells), INSL3 in the male testis (Leydig cells), KLK3 and KLK4 in the male prostate (glandular cells), MPO and ACP5 in immune cells. This list of proteins would be potentially useful for understanding age-related changes in the plasma proteome and inter-tissue networks.

The prevalence and associated factors of myocardial involvement in Duchenne muscular dystrophy patients in the first decade of life.

The prevalence and associated factors of myocardial involvement in Duchenne muscular dystrophy patients in the first decade of life.

Differentiation of patient-specific void urine-derived human induced pluripotent stem cells to fibroblasts and skeletal muscle myocytes

Cell-based therapy is a major focus for treatment of stress urinary incontinence (SUI). However, derivation of primary cells requires tissue biopsies, which often have adverse effects on patients. A recent study used human induced pluripotent stem cells (iPSC)-derived smooth muscle myocytes for urethral sphincter regeneration in rats. Here, we establish a workflow using iPSC-derived fibroblasts and skeletal myocytes for urethral tissue regeneration: (1) Cells from voided urine of women were reprogrammed into iPSC. (2) The iPSC line U1 and hESC line H9 (control) were differentiated into fibroblasts expressing FSP1, TE7, vinculin, vimentin, αSMA, fibronectin and paxillin. (3) Myogenic differentiation of U1 and H9 was induced by small molecule CHIR99021 and confirmed by protein expression of myogenic factors PAX7, MYOD, MYOG, and MF20. Striated muscle cells enriched by FACS expressed NCAM1, TITIN, DESMIN, TNNT3. (4) Human iPSC-derived fibroblasts and myocytes were engrafted into the periurethral region of RNU rats. Injected cells were labelled with ferric nanoparticles and traced by Prussian Blue stain, human-specific nuclear protein KU80, and human anti-mitochondria antibody. This workflow allows the scalable derivation, culture, and in vivo tracing of patient-specific fibroblasts and myocytes, which can be assessed in rat SUI models to regenerate urethral damages and restore continence.

Nuclear mechanosignaling in striated muscle diseases.

Mechanosignaling describes processes by which biomechanical stimuli are transduced into cellular responses. External biophysical forces can be transmitted via structural protein networks that span from the cellular membrane to the cytoskeleton and the nucleus, where they can regulate gene expression through a series of biomechanical and/or biochemical mechanosensitive mechanisms, including chromatin remodeling, translocation of transcriptional regulators, and epigenetic factors. Striated muscle cells, including cardiac and skeletal muscle myocytes, utilize these nuclear mechanosignaling mechanisms to respond to changes in their intracellular and extracellular mechanical environment and mediate gene expression and cell remodeling. In this brief review, we highlight and discuss recent experimental work focused on the pathway of biomechanical stimulus propagation at the nucleus-cytoskeleton interface of striated muscles, and the mechanisms by which these pathways regulate gene regulation, muscle structure, and function. Furthermore, we discuss nuclear protein mutations that affect mechanosignaling function in human and animal models of cardiomyopathy. Furthermore, current open questions and future challenges in investigating striated muscle nuclear mechanosignaling are further discussed.

Differentiation of Myoblasts in Culture: Focus on Serum and Gamma-Aminobutyric Acid

There are many facts about the possible role of gamma-aminobutyric acid (GABA) in the development and differentiation of cells not only in nervous but also in muscle tissue. In the present study, a primary culture of rat skeletal muscle myocytes was used to evaluate the correlation between the content of GABA in the cytoplasm and the processes of myocyte division and their fusion into myotubes. The effect of exogenous GABA on the processes of culture development was also estimated. Since the classical protocol for working with myocyte cultures involves the use of fetal bovine serum (FBS) to stimulate cell division (growth medium) and horse serum (HS) to activate the differentiation process (differentiation medium), the studies were carried out both in the medium with FBS and with HS. It was found that cells grown in medium supplemented with FBS contain more GABA compared to cultures growing in medium supplemented with HS. Addition of exogeneous GABA leads to a decrease in the number of myotubes formed in both media, while the addition of an amino acid to the medium supplemented with HS had a more pronounced inhibitory effect. Thus, we have obtained data indicating that GABA is able to participate in the early stages of skeletal muscle myogenesis by modulating the fusion process.

