Skeletal Myotubes Research Articles

Abstract 13103: Increased Mitochondrial Oxidative Stress and Decreased Mitochondrial Respiration in an in vitro Model of Ischemia Reperfusion Injury

Introduction: Peripheral artery disease (PAD) patients experience ischemia during activity and reperfusion of ischemic skeletal muscle during rest. Cycles of ischemia and reperfusion (I/R) result in a compromised metabolic myopathy that adds to the pathophysiology of PAD. We have recently developed an I/R in vitro model that can mimic the skeletal muscle damage seen in the PAD gastrocnemius. We hypothesized that cycles of I/R will increase mitochondrial oxidative stress and mitochondrial respiration in skeletal muscle myotubes. Methods: Primary human skeletal muscle cells derived from PAD patients and non-PAD-control gastrocnemius biopsy samples were differentiated in myotubes and exposed to constant normoxia, cycles of normoxia-hypoxia, or cycles of normoxia-hypoxia-hyperoxia, to mimic I/R, with the OxyCyler C42. A substrate inhibitor protocol was used to measure mitochondrial hydrogen peroxide and respiration using an Oroboros fluorespirometer. Mitochondrial gene and protein expressions and transmitted electron microscopy were used to identify changes in mitochondrial function and morphology. Results: The oxygen consumption rate was significantly reduced in the PAD myotubes compared to control myotubes followed by significant increase of H 2 O 2 production. The mRNA and protein expression of several mitochondrial energy metabolism components were significantly reduced in the PAD myotubes. Cycling of normoxia-hypoxia and normoxia-hypoxia-hyperoxia significantly altered the mitochondrial function and morphology in both control and PAD myotubes. Conclusions: Our in vitro model of I/R injury can alter oxidative stress, mitochondrial function and morphology in control and PAD myotubes. Therefore, the pathophysiology of PAD can be modeled in vitro , which allows for a method to test novel therapeutics to treat PAD.

In vitro and in silico characterization of adiponectin-receptor agonist dipeptides

The aim of this study is to develop a dipeptide showing an adiponectin receptor 1 (AdipoR1) agonistic effect in skeletal muscle L6 myotubes. Based on the structure of the AdipoR1 agonist, AdipoRon, 15 synthetic dipeptides were targeted to promote glucose uptake in L6 myotubes. Tyr-Pro showed a significant increase in glucose uptake among the dipeptides, while other dipeptides, including Pro-Tyr, failed to exert this effect. Tyr-Pro induces glucose transporter 4 (Glut4) expression in the plasma membrane, along with adenosine monophosphate-activated protein kinase (AMPK) activation. In AdipoR1-knocked down cells, the promotion by Tyr-Pro was ameliorated, indicating that Tyr-Pro may directly interact with AdipoR1 as an agonist, followed by the activation of AMPK/Glut4 translocation in L6 myotubes. Molecular dynamics simulations revealed that a Tyr-Pro molecule was stably positioned in the two potential binding pockets (sites 1 and 2) of the seven-transmembrane receptor, AdipoR1, anchored in a virtual 1-palmitoyl-2-oleoyl-phosphatidylcholine membrane. In conclusion, we demonstrated the antidiabetic function of the Tyr-Pro dipeptide as a possible AdipoR1 agonist.

Establishment of myoblast cell line and identification of key genes regulating myoblast differentiation in a marine teleost, Sebastes schlegelii.

Skeletal myoblasts are activated satellite cells capable of proliferation and differentiation. Studies on mammalian myoblast differentiation and myogenesis could be carried out in vitro thanks to the availability of mouse myoblast cell line C2C12. Lacking of muscle cell line hinders the studies of teleost fish myogenesis. Here, we established a continuous skeletal muscle cell line from juvenile rockfish (Sebastes schlegelii) muscle using explant method and subcultured more than 50 passages for over 150days. Stable expression of myoblast-specific marker, MyoD (myoblast determination protein) and the potential of differentiation into multi-nucleated skeletal myotubes upon induction suggested the cell line were predominately composed of myoblasts. Transcriptome analysis revealed a total of 4375 genes differentially expressed at four time points after the switch to differentiation medium, which were mainly involved in proliferation and differentiation of myoblasts. KIF22 (kinesin family member 22) and POLA1 (DNA polymerase alpha 1) were identified as the key genes involved in fish myoblast proliferation whereas MYL3 (myosin light chain 3) and TNNT2 (troponin T2) were determined as the crucial genes responsible for differentiation. In all, the continuous myoblasts cultured in this study provided a cell platform for future studies on marine fish myoblast differentiation and myogenesis. The molecular process of myoblast differentiation revealed in this study will open a window into the understanding of indeterminate muscle growth of large teleost.

