Primary Human Skeletal Muscle Research Articles

Extracellular vesicles may provide an alternative detoxification pathway during skeletal muscle myoblast ageing.

Skeletal muscle (SM) acts as a secretory organ, capable of releasing myokines and extracellular vesicles (SM-EVs) that impact myogenesis and homeostasis. While age-related changes have been previously reported in murine SM-EVs, no study has comprehensively profiled SM-EV in human models. To this end, we provide the first comprehensive comparison of SM-EVs from young and old human primary skeletal muscle cells (HPMCs) to map changes associated with SM ageing. HPMCs, isolated from young (24±1.7 years old) and older (69±2.6 years old) participants, were immunomagnetically sorted based on the presence of the myogenic marker CD56 (N-CAM) and cultured as pure (100% CD56+) or mixed populations (MP: 90% CD56+). SM-EVs were isolated using an optimised protocol combining ultrafiltration and size exclusion chromatography (UF+SEC) and their biological content was extensively characterised using Raman spectroscopy (RS) and liquid chromatography mass spectrometry (LC-MS). Minimal variations in basic EV parameters (particle number, size, protein markers) were observed between young and old populations. However, biochemical fingerprinting by RS highlighted increased protein (amide I), lipid (phospholipids and phosphatidylcholine) and hypoxanthine signatures for older SM-EVs. Through LC-MS, we identified 84 shared proteins with functions principally related to cell homeostasis, muscle maintenance and transcriptional regulation. Significantly, SM-EVs from older participants were comparatively enriched in proteins involved in oxidative stress and DNA/RNA mutagenesis, such as E3 ubiquitin-protein ligase TTC3 (TTC3), little elongation complex subunit 1 (ICE1) and Acetyl-CoA carboxylase 1 (ACACA). These data suggest SM-EVs could provide an alternative pathway for homeostasis and detoxification during SM ageing.

Soluble guanylate cyclase activator vericiguat prevents anthracycline-mediated cardiotoxicity and sarcopenia through cGMP-NLRP3-cytokine pathway: Potential application in patients with cancer.

e24015 Background: Anthracycline-induced cardiomyopathies and sarcopenia are frequently seen in cancer patients, affecting their overall survival and quality of life; therefore, new cardioprotective strategies are needed in cardioncology. Vericiguat is a new oral guanylate cyclase activator that reduces heart failure hospitalizations or cardiovascular death through improvement of smooth muscle cell relaxation and reduction of myocardial fibrosis and inflammation. In this study, we highlight on the potential cardioprotective properties of vericiguat against anthracycline.mediated cardiotoxicity and sarcopenia. Methods: Human cardiomyocytes (AC-16 cells) and Primary Skeletal Muscle Cells (HSkMC cells) were exposed for 24h h to doxorubicin (DOXO) at 0,3 and 1 µM with or withour a pre-treatment with Vericiguat at 0.1 or 1 or 10 µM for 1h. Mithocondrial cell viability, LDH and Citocrome C release were performed to study cytoprotective properties. TUNEL assay, cGMP and NLRP-3, Myd-88 intracellular levels were quantified through colorimetric and selective ELISA methods. IL-1β, IL-6, IL-8, CXCL-2, TGF-β and IL-18 were also analyzed in cardiac and muscle cell lysates after treatments. Results: Vericiguat exerts a significant cytoprotective and anti-apoptotic effect in cardiac and muscle cell lines during exposure to DOXO. A drastic increase in cGMP expression and reduction in NLRP-3, Myd-88 levels were also seen in Vericiguat-DOXO groups vs DOXO groups ( p < 0.001). Pro-inflammatory and cytotoxic cytokines were also downregulated after Vericiguat treatment during DOXO exposure. Conclusions: Vericiguat significantly reduced cardiotoxic effects and sarcopenia induced by DOXO therapy though enhancement of cGMP levels and reduction of NLRP-3/Myd-88/cytokines pathways. These findings provide a framework for future studies aiming to assess vericiguat for the primary prevention of anthracycline-mediated cardiotoxicity and sarcopenia.