Extracellular Vesicles as Communicators of Senescence in Musculoskeletal Aging.

Extracellular vesicles (EVs), including exosomes and microvesicles, are released by numerous cell types. EVs are now acknowledged as playing a critical role in cell-cell communication in healthy aging as well as in age-related diseases. Recently it was shown that senescence, a key hallmark of aging, increases the secretion of EVs. Moreover, EVs can transport proteins and microRNAs (miRNAs) that are key components of the senescence-associated secretory phenotype (SASP). Here we review evidence that SASP-related miRNAs are involved in musculoskeletal degeneration with aging. Specifically, senescence-related miRNAs are elevated in EVs released by skeletal muscle myocytes and fibro-adipogenic progenitor cells with aging and disuse atrophy, respectively. Many of these same senescence-related miRNAs are detected in EVs from the synovial fluid of patients with osteoarthritis, and these miRNAs can contribute to cartilage degeneration. Finally, senescence-associated miRNAs are secreted from bone marrow-derived stem (stromal) cells impacting neighboring hematopoietic stem cells and circulating in the blood. The senescence-associated miRNA mir-34a, which is known to target Wnt and Notch pathways as well as the cell survival factors Sirt1 and Bcl2, is detected in EVs from human and animal subjects with muscle atrophy, bone loss, and osteoarthritis. These findings suggest that suppressing the secretion of EV-derived, senescence-related miRNAs, such as miR-34a, or increasing levels of competing endogenous long noncoding RNAs, such as MALAT1 that inhibit miR-34a, may help to improve musculoskeletal function with aging. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Quercetin ameliorated insulin resistance via regulating METTL3-mediated N6-methyladenosine modification of PRKD2 mRNA in skeletal muscle and C2C12 myocyte cell line

Background and aimsN6-Methyladenosine (m6A) modification is involved in many pathological processes, including insulin resistance (IR). Quercetin (Que), a bioactive compound with strong antioxidant activity, has potential therapeutic effects on IR-related metabolic diseases. The aim of this study is to investigate the roles of m6A and Que in hyperinsulinemia. Methods and resultsMale C57Bl/6 mice received a high-fat diet (HFD) for 8 weeks to establish an IR model. Que treatment reduced the body weight, blood glucose, plasma triglycerides (TG) and serum insulin, ameliorated IR, and decreased oxidative stress in HFD-fed mice. Cellular IR model was established in C2C12 cells by palmitic acid (PA) stimulation, and a noncytotoxic dose of Que was found to promote glucose uptake and inhibit oxidative stress. Moreover, methyltransferase-like 3 (METTL3) and serine–threonine kinase protein kinase D2 (PRKD2) was downregulated in skeletal muscle of HFD-fed mouse and in PA-induced C2C12 cells. The online bioinformatic tool SRAMP revealed that there were multiple m6A modification sites in the PRKD2 mRNA sequence. Downregulation of METTL3 enhanced PRKD2 expression by reducing m6A level and promoting mRNA stability in PRKD2 mRNA transcript. Que decreased m6A, METTL3, and phosphorylated insulin receptor substrate 1 (p-IRS1) levels, increased the protein expression of PRKD2, glucose transporter type 4 (GLUT4) and p-AKT, promoted glucose uptake, and reduced oxidative stress in PA-induced C2C12 cells. Moreover, METTL3 overexpression or PRKD2 silence reversed the inhibitory effects of Que on the levels of MDA and p-IRS1 and the promotive effects on glucose uptake, superoxide dismutase (SOD), GSH and GLUT4 and p-AKT levels. ConclusionQue promoted glucose uptake, repressed oxidative stress and improved IR through METTL3-mediated m6A of PRKD2 mRNA.

Skeletal Muscle Myofibers Directly Contribute to LPS-Induced Systemic Inflammatory Tone.