Inhibitory Effect of the Glycerophosphate Moiety of Lipoteichoic Acid from Lactic Acid Bacteria on Dexamethasone-Induced Atrogin-1 Expression in C2C12 Myotubes.

Atrogin-1, which is an important regulator of ubiquitin-mediated protein degradation in skeletal muscle, is a major marker of muscle loss and disuse muscle atrophy. To investigate which components of lactic acid bacteria (LAB) suppress dexamethasone (DEX)-induced atrogin-1 expression, mouse skeletal muscle C2C12 myotubes were treated with DEX in the presence or absence of components of LAB. Heat-killed cells and lipoteichoic acid (LTA) derived from five LAB strains significantly suppressed DEX-induced atrogin-1 expression. The glycerophosphate (GroP) fraction prepared from chemically-degraded LTA and sn-glycerol-1-phosphate suppressed DEX-induced atrogin-1 expression, whereas the glycolipid anchor fraction of LTA did not. Heat-killed cells obtained by culturing under low-Mn2+ conditions, which generated fewer poly-GroP polymers in LTA, displayed significantly lower inhibitory activity compared to heat-killed cells grown under normal conditions. These results suggested that LTA of LAB contributed to suppressing atrogin-1 expression and that the GroP moiety of LTA was responsible for its inhibitory activity.

Calsequestrin 1 Is an Active Partner of Stromal Interaction Molecule 2 in Skeletal Muscle.

Calsequestrin 1 (CASQ1) in skeletal muscle buffers and senses Ca2+ in the sarcoplasmic reticulum (SR). CASQ1 also regulates store-operated Ca2+ entry (SOCE) by binding to stromal interaction molecule 1 (STIM1). Abnormal SOCE and/or abnormal expression or mutations in CASQ1, STIM1, or STIM2 are associated with human skeletal, cardiac, or smooth muscle diseases. However, the functional relevance of CASQ1 along with STIM2 has not been studied in any tissue, including skeletal muscle. First, in the present study, it was found by biochemical approaches that CASQ1 is bound to STIM2 via its 92 N-terminal amino acids (C1 region). Next, to examine the functional relevance of the CASQ1-STIM2 interaction in skeletal muscle, the full-length wild-type CASQ1 or the C1 region was expressed in mouse primary skeletal myotubes, and the myotubes were examined using single-myotube Ca2+ imaging experiments and transmission electron microscopy observations. The CASQ1-STIM2 interaction via the C1 region decreased SOCE, increased intracellular Ca2+ release for skeletal muscle contraction, and changed intracellular Ca2+ distributions (high Ca2+ in the SR and low Ca2+ in the cytosol were observed). Furthermore, the C1 region itself (which lacks Ca2+-buffering ability but has STIM2-binding ability) decreased the expression of Ca2+-related proteins (canonical-type transient receptor potential cation channel type 6 and calmodulin 1) and induced mitochondrial shape abnormalities. Therefore, in skeletal muscle, CASQ1 plays active roles in Ca2+ movement and distribution by interacting with STIM2 as well as Ca2+ sensing and buffering.

Restoring the Cell Cycle and Proliferation Competence in Terminally Differentiated Skeletal Muscle Myotubes.