Enhanced Human Skeletal Muscle Cell Differentiation via the Inhibition of 15-Hydroxyprostaglandin Dehydrogenase

Musculoskeletal diseases, including sarcopenia, are increasingly affecting billions globally. Individuals afflicted with such diseases experience impaired ability to regenerate muscle following injury or volumetric loss. Regeneration is vital for maintaining healthy muscle function and muscle cell differentiation plays a significant role. Our previous studies show that Prostaglandin E2 (PGE2) increases mouse muscle cell differentiation and improves muscle function. Adequate levels of PGE2 in muscle are therefore required for proper regeneration. 15-Hydroxyprostaglandin dehydrogenase (15-PDGH) is an enzyme which degrades PGE2. Previously, studies in rat muscle cells that were treated with SW0033291 (referred hereafter as SW), an inhibitor of 15-PDGH, showed that muscle cell differentiation was enhanced. There is no published study of SW effects on human skeletal muscle myogenesis. Our hypothesis is that when human skeletal muscle cells are treated with SW, the differentiation will be enhanced. To study the effects of SW on human muscle cell differentiation, first the cytotoxicity of SW on human primary skeletal muscle cells (HPSMC) from a young healthy woman were treated with 25, 50, 100, 500 and 1000nM SW for 24h and 48h. No cytotoxicity of SW was detected in all SW-treated groups compared to control group (p>0.05). The effects of SW on myogenic differentiation were measured on day 4 using the methodology of Fusion Index (FI). To study the molecular mechanisms of SW on myogenesis, the expression of myogenic regulator genes (MyoD, MyoG and Mhc) were analyzed by RT-qPCR after the treatment of SW. At day 4 of differentiation, compared to control, no obvious difference of FI was found in 25, 50, 100, and 500nM SW-treated groups (p > 0.05), while a significant increase in FI was found in the 1000nM (p < 0.05) SW0033291-treated group (p<0.05). These results suggest that SW enhances HPMC differentiation. Compared to control, in 25, 50, 100, and 500nM SW-treated groups, the expression of all three genes was not significantly changed (p > 0.05). In the 1000nM SW-treated group, the expression of the myogenic markers MyoD, MyoG and Mhc increased significantly (p < 0.05) by 1.83±0.21-fold, 1.47±0.18-fold, and 2.09±0.37-fold, respectively. These findings confirm our original hypothesis that SW enhances HPSMC differentiation. In conclusion, these results show initial promising application for maintaining proper PGE2 levels and thus allowing for proper human muscle cell differentiation and potential clinical applications in a wide range of musculoskeletal disorders. This work was directly supported by NIH-National Institutes of Aging P01AG039355 (MB) and the George W. and Hazel M. Jay and Evanston Research Endowments (MB). Authors were supported by NIH Grants: National Institutes of Aging (NINDS) 2-R01NS105621; NIA-R01AG056504, NIA-2R01AG060341, National Institutes of Diabetes, Digestive, and Kidney Diseases Kidney (NIDDK)-1R01DK119066 to MB, and National Institutes of Neurological Disorders and Stroke (NINDS) 2-R01NS105621 to MB. Also, NIH/NIDC 1R01DE031872-01 (VV). 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.

The sGCa Vericiguat Exhibit Cardioprotective and Anti-Sarcopenic Effects through NLRP-3 Pathways: Potential Benefits for Anthracycline-Treated Cancer Patients.

Anthracycline-induced cardiomyopathies and sarcopenia are frequently seen in cancer patients, affecting their overall survival and quality of life; therefore, new cardioprotective and anti-sarcopenic strategies are needed. Vericiguat is a new oral guanylate cyclase activator that reduces heart failure hospitalizations or cardiovascular death. This study highlighted the potential cardioprotective and anti-sarcopenic properties of vericiguat during anthracycline therapy. Human cardiomyocytes and primary skeletal muscle cells were exposed to doxorubicin (DOXO) with or without a pre-treatment with vericiguat. Mitochondrial cell viability, LDH, and Cytochrome C release were performed to study cytoprotective properties. Intracellular Ca++ content, TUNEL assay, cGMP, NLRP-3, Myd-88, and cytokine intracellular levels were quantified through colorimetric and selective ELISA methods. Vericiguat exerts significant cytoprotective and anti-apoptotic effects during exposure to doxorubicin. A drastic increase in cGMP expression and reduction in NLRP-3, MyD-88 levels were also seen in Vericiguat-DOXO groups vs. DOXO groups (p < 0.001) in both cardiomyocytes and human muscle cells. GCa vericiguat reduces cytokines and chemokines involved in heart failure and sarcopenia. The findings that emerged from this study could provide the rationale for further preclinical and clinical investigations aimed at reducing anthracycline cardiotoxicity and sarcopenia in cancer patients.

Sulforaphane Ameliorates High-Fat-Diet-Induced Metabolic Abnormalities in Young and Middle-Aged Obese Male Mice.