The abundance, anatomical distribution, and vascularity of skeletal muscle make it a potentially important contributor to local cytokine production and systemic cytokine abundance during inflammatory events. An orchestrated balance between the production of pro- and anti-inflammatory mediators is necessary for proper immune function, yet the contribution of the body’s largest organ system, comprised primarily of skeletal muscle myocytes that fuse to form myofibers, to this process is largely unknown. Endotoxin (lipopolysaccharide, LPS) stimulates toll-like receptor 4 (TLR4) to induce the production of several pro-inflammatory cytokines, including interleukin-6 (IL-6) and C-C motif chemokine ligand 2 (CCL2), by a of myriad cell types. We sought to quantify the influence of myofibers on systemic cytokine concentrations following an acute endotoxemia challenge. To accomplish this, we generated muscle specific conditional knockouts for TLR4 (TLR4SMKO), IL-6 (IL6SMKO), and CCL2 (CCL2SMKO). We administered low concentrations of intravenous LPS (IV LPS) to these receptor and effector knockout mice and collected samples after 3 h. Using gene expression analysis of gastrocnemius muscle and serum cytokine measurements after IV LPS, we determined that deletion of myofiber IL-6 or CCL2 led to a 93% and 57% reduction of these specific cytokines in the systemic circulation, respectively. Myofiber specific TLR4 deletion decreased the expression of IL-6, CCL2, and C-X-C motif chemokine ligand 1 (CXCL1) in the gastrocnemius muscle. These data indicate the critical involvement and direct contribution of myofibers during the early systemic inflammatory cytokine response to endotoxin.

Effects of moderate aerobic exercise, low-level laser therapy, or their combination on muscles pathology, oxidative stress and irisin levels in the mdx mouse model of Duchenne muscular dystrophy.

This study aimed to investigate how the combined use of low-level laser therapy (LLLT) and exercise, to reduce the possible side effects and/or increase the benefits of exercise, would affect oxidative stress, utrophin, irisin peptide, and skeletal, diaphragmatic, and cardiac muscle pathologies. In our study, 20 mdx mice were divided into four groups. Groups; sedentary and placebo LLLT (SC), sedentary and LLLT (SL), 30-min swimming exercise (Ex), and 30-min swimming exercise and LLLT (ExL). After 8weeks of swimming exercise, muscle tests, biochemically; oxidative stress index (OSI), utrophin and irisin levels were measured. Skeletal, diaphragmatic and cardiac muscle histopathological scores, skeletal and cardiac muscle myocyte diameters were determined under the light and electron microscope. While only irisin levels were increased in group SL compared to SC, it was determined that OSI, heart muscle histopathological scores decreased and irisin levels increased in both exercise groups (p < 0.05). In addition, in the ExL group, an increase in rotarod and utrophin levels, and a decrease in muscle and diaphragm muscle histopathological scores were observed (p < 0.05). It was determined that the application of swimming exercise in the mdx mouse model increased the irisin level in the skeletal muscle, while reducing the OSI, degeneration in the heart muscle, inflammation and cardiopathy. When LLLT was applied in addition to exercise, muscle strength, skeletal muscle utrophin levels increased, and skeletal and diaphragmatic muscle degeneration and inflammation decreased. In addition, it was determined that only LLLT application increased the level of skeletal muscle irisin.

Infrared laser exposure in combination with branchedchain amino acid stimulates physiological adaptation of skeletal muscles

Laser exposure stimulates cell proliferation and tissue repair. Branched-chain amino acids (BCAA) are widely used in sports medicine as a stimulator of anabolic processes. However, there is no evidence of the effect of combined laser and BCAA application on skeletal muscle morphometric characteristics during exercise in the training process.Purpose: to study the effect of infrared laser exposure in combination with amino acid at myosatellitocytes and skeletal muscle myocytes during swimming training.Material and methods. The experiment was conducted on 30 Wistar rats: Group 1 – intact, Group 2 – dynamic control (trainings with endurance swimming), Group 3 – trainings and BCAA, Group 4 – trainings and laser exposure at hip muscles (970 nm, 1 W, 60 s), Group 5 – combined exposure to laser and BCAA. Trainings were three times a week for 6 weeks; laser session was after each training. Samples of the bipedal thigh muscle were fixed with formalin; histological sections were stained with hematoxylin-eosin. The morphometric analysis of the digital image of objects with statistical processing by Mann – Whitney method was made.Results. Laser exposure combined with BCAA during trainings increased the nuclei area and the number of myosatellites and myocytes; it also enlarged the cross section of muscle fibers which was more pronounced if to compare with isolated laser irradiation of the muscle or BCAA.Conclusions. Infrared laser exposure in combination with branched-chain amino acids effectively stimulates regeneration due to hyperplasia and hypertrophy of skeletal muscular tissue, thus providing physiological adaptation in the training process.