Terminal differentiation is an ill-defined, insufficiently characterized, nonproliferation state. Although it has been classically deemed irreversible, it is now clear that at least several terminally differentiated (TD) cell types can be brought back into the cell cycle. We are striving to uncover the molecular bases of terminal differentiation, whose fundamental understanding is a goal in itself. In addition, the field has sought to acquire the ability to make TD cells proliferate. Attaining this end would probe the very molecular mechanisms we are trying to understand. Equally important, it would be invaluable in regenerative medicine, for tissues depending on TD cells and devoid of significant self-repair capabilities. The skeletal muscle has long been used as a model system to investigate the molecular foundations of terminal differentiation. Here, we summarize more than 50 years of studies in this field.

MYOTONIC DYSTROPHY: EP.235 MyoScreen, a robust muscle-based assay platform validated for screening pharmacologicals against myotonic dystrophy

Development of a predictive in vitro model for deciphering the mechanisms involved in Myotonic Dystrophy type 1 (DM1) and accelerating the identification of new therapies remains a crucial unmet need. Our objective was to provide such an in vitro DM1 model capitalizing on the use of MyoScreen™, a well-established high-throughput screening platform integrating human primary skeletal myotubes that demonstrate mature sarcomeric organization and expected responses to chemical/electrical stimulation and pharmacologically relevant drugs. Primary myoblasts from three DM1 patients (blood CTG repeats from 300-1300) and one healthy donor were obtained, amplified and differentiated into aligned myotubes. Main hallmarks of the disease, such as DMPK mRNA foci colocalizing with MBNL1 protein aggregates, were present in the DM1 myotubes and quantified using high-content analysis. No direct relationship between the CTG expansion size and foci number was noted. The splicing profile of several genes involved in the Excitation-Contraction (EC) coupling machinery are altered in DM1. Thus, calcium flux upon chemical induction of EC coupling was investigated and defects in calcium transients were detected in two DM1 donors. To further characterize DM1 myotubes, Cell Profiling was applied to extract over 300 MBNL1 expression features from the generated images. We found this to be a powerful method to discriminate between DM1 and healthy myotube populations. A corresponding machine learning model was built to measure the efficacy of a (CAG)7 ASO (antisense oligonucleotide) to reverse the DM1-associated characteristics. A dose-dependent rescue by up to 60% was observed in DM1 donors. Overall, the DM1 MyoScreen platform will be useful for predictive preclinical testing to rapidly identify new therapeutic targets and select candidate therapeutics targeting skeletal muscle including ASO conjugates, compounds, siRNAs and AAVs (adenovirus associated vectors) producing antisense RNAs and proteins.

Functional significance of gain-of-function H19 lncRNA in skeletal muscle differentiation and anti-obesity effects

BackgroundExercise training is well established as the most effective way to enhance muscle performance and muscle building. The composition of skeletal muscle fiber type affects systemic energy expenditures, and perturbations in metabolic homeostasis contribute to the onset of obesity and other metabolic dysfunctions. Long noncoding RNAs (lncRNAs) have been demonstrated to play critical roles in diverse cellular processes and diseases, including human cancers; however, the functional importance of lncRNAs in muscle performance, energy balance, and obesity remains elusive. We previously reported that the lncRNA H19 regulates the poly-ubiquitination and protein stability of dystrophin (DMD) in muscular dystrophy.MethodsHere, we identified mouse/human H19-interacting proteins using mouse/human skeletal muscle tissues and liquid chromatography–mass spectrometry (LC-MS). Human induced pluripotent stem-derived skeletal muscle cells (iPSC-SkMC) from a healthy donor and Becker Muscular Dystrophy (BMD) patients were utilized to study DMD post-translational modifications and associated proteins. We identified a gain-of-function (GOF) mutant of H19 and characterized the effects on myoblast differentiation and fusion to myotubes using iPSCs. We then conjugated H19 RNA gain-of-function oligonucleotides (Rgof) with the skeletal muscle enrichment peptide agrin (referred to as AGR-H19-Rgof) and evaluated AGR-H19-Rgof’s effects on skeletal muscle performance using wild-type (WT) C57BL/6 J mice and its anti-obesity effects using high-fat diet (HFD)- and leptin deficiency-induced obese mouse models.ResultsWe demonstrated that both human and mouse H19 associated with DMD and that the H19 GOF exhibited enhanced interaction with DMD compared to WT H19. DMD was found to associate with serine/threonine-protein kinase MRCK alpha (MRCKα) and α-synuclein (SNCA) in iPSC-SkMC derived from BMD patients. Inhibition of MRCKα and SNCA-mediated phosphorylation of DMD antagonized the interaction between H19 and DMD. These signaling events led to improved skeletal muscle cell differentiation and myotube fusion. The administration of AGR-H19-Rgof improved the muscle mass, muscle performance, and base metabolic rate of WT mice. Furthermore, mice treated with AGR-H19-Rgof exhibited resistance to HFD- or leptin deficiency-induced obesity.ConclusionsOur study suggested the functional importance of the H19 GOF mutant in enhancing muscle performance and anti-obesity effects.