Type 2 diabetes (T2D) is still a fast-growing health problem globally. It is evident that chronic insulin resistance (IR) and progressive loss of β-cell mass and function are key features of T2D etiology. Obesity is a leading pathogenic factor for developing IR. The aim of the present study was to determine whether sulforaphane (SFN), a natural compound derived from cruciferous vegetables, can prevent (prevention approach) or treat (treatment approach) obesity and IR in mouse models. We show that dietary intake of SFN (0.5 g/kg of HFD) for 20 weeks suppressed high-fat diet (HFD)-induced fat accumulation by 6.04% and improved insulin sensitivity by 23.66% in young male mice. Similarly, dietary provision of SFN (0.25 g/kg) significantly improved blood lipid profile, glucose tolerance, and insulin sensitivity of the middle-aged male mice while it had little effects on body composition as compared with the HFD group. In the treatment study, oral administration of SFN (40 mg/kg) induced weight loss and improved insulin sensitivity and plasma lipid profile in the diet-induced-obesity (DIO) male mice. In all three studies, the metabolic effects of SFN administration were not associated with changes in food intake. In vitro, SFN increased glucose uptake in C2C12 myotubes and increased fatty acid and pyruvate oxidation in primary human skeletal muscle cells. Our results suggest that SFN may be a naturally occurring insulin-sensitizing agent that is capable of improving the metabolic processes in HFD-induced obesity and IR and thereby may be a promising compound for T2D prevention.

Small extracellular vesicle microRNAs in pediatric myasthenia gravis plasma and skeletal muscle.

The diagnosis of myasthenia gravis (MG) in children remains difficult. Circulating small extracellular vesicle (sEV)-derived miRNAs (sEV-miRNAs) have been recognized as biomarkers of various diseases and can be excreted by different cell types. These biomarker candidates also play a vital role in autoimmune diseases via intercellular communication. In the present study, we used sEV isolation and purification methods to extract the plasma-derived sEV-miRNAs from children with MG and healthy controls. A small RNA sequencing analysis confirmed the miRNA expression features in plasma-derived sEVs from MG patients. The miRNA expression analysis in vitro was determined using microarray analysis. The enrichment and network analyses of altered sEV-miRNAs were performed using miRNA databases and Database for Annotation, Visualization, and Integrated Discovery website. Quantitative real-time polymerase chain reaction was performed for validation of sEV-miRNA. The diagnostic power of altered sEV-miRNAs was evaluated using receiver operating characteristic curve analyses. Twenty-four sEV-miRNAs with altered expression level were identified between groups by DESeq2 method. The miRNAs were extracted from the sEVs, which were isolated from human primary skeletal muscle cell culture treated with mAb198. The target genes and enriched pathways of sEV-miRNAs partially overlapped between cell supernatant and plasma samples. The significantly downregulated miR-143-3p was validated in quantitative real-time polymerase chain reaction analysis. For the first time, we report that plasma-derived sEV-miRNAs may act as novel circulating biomarkers and therapeutic targets in pediatric MG.

Amorphous calcium carbonate enhances osteogenic differentiation and myotube formation of human bone marrow derived mesenchymal stem cells and primary skeletal muscle cells under microgravity conditions

Astronauts are exposed to severely stressful physiological conditions due to microgravity and increased space radiation. Space environment affects every organ and cell in the body and the significant adverse effects of long-term weightlessness include muscle atrophy and deterioration of the skeleton (spaceflight osteopenia). Amorphous Calcium Carbonate (ACC) emerges as a promising candidate for prevention of these effects, owing to its unique physicochemical properties and its potential to address the intricately linked nature of bone-muscle crosstalk. Reported here are two studies carried out on the International Space Station (ISS). The first, performed in 2018 as a part of the Ramon-Spacelab project, was a preliminary experiment, in which stromal murine cells were differentiated into osteoblasts when ACC was added to the culture medium. A parallel experiment was done on Earth as a control. The second study was part of Axiom-1‘s Rakia project mission launched to the ISS on 2022 utilizing organ-on-a-chip methodology with a specially designed autonomous module. In this experiment, human bone-marrow derived mesenchymal stem cells (hBM-MSCs) and human primary muscle cells were cultured in the presence or absence of ACC, in duplicates. The results showed that ACC enhanced differentiation of human primary skeletal muscle cells into myotubes. Similarly, hBM-MSCs were differentiated significantly better into osteocytes in the presence of ACC leading to increased calcium deposits. The results, combined with previous data, support the use of ACC as an advantageous supplement for preventing muscle and bone deterioration in outer space conditions, facilitating extended extraterrestrial voyages and colonization.

Sex-specific increases in myostatin and SMAD3 contribute to obesity-related insulin resistance in human skeletal muscle and primary human myotubes.