Fatigue in CKD: Epidemiology, Pathophysiology, and Treatment.

Fatigue is a commonly reported and debilitating symptom among patients with CKD, yet little is known about its epidemiology, pathogenesis, and treatment. Various measurement tools have been used in published studies to identify and quantify fatigue. These include several single-item measures embedded in longer questionnaires for assessing depression, quality of life, or symptom burden in patients with kidney disease. Approximately 70% of patients with CKD report fatigue, with up to 25% reporting severe symptoms. Patient-reported fatigue is associated with death, dialysis initiation, and hospitalization among individuals with CKD. The pathophysiology is multifactorial and likely includes decreased oxygen delivery and increased reliance on anaerobic metabolism, thus generating lactic acidosis in response to exertion; the effects of chronic metabolic acidosis and hyperphosphatemia on skeletal muscle myocytes; protein-energy wasting and sarcopenia; and depression. Physical activity has been shown to improve fatigue in some small but promising trials, and so should be recommended, given the additional benefits of exercise. Targeting higher hemoglobin levels with erythropoiesis-stimulating agents may improve fatigue, but potential adverse cardiovascular effects preclude their use to solely treat fatigue without the presence of another indication. Current guidelines recommend cautious individualization of hemoglobin targets for those at low cardiovascular risk who still experience fatigue or functional limitation despite a hemoglobin level of 10 g/dl. Sodium bicarbonate supplementation for the treatment of metabolic acidosis may also improve functional status. Selective serotonin reuptake inhibitors have not been consistently shown to improve fatigue in patients with kidney disease, but an ongoing trial will evaluate the effect of alternative antidepressant drug and behavioral activation therapy on fatigue in patients with CKD. Overall, more research is needed to further clarify underlying mechanisms of fatigue and identify effective, targeted treatments for patients with CKD.

Plasma membrane disruption (PMD) formation and repair in mechanosensitive tissues.

Mammalian cells employ an array of biological mechanisms to detect and respond to mechanical loading in their environment. One such mechanism is the formation of plasma membrane disruptions (PMD), which foster a molecular flux across cell membranes that promotes tissue adaptation. Repair of PMD through an orchestrated activity of molecular machinery is critical for cell survival, and the rate of PMD repair can affect downstream cellular signaling. PMD have been observed to influence the mechanical behavior of skin, alveolar, and gut epithelial cells, aortic endothelial cells, corneal keratocytes and epithelial cells, cardiac and skeletal muscle myocytes, neurons, and most recently, bone cells including osteoblasts, periodontal ligament cells, and osteocytes. PMD are therefore positioned to affect the physiological behavior of a wide range of vertebrate organ systems including skeletal and cardiac muscle, skin, eyes, the gastrointestinal tract, the vasculature, the respiratory system, and the skeleton. The purpose of this review is to describe the processes of PMD formation and repair across these mechanosensitive tissues, with a particular emphasis on comparing and contrasting repair mechanisms and downstream signaling to better understand the role of PMD in skeletal mechanobiology. The implications of PMD-related mechanisms for disease and potential therapeutic applications are also explored.

Myokines and Heart Failure: Challenging Role in Adverse Cardiac Remodeling, Myopathy, and Clinical Outcomes.