Integrated multiomics analysis identifies molecular landscape perturbations during hyperammonemia in skeletal muscle and myotubes

Ammonia is a cytotoxic molecule generated during normal cellular functions. Dysregulated ammonia metabolism, which is evident in many chronic diseases such as liver cirrhosis, heart failure, and chronic obstructive pulmonary disease, initiates a hyperammonemic stress response in tissues including skeletal muscle and in myotubes. Perturbations in levels of specific regulatory molecules have been reported, but the global responses to hyperammonemia are unclear. In this study, we used a multiomics approach to vertically integrate unbiased data generated using an assay for transposase-accessible chromatin with high-throughput sequencing, RNA-Seq, and proteomics. We then horizontally integrated these data across different models of hyperammonemia, including myotubes and mouse and human muscle tissues. Changes in chromatin accessibility and/or expression of genes resulted in distinct clusters of temporal molecular changes including transient, persistent, and delayed responses during hyperammonemia in myotubes. Known responses to hyperammonemia, including mitochondrial and oxidative dysfunction, protein homeostasis disruption, and oxidative stress pathway activation, were enriched in our datasets. During hyperammonemia, pathways that impact skeletal muscle structure and function that were consistently enriched were those that contribute to mitochondrial dysfunction, oxidative stress, and senescence. We made several novel observations, including an enrichment in antiapoptotic B-cell leukemia/lymphoma 2 family protein expression, increased calcium flux, and increased protein glycosylation in myotubes and muscle tissue upon hyperammonemia. Critical molecules in these pathways were validated experimentally. Human skeletal muscle from patients with cirrhosis displayed similar responses, establishing translational relevance. These data demonstrate complex molecular interactions during adaptive and maladaptive responses during the cellular stress response to hyperammonemia.

Insulin signaling pathway assessment by enhancing antioxidant activity due to morin using in vitro rat skeletal muscle L6 myotubes cells.

Plant-derived phytochemicals such as flavonoids have been explored to be powerful antioxidants that protect against oxidative stress-related diseases. In the present study, Morin, a flavonoid compound was studied for its antioxidant and antidiabetic properties in relation to oxidative stress in insulin resistant models conducted in rat skeletal muscle L6 cell line model. Evaluation of antioxidant property of morin was assayed using in vitro methods such as cell viability by MTT assay, estimation of SOD and CAT activity and NO scavenging activity. The anti-oxidative nature of morin on L6 cell line was conducted by the DCF-DA fluorescent activity. Glucose uptake in morin treated L6 myotubes are accessed by 2-NBDG assay in the presence or absence of IRTK and PI3K inhibitors. Further glycogen content estimation due to the morin treatment in L6 myotubes was performed. Antioxidant and insulin signaling pathway gene expression was examined over RT-PCR analysis. Morin has a negligible cytotoxic effect at doses of 20, 40, 60, 80, and 100µM concentration according to cell viability assay. Morin revealed that the levels of the antioxidant enzymes SOD and CAT in L6 myotubes had increased. When the cells were subjected to the nitro blue tetrazolium assay, morin lowered reactive oxygen species (ROS) formation at 60µM concentration displaying 39% ROS generation in oxidative stress condition. Lesser NO activity and a drop in green fluorescence emission in the DCFDA assay, demonstrating its anti-oxidative nature by reducing ROS formation in vitro. Glucose uptake by the L6 myotube cells using 2-NBDG, and with IRTK and PI3K inhibitors (genistein and wortmannin) showed a significant increase in glucose uptake by the cells which shows the up regulated GLUT-4 movement from intracellular pool to the plasma membrane. Morin (60µM) significantly enhanced the expression of antioxidant genes GPx, GST and GCS as well as insulin signalling genes IRTK, IRS-1, PI3K, GLUT-4, GSK-3β and GS in L6 myotubes treated cells. Morin has the ability to act as an anti-oxidant by lowering ROS levels and demonstrating insulin mimetic activity by reversing insulin resistance associated with oxidative stress.