The purpose of the present study was to determine the effects of obesity and biological sex on myostatin expression in humans and to examine the direct effects of myostatin, SMAD2, and SMAD3 on insulin signaling in primary human skeletal muscle cells (HSkMCs). For cohort 1, 15 lean [body mass index (BMI): 22.1 ± 0.5 kg/m2; n = 8 males; n = 7 females] and 14 obese (BMI: 40.6 ± 1.4 kg/m2; n = 7 males; n = 7 females) individuals underwent skeletal muscle biopsies and an oral glucose tolerance test. For cohort 2, 14 young lean (BMI: 22.4 ± 1.9 kg/m2; n = 6 males; n = 8 females) and 14 obese (BMI: 39.3 ± 7.9 kg/m2; n = 6 males; n = 8 females) individuals underwent muscle biopsies for primary HSkMC experiments. Plasma mature myostatin (P = 0.041), skeletal muscle precursor myostatin (P = 0.048), and skeletal muscle SMAD3 (P = 0.029) were elevated in obese females compared to lean females, and plasma mature myostatin (r = 0.58, P = 0.029) and skeletal muscle SMAD3 (r = 0.56, P = 0.037) were associated with insulin resistance in females but not males. Twenty-four hours of myostatin treatment impaired insulin signaling in primary HSkMCs derived from females (P < 0.024) but not males. Overexpression of SMAD3, but not SMAD2, impaired insulin-stimulated AS160 phosphorylation in HSkMCs derived from lean females (-27%, P = 0.040), whereas silencing SMAD3 improved insulin-stimulated AS160 phosphorylation and insulin-stimulated glucose uptake (25%, P < 0.014) in HSkMCs derived from obese females. These results suggest for the first time that myostatin-induced impairments in skeletal muscle insulin signaling are sex specific and that increased body fat in females is associated with detrimental elevations in myostatin and SMAD3, which contribute to obesity-related insulin resistance.NEW & NOTEWORTHY Obesity is considered a main risk factor for the development of insulin resistance and type 2 diabetes. The present study utilizes in vivo and in vitro experiments in human skeletal muscle to demonstrate for the first time that females are inherently more susceptible to myostatin-induced insulin resistance, which is further enhanced with obesity due to increased myostatin and SMAD3 expression.

Sex-Chromosome-Related Dimorphism in Steroidogenic Enzymes and Androgen Receptor in Response to Testosterone Treatment: An In Vitro Study on Human Primary Skeletal Muscle Cells.

Gender-related methodology in biomedical sciences receives considerable attention, with numerous studies highlighting biological differences between cisgender males and females. These differences influence the clinical symptoms of various diseases and impact therapeutic approaches. In this in vitro study, we investigate the potential role of sex-chromosome-related dimorphism on steroidogenic enzymes, androgen receptor (AR) expression, and cellular translocation in primary human skeletal muscle cells before and after exposure to testosterone. We analyzed 46XY and 46XX cells for 17β-hydroxysteroid dehydrogenase (17β-HSD), 5α-reductase (5α-R2), aromatase (Cyp-19), and AR gene expression. We also compared AR expression and intracellular translocation after increasing exposure to testosterone. At baseline, we observed higher mRNA expression for 5α-R2 and AR in 46XY cells and higher Cyp-19 mRNA expression in 46XX cells. Following testosterone exposure, we observed an increase in AR expression and translocation in 46XX cells, even at the lowest dose of 0.5 nM, while significant changes in 46XY cells were observed only from 10 nM. Our in vitro results demonstrate that the diverse sex chromosome assets reflect important differences in muscle steroidogenesis. They support the concept that chromosomal disparities between males and females, even in vitro, lead to pivotal variations in cellular physiology and response. This understanding represents a crucial starting point in gender medicine, ensuring a precise approach in clinical practice, sports, and exercise settings and facilitating the translation of in vitro data to in vivo applicability.

Muscle miRNAs are influenced by sex at baseline and in response to exercise

BackgroundSex differences in microRNA (miRNA) expression profiles have been found across multiple tissues. Skeletal muscle is one of the most sex-biased tissues of the body. MiRNAs are necessary for development and have regulatory roles in determining skeletal muscle phenotype and have important roles in the response to exercise in muscle. Yet there is limited research into the role and regulation of miRNAs in the skeletal muscle at baseline and in response to exercise, a well-known modulator of miRNA expression. The aim of this study was to investigate the effect of sex on miRNA expression in the skeletal muscle at baseline and after an acute bout of high-intensity interval exercise. A total of 758 miRNAs were measured using Taqman®miRNA arrays in the skeletal muscle of 42 healthy participants from the Gene SMART study (23 males and 19 females of comparable fitness levels and aged 18–45 years), of which 308 were detected. MiRNAs that differed by sex at baseline and whose change in expression following high-intensity interval exercise differed between the sexes were identified using mixed linear models adjusted for BMI and Wpeak. We performed in silico analyses to identify the putative gene targets of the exercise-induced, sex-specific miRNAs and overrepresentation analyses to identify enriched biological pathways. We performed functional assays by overexpressing two sex-biased miRNAs in human primary muscle cells derived from male and female donors to understand their downstream effects on the transcriptome.ResultsAt baseline, 148 miRNAs were differentially expressed in the skeletal muscle between the sexes. Interaction analysis identified 111 miRNAs whose response to an acute bout of high-intensity interval exercise differed between the sexes. Sex-biased miRNA gene targets were enriched for muscle-related processes including proliferation and differentiation of muscle cells and numerous metabolic pathways, suggesting that miRNAs participate in programming sex differences in skeletal muscle function. Overexpression of sex-biased miRNA-30a and miRNA-30c resulted in profound changes in gene expression profiles that were specific to the sex of the cell donor in human primary skeletal muscle cells.ConclusionsWe uncovered sex differences in the expression levels of muscle miRNAs at baseline and in response to acute high-intensity interval exercise. These miRNAs target regulatory pathways essential to skeletal muscle development and metabolism. Our findings highlight that miRNAs play an important role in programming sex differences in the skeletal muscle phenotype.