Heart failure (HF) is a global medical problem that characterizes poor prognosis and high economic burden for the health system and family of the HF patients. Although modern treatment approaches have significantly decreased a risk of the occurrence of HF among patients having predominant coronary artery disease, hypertension, and myocarditis, the mortality of known HF continues to be unacceptably high. One of the most important symptoms of HF that negatively influences tolerance to physical exercise, well-being, social adaptation, and quality of life is deep fatigue due to HF-related myopathy. Myopathy in HF is associated with weakness of the skeletal muscles, loss of myofibers, and the development of fibrosis due to microvascular inflammation, metabolic disorders, and mitochondrial dysfunction. The pivotal role in the regulation of myocardial and skeletal muscle rejuvenation, attenuation of muscle metabolic homeostasis, and protection against ischemia injury and apoptosis belongs to myokines. Myokines are defined as a wide spectrum of active molecules that are directly synthesized and released by both cardiac and skeletal muscle myocytes and regulate energy homeostasis in autocrine/paracrine manner. In addition, myokines have a large spectrum of pleiotropic capabilities that are involved in the pathogenesis of HF including cardiac remodeling, muscle atrophy, and cardiac cachexia. The aim of the narrative review is to summarize the knowledge with respect to the role of myokines in adverse cardiac remodeling, myopathy, and clinical outcomes among HF patients. Some myokines, such as myostatin, irisin, brain-derived neurotrophic factor, interleukin-15, fibroblast growth factor-21, and growth differential factor-11, being engaged in the regulation of the pathogenesis of HF-related myopathy, can be detected in peripheral blood, and the evaluation of their circulating levels can provide new insights to the course of HF and stratify patients at higher risk of poor outcomes prior to sarcopenic stage.

Angiogenic potential is reduced in skeletal muscle of aged women.

The risk of cardiovascular disease and associated skeletal muscle microvascular rarefaction is enhanced in women after menopause, yet knowledge about the angiogenic potential in ageing women is generally sparse. Aged healthy and sedentary women were found to present a markedly impaired capacity for proliferation of skeletal muscle derived microvascular endothelial cells compared to young women. Vascular endothelial growth factor (VEGF) levels in skeletal muscle myocytes and release of VEGF from myocytes tended to be lower in aged compared to young women. The aged women did not show a detectable increase in skeletal muscle capillarization with 8weeks of intense aerobic cycle training. Combined, the findings indicate that aged women have a reduced potential for capillary growth in skeletal muscle which, with ageing, may lead to age-induced microvascular rarefaction. Skeletal muscle angiogenic potential was examined in cell cultures derived from aged and young women, and the effect of 8weeks of intense cycle training on muscle capillary growth was determined in the group of aged women. Basal muscle samples were obtained from healthy sedentary aged (n=12; 64±4.2years) and young women (n=5; 24±3.2years) for endothelial cell and skeletal muscle myocyte isolation and experiments. In addition, the aged women completed an 8-week training intervention. Peak oxygen uptake and muscle samples for histology and protein determination were obtained before and after the training period. Before training, muscle microdialysate was collected from the aged women at rest and during exercise. In Part 1 of the experiments, growth-supplement stimulated proliferation of endothelial cells was ∼75% lower in cells from aged compared to young women (P<0.001). There was a tendency for a lower vascular endothelial growth factor (VEGF) concentration in muscle conditioned media (P=0.0696) and for a lower VEGF content in the myocytes (P=0.0705) from aged compared to young women. Endothelial proliferation was found to be highly dependent on mitochondrial function. Acute exercise resulted in a modest (1.3-fold; P=0.0073) increase in muscle interstitial VEGF protein in the aged women. In Part 2, 8weeks of intense training did not change muscle capillarization (P≥0.1502) in the aged women, but led to an increased amount of muscle VEGF (P=0.0339). In conclusion, aged women have impaired angiogenic potential, which is associated with a compromised response both at the skeletal muscle myocyte and microvascular endothelial cell level.