Human HDL subclasses modulate energy metabolism in skeletal muscle cells

In addition to its antiatherogenic role, HDL reportedly modulates energy metabolism at the whole-body level. HDL functionality is associated with its structure and composition, and functional activities can differ between HDL subclasses. Therefore, we studied if HDL2 and HDL3, the two major HDL subclasses, are able to modulate energy metabolism of skeletal muscle cells. Differentiated mouse and primary human skeletal muscle myotubes were used to investigate the influences of human HDL2 and HDL3 on glucose and fatty uptake and oxidation. HDL-induced changes in lipid distribution and mRNA expression of genes related to energy substrate metabolism, mitochondrial function, and HDL receptors were studied with human myotubes. Additionally, we examined the effects of apoA-I and discoidal, reconstituted HDL particles on substrate metabolism. In mouse myotubes, HDL subclasses strongly enhanced glycolysis upon high and low glucose concentrations. HDL3 caused a minor increase in ATP-linked respiration upon glucose conditioning but HDL2 improved complex I–mediated mitochondrial respiration upon fatty acid treatment. In human myotubes, glucose metabolism was attenuated but fatty acid uptake and oxidation were markedly increased by both HDL subclasses, which also increased mRNA expression of genes related to fatty acid metabolism and HDL receptors. Finally, both HDL subclasses induced incorporation of oleic acid into different lipid classes. These results, demonstrating that HDL subclasses enhance fatty acid oxidation in human myotubes but improve anaerobic metabolism in mouse myotubes, support the role of HDL as a circulating modulator of energy metabolism. Exact mechanisms and components of HDL causing the change, require further investigation.

Development of capsid- and genome-modified optimized AAVrh74 vectors for muscle gene therapy

The first generation of adeno-associated virus (AAV) vectors composed of the naturally occurring capsids and genomes, although effective in some instances, are unlikely to be optimal for gene therapy in humans. The use of the first generation of two different AAV serotype vectors (AAV9 and AAVrh74) in four separate clinical trials failed to be effective in patients with Duchenne muscular dystrophy, although some efficacy was observed in a subset of patients with AAVrh74 vectors leading to US Food and Drug Administration approval (Elevidys). In two trials with the first generation of AAV9 vectors, several serious adverse events were observed, including the death of a patient in one trial, and more recently, in the death of a second patient in an N-of-1 clinical trial. In a fourth trial with the first generation of AAVrh74 vectors, myositis and myocarditis were also observed. Here, we report that capsid- and genome-modified optimized AAVrh74 vectors are significantly more efficient in transducing primary human skeletal muscle cells invitro and in all major muscle tissues invivo following systemic administration in a murine model. The availability of optimized AAVrh74 vectors promises to be safe and effective in the potential gene therapy of muscle diseases in humans.

Apabetalone, a Clinical-Stage, Selective BET Inhibitor, Opposes DUX4 Target Gene Expression in Primary Human FSHD Muscle Cells.

Facioscapulohumeral dystrophy (FSHD) is a muscle disease caused by inappropriate expression of the double homeobox 4 (DUX4) gene in skeletal muscle, and its downstream activation of pro-apoptotic transcriptional programs. Inhibitors of DUX4 expression have the potential to treat FSHD. Apabetalone is a clinical-stage bromodomain and extra-terminal (BET) inhibitor, selective for the second bromodomain on BET proteins. Using primary human skeletal muscle cells from FSHD type 1 patients, we evaluated apabetalone for its ability to counter DUX4's deleterious effects and compared it with the pan-BET inhibitor JQ1, and the p38 MAPK inhibitor-and DUX4 transcriptional repressor-losmapimod. We applied RNA-sequencing and bioinformatic analysis to detect treatment-associated impacts on the transcriptome of these cells. Apabetalone inhibited the expression of DUX4 downstream markers, reversing hallmarks of FSHD gene expression in differentiated muscle cells. JQ1, but not apabetalone, was found to induce apoptosis. While both BET inhibitors modestly impacted differentiation marker expression, they did not affect myotube fusion. Losmapimod also reduced expression of DUX4 target genes but differed in its impact on FSHD-associated pathways. These findings demonstrate that apabetalone inhibits DUX4 target gene expression and reverses transcriptional programs that contribute to FSHD pathology, making this drug a promising candidate therapeutic for FSHD.