1328-P: Infant Mesenchymal Stem Cell Insulin Sensitivity Is Associated with Maternal Plasma-Free Fatty Acids, Independent of Obesity Status: The Healthy Start Study

Introduction: Maternal obesity is associated with greater neonatal adiposity and increased offspring risk for type 2 diabetes (T2D) later in life. We have previously reported that mesenchymal stem cells (MSCs) isolated from umbilical cord tissue maintain similar metabolic phenotype to the infant, particularly when differentiated to adipocytes or myocytes. A well-established driver in the pathogenesis of T2D is skeletal muscle insulin resistance. Therefore, we hypothesized that MSCs from infants born to mothers with obesity would display insulin resistance when differentiated to myocytes. Objectives: The objective was to determine the effect of maternal obesity on offspring MSC insulin sensitivity (Si). Methods: MSCs were isolated from umbilical cord tissue from offspring born to either normal weight (BMI 21.1 ± 0.9, n = 9) or obese (BMI 32.9 ± 2.1, n = 10) mothers enrolled in the Healthy Start pre-birth cohort study in Colorado. Following differentiation into skeletal muscle myocytes, MSCs were serum starved and incubated with 14C-glucose, in the presence or absence of insulin. The rate of insulin-mediated 14C-glucose incorporation into glycogen, was used as an index of offspring Si. Results: Offspring Si did not correlate with maternal BMI (r = 0.26), nor was BMI different between groups when median-stratified by offspring Si. However, maternal free fatty acid levels positively correlated with offspring Si (r = 0.63, p &amp;lt; 0.05) and were significantly elevated in the high Si group (low Si vs. high Si; 332.1 ± 30.9 vs. 483.3 ± 48.7 uEq/L, p &amp;lt; 0.05). Conclusions: Offspring Si was not related to the BMI of the mother. Moreover, offspring Si was higher in cells from infants born to mothers with higher free fatty acids during pregnancy. Considering the role of insulin in somatic growth pathways, this phenomenon may serve to increase available nutrient storage capacity during early development. Disclosure A.B. Chaves: None. J.A. Johnson: None. D. Zheng: None. D. Dabelea: None. J.A. Houmard: None. K.E. Boyle: None. Funding American Heart Association (14PRE18230008); Nutrition Obesity Research Center (P30DK048520); National Institutes of Health (R01DK076648); Colorado National Center for Advancing Translational Sciences (UL1TR001082)

Testosterone supplementation improves insulin responsiveness in HFD fed male T2DM mice and potentiates insulin signaling in the skeletal muscle and C2C12 myocyte cell line.

BackgroundType 2 Diabetes Mellitus (T2DM) is characterised by hyperglycemia due to the incidence of insulin resistance. Testosterone supplementation has been shown to have a positive co-relation with improved glycemic control in T2DM males. Clinical studies have reported that Androgen Replacement Therapy (ART) to hypogonadic males with T2DM resulted in improved glycemic control and metabolic parameters, but, these studies did not address in detail how testosterone acted on the key glucose homeostatic organs.MethodIn this study, we delineate the effect of testosterone supplementation to high-fat diet (HFD) induced T2DM in male C57BL6J mice and the effect of testosterone supplementation on the skeletal muscle insulin responsiveness. We also studied the effect of testosterone on the insulin signaling pathway proteins in C2C12 myocyte cells to validate the in vivo findings.ResultsWe found that testosterone had a potentiating effect on the skeletal muscle insulin signaling pathway to improve glycaemic control. We demonstrate that, in males, testosterone improves skeletal muscle insulin responsiveness by potentiating the PI3K-AKT pathway. The testosterone treated animals showed significant increase in the skeletal muscle Insulin Receptor (IR), p85 subunit of PI3K, P-GSK3α (Ser-21), and P-AKT (Ser-473) levels as compared to the control animals; but there was no significant change in total AKT and GSK3α. Testosterone supplementation inhibited GSK3α in the myocytes in a PI3K/AKT pathway dependent manner; on the other hand GSK3β gene expression was reduced in the skeletal muscle upon testosterone supplementation.ConclusionTestosterone increases insulin responsiveness by potentiating insulin signaling in the skeletal muscle cells, which is in contrast to the increased insulin resistance in the liver of testosterone treated T2DM male animals.