Partial skeletal muscle-specific Drp1 knockout enhances insulin sensitivity in diet-induced obese mice, but not in lean mice

ObjectiveDynamin-related protein 1 (Drp1) is the key regulator of mitochondrial fission. We and others have reported a strong correlation between enhanced Drp1 activity and impaired skeletal muscle insulin sensitivity. This study aimed to determine whether Drp1 directly regulates skeletal muscle insulin sensitivity and whole-body glucose homeostasis. MethodsWe employed tamoxifen-inducible skeletal muscle-specific heterozygous Drp1 knockout mice (mDrp1+/−). Male mDrp1+/− and wildtype (WT) mice were fed with either a high-fat diet (HFD) or low-fat diet (LFD) for four weeks, followed by tamoxifen injections for five consecutive days, and remained on their respective diet for another four weeks. In addition, we used primary human skeletal muscle cells (HSkMC) from lean, insulin-sensitive, and severely obese, insulin-resistant humans and transfected the cells with either a Drp1 shRNA (shDrp1) or scramble shRNA construct. Skeletal muscle and whole-body insulin sensitivity, skeletal muscle insulin signaling, mitochondrial network morphology, respiration, and H2O2 production were measured. ResultsPartial deletion of the Drp1 gene in skeletal muscle led to improved whole-body glucose tolerance and insulin sensitivity (P < 0.05) in diet-induced obese, insulin-resistant mice but not in lean mice. Analyses of mitochondrial structure and function revealed that the partial deletion of the Drp1 gene restored mitochondrial dynamics, improved mitochondrial morphology, and reduced mitochondrial Complex I- and II-derived H2O2 (P < 0.05) under the condition of diet-induced obesity. In addition, partial deletion of Drp1 in skeletal muscle resulted in elevated circulating FGF21 (P < 0.05) and in a trend towards increase of FGF21 expression in skeletal muscle tissue (P = 0.095). In primary myotubes derived from severely obese, insulin-resistant humans, ShRNA-induced-knockdown of Drp1 resulted in enhanced insulin signaling, insulin-stimulated glucose uptake and reduced cellular reactive oxygen species (ROS) content compared to the shScramble-treated myotubes from the same donors (P < 0.05). ConclusionThese data demonstrate that partial loss of skeletal muscle-specific Drp1 expression is sufficient to improve whole-body glucose homeostasis and insulin sensitivity under obese, insulin-resistant conditions, which may be, at least in part, due to reduced mitochondrial H2O2 production. In addition, our findings revealed divergent effects of Drp1 on whole-body metabolism under lean healthy or obese insulin-resistant conditions in mice.

Kaempferol improves glucose uptake in skeletal muscle via an AMPK-dependent mechanism

Insulin resistance is a hallmark of type-2 diabetes (T2D) pathogenesis. Because skeletal muscle (SkM) is the major tissue for insulin-mediated glucose disposal, insulin resistance in SkM is considered a major risk factor for developing T2D. Thus, the identification of compounds that enhance the ability of SkM to take up glucose is a promising strategy for preventing T2D. Our previous work showed that kaempferol, a flavonol present in many foods, improves insulin sensitivity in obese mice, however, the mechanism underlying this beneficial action remains unclear. Here, we show that kaempferol directly stimulates glucose uptake and prevents lipotoxicity-impaired glucose uptake in primary human SkM. Kaempferol stimulates Akt phosphorylation in a time-dependent manner in human SkM cells. The effect of kaempferol on glucose uptake was blunted by inhibition of glucose transporter 4, phosphoinositide 3-kinase (PI3K), or AMPK. In addition, kaempferol induced AMPK phosphorylation, and inhibition of AMPK prevented kaempferol-stimulated Akt phosphorylation. In vivo, kaempferol administration induced rapid glucose disposal accompanied with increased Akt and AMPK phosphorylation in SkM tissue of the mice. Taken together, these findings suggest that kaempferol stimulates glucose uptake in SkM via an AMPK/Akt dependent mechanism, and it may be a viable therapeutic agent for insulin resistance.

Finnish-specific AKT2 gene variant leads to impaired insulin signalling in myotubes.

Finnish-specific gene variant p.P50T/AKT2 (MAF=1.1%), is associated with insulin resistance and increased predisposition to type 2 diabetes. Here, we have investigated in vitro the impact of the gene variant on glucose metabolism and intracellular signalling in human primary skeletal muscle cells, that were established from 14 male p.P50T/AKT2 variant carriers and 14 controls. Insulin-stimulated glucose uptake and glucose incorporation into glycogen were detected with 2-[1,2-3H]-deoxy-D-glucose and D-[14C]-glucose, respectively, and the rate of glycolysis was measured with a Seahorse XFe96 analyzer. Insulin signalling was investigated with western blotting. Binding of variant and control AKT2-PH domains to phosphatidylinositol (3,4,5)-trisphosphate ((PI(3,4,5)P3)) was assayed using PIP Strips™ Membranes. Tyrosine (PTK) and serine-threonine (STK) kinase assay was performed using the PamGene® kinome profiling system. Insulin-stimulated glucose uptake and glycogen synthesis in myotubes in vitro were not significantly affected by the genotype. However, insulin-stimulated glycolytic rate was impaired in variant myotubes. Western blot analysis showed that insulin-stimulated phosphorylation of Akt-Thr308, AS160-Thr642 and GSK3β-Ser9 was reduced in variant myotubes compared to controls. Binding of variant AKT2-PH domain to PI(3,4,5)P3 was reduced as compared to control protein. PamGene® kinome profiling revealed multiple differentially phosphorylated kinase substrates, e.g. calmodulin, between the genotypes. Further in silico upstream kinase analysis predicted a large-scale impairment in activities of kinases participating for example in intracellular signal transduction, protein translation and cell cycle events. In conclusion, myotubes from p.P50T/AKT2 variant carriers show multiple signalling alterations which may contribute to predisposition to insulin resistance and T2D in the carriers of this signalling variant.

Improving physiological relevance of cell culture: the possibilities, considerations, and future directions of the ex vivo coculture model.

In vitro models provide an important platform for the investigation of cellular growth and atrophy to inform, or extend mechanistic insights from, logistically challenging in vivo trials. Although these models allow for the identification of candidate mechanistic pathways, many models involve supraphysiological dosages, nonphysiological conditions, or experimental changes relating to individual proteins or receptors, all of which limit translation to human trials. To overcome these drawbacks, the use of ex vivo human plasma and serum has been used in cellular models to investigate changes in myotube hypertrophy, cellular protein synthesis, anabolic and catabolic markers in response to differing age, disease states, and nutrient status. However, there are currently no concurrent guidelines outlining the optimal methodology for this model. This review discusses the key methodological considerations surrounding the use of ex vivo plasma and serum with a focus in application to skeletal muscle cell lines (i.e., C2C12, L6, and LHCN-M2) and human primary skeletal muscle cells (HSMCs) as a means to investigate molecular signaling in models of atrophy and hypertrophy, alongside future directions.

Pyruvate dehydrogenase B regulates myogenic differentiation via the FoxP1-Arih2 axis.

Sarcopenia, the age-related decline in skeletal muscle mass and function, diminishes life quality in elderly people. Improving the capacity of skeletal muscle differentiation is expected to counteract sarcopenia. However, the mechanisms underlying skeletal muscle differentiation are complex, and effective therapeutic targets are largely unknown. The human Gene Expression Omnibus database, aged mice and primary skeletal muscle cells were used to assess the expression level of pyruvate dehydrogenase B (PDHB) in human and mouse aged state. d-Galactose (d-gal)-induced sarcopenia mouse model and two classic cell models (C2C12 and HSkMC) were used to assess the myogenic effect of PDHB and the underlying mechanisms via immunocytochemistry, western blotting, quantitative real-time polymerase chain reaction, RNA interference or overexpression, dual-luciferase reporter assay, RNA sequencing and untargeted metabolomics. We identified that a novel target PDHB promoted myogenic differentiation. PDHB expression decreased in aged mouse muscle relative to the young state (-50% of mRNA level, P<0.01) and increased during mouse and primary human muscle cell differentiation (+3.97-fold, P<0.001 and +3.79-fold, P<0.001). Knockdown or overexpression of PDHB modulated the expression of genes related to muscle differentiation, namely, myogenic factor 5 (Myf5) (-46%, P<0.01 and -27%, P<0.05; +1.8-fold, P<0.01), myogenic differentiation (MyoD) (-55%, P<0.001 and -34%, P<0.01; +2.27-fold, P<0.001), myogenin (MyoG) (-60%, P<0.001 and -70%, P<0.001; +5.46-fold, P<0.001) and myosin heavy chain (MyHC) (-70%, P<0.001 and -69%, P<0.001; +3.44-fold, P<0.001) in both C2C12 cells and HSkMC. Metabolomic and transcriptomic analyses revealed that PDHB knockdown suppressed pyruvate metabolism (P<0.001) and up-regulated ariadne RBR E3 ubiquitin protein ligase 2 (Arih2) (+7.23-fold, P<0.001) in cellular catabolic pathways. The role of forkhead box P1 (FoxP1) (+4.18-fold, P<0.001)-mediated Arih2 transcription was the key downstream regulator of PDHB in muscle differentiation. PDHB overexpression improved d-gal-induced muscle atrophy in mice, which was characterized by significant increases in grip strength, muscle mass and mean muscle cross-sectional area (1.19-fold to 1.5-fold, P<0.01, P<0.05 and P<0.001). The comprehensive results show that PDHB plays a sarcoprotective role by suppressing the FoxP1-Arih2 axis and may serve as a therapeutic target in sarcopenia.

Energy metabolism in skeletal muscle cells from donors with different body mass index.

Obesity and physical inactivity have a profound impact on skeletal muscle metabolism. In the present work, we have investigated differences in protein expression and energy metabolism in primary human skeletal muscle cells established from lean donors (BMI<25kg/m2) and individuals with obesity (BMI>30kg/m2). Furthermore, we have studied the effect of fatty acid pretreatment on energy metabolism in myotubes from these donor groups. Alterations in protein expression were investigated using proteomic analysis, and energy metabolism was studied using radiolabeled substrates. Gene Ontology enrichment analysis showed that glycolytic, apoptotic, and hypoxia pathways were upregulated, whereas the pentose phosphate pathway was downregulated in myotubes from donors with obesity compared to myotubes from lean donors. Moreover, fatty acid, glucose, and amino acid uptake were increased in myotubes from individuals with obesity. However, fatty acid oxidation was reduced, glucose oxidation was increased in myotubes from subjects with obesity compared to cells from lean. Pretreatment of myotubes with palmitic acid (PA) or eicosapentaenoic acid (EPA) for 24h increased glucose oxidation and oleic acid uptake. EPA pretreatment increased the glucose and fatty acid uptake and reduced leucine fractional oxidation in myotubes from donors with obesity. In conclusion, these results suggest that myotubes from individuals with obesity showed increased fatty acid, glucose, and amino acid uptake compared to cells from lean donors. Furthermore, myotubes from individuals with obesity had reduced fatty acid oxidative capacity, increased glucose oxidation, and a higher glycolytic reserve capacity compared to cells from lean donors. Fatty acid pretreatment enhances glucose metabolism, and EPA reduces oleic acid and leucine fractional oxidation in myotubes from donor with obesity, suggesting increased metabolic flexibility after EPA treatment.

Effect of Insulin and Pioglitazone on Protein Phosphatase 2A Interaction Partners in Primary Human Skeletal Muscle Cells Derived from Obese Insulin-Resistant Participants.

Skeletal muscle insulin resistance is a major contributor to type-2 diabetes (T2D). Pioglitazone is a potent insulin sensitizer of peripheral tissues by targeting peroxisome proliferator-activated receptor gamma. Pioglitazone has been reported to protect skeletal muscle cells from lipotoxicity by promoting fatty acid mobilization and insulin signaling. However, it is unclear whether pioglitazone increases insulin sensitivity through changes in protein-protein interactions involving protein phosphatase 2A (PP2A). PP2A regulates various cell signaling pathways such as insulin signaling. Interaction of the catalytic subunit of PP2A (PP2Ac) with protein partners is required for PP2A specificity and activity. Little is known about PP2Ac partners in primary human skeletal muscle cells derived from lean insulin-sensitive (Lean) and obese insulin-resistant (OIR) participants. We utilized a proteomics method to identify PP2Ac interaction partners in skeletal muscle cells derived from Lean and OIR participants, with or without insulin and pioglitazone treatments. In this study, 216 PP2Ac interaction partners were identified. Furthermore, 26 PP2Ac partners exhibited significant differences in their interaction with PP2Ac upon insulin treatments between the two groups. Multiple pathways and molecular functions are significantly enriched for these 26 interaction partners, such as nonsense-mediated decay, metabolism of RNA, RNA binding, and protein binding. Interestingly, pioglitazone restored some of these abnormalities. These results provide differential PP2Ac complexes in Lean and OIR in response to insulin/pioglitazone, which may help understand molecular mechanisms underpinning insulin resistance and the insulin-sensitizing effects of pioglitazone treatments, providing multiple targets in various pathways to reverse insulin resistance and prevent and/or manage T2D with less drug side effects.