Pathological Mechanism of a Constitutively Active Form of Stromal Interaction Molecule 1 in Skeletal Muscle.

Stromal interaction molecule 1 (STIM1) is the main protein that, along with Orai1, mediates store-operated Ca2+ entry (SOCE) in skeletal muscle. Abnormal SOCE due to mutations in STIM1 is one of the causes of human skeletal muscle diseases. STIM1-R304Q (a constitutively active form of STIM1) has been found in human patients with skeletal muscle phenotypes such as muscle weakness, myalgia, muscle stiffness, and contracture. However, the pathological mechanism(s) of STIM1-R304Q in skeletal muscle have not been well studied. To examine the pathological mechanism(s) of STIM1-R304Q in skeletal muscle, STIM1-R304Q was expressed in mouse primary skeletal myotubes, and the properties of the skeletal myotubes were examined using single-myotube Ca2+ imaging, transmission electron microscopy (TEM), and biochemical approaches. STIM1-R304Q did not interfere with the terminal differentiation of skeletal myoblasts to myotubes and retained the ability of STIM1 to attenuate dihydropyridine receptor (DHPR) activity. STIM1-R304Q induced hyper-SOCE (that exceeded the SOCE by wild-type STIM1) by affecting both the amplitude and the onset rate of SOCE. Unlike that by wild-type STIM1, hyper-SOCE by STIM1-R304Q contributed to a disturbance in Ca2+ distribution between the cytosol and the sarcoplasmic reticulum (SR) (high Ca2+ in the cytosol and low Ca2+ in the SR). Moreover, the hyper-SOCE and the high cytosolic Ca2+ level induced by STIM1-R304Q involve changes in mitochondrial shape. Therefore, a series of these cellular defects induced by STIM1-R304Q could induce deleterious skeletal muscle phenotypes in human patients carrying STIM1-R304Q.

Transdifferentiation of Human Fibroblasts into Skeletal Muscle Cells: Optimization and Assembly into Engineered Tissue Constructs through Biological Ligands.

Simple SummaryEngineered human skeletal muscle tissue is a platform tool that can help scientists and physicians better understand human physiology, pharmacology, and disease modeling. Over the past few years this area of research has been actively being pursued by many labs worldwide. Significant challenges remain, including accessing an adequate cell source, and achieving proper physiological-like architecture of the engineered tissue. To address cell resourcing we aimed at further optimizing a process called transdifferentiation which involves the direct conversion of fibroblasts into skeletal muscle cells. The opportunity here is that fibroblasts are readily available and can be expanded sufficiently to meet the needs of a tissue engineering approach. Additionally, we aimed to demonstrate the applicability of transdifferentiation in assembling tissue engineered skeletal muscle. We implemented a screening process of protein ligands in an effort to refine transdifferentiation, and identified that most proteins resulted in a deficit in transdifferentiation efficiency, although one resulted in robust expansion of cultured cells. We were also successful in assembling engineered constructs consisting of transdifferentiated cells. Future directives involve demonstrating that the engineered tissues are capable of contractile and functional activity, and pursuit of optimizing factors such as electrical and chemical exposure, towards achieving physiological parameters observed in human muscle.The development of robust skeletal muscle models has been challenging due to the partial recapitulation of human physiology and architecture. Reliable and innovative 3D skeletal muscle models recently described offer an alternative that more accurately captures the in vivo environment but require an abundant cell source. Direct reprogramming or transdifferentiation has been considered as an alternative. Recent reports have provided evidence for significant improvements in the efficiency of derivation of human skeletal myotubes from human fibroblasts. Herein we aimed at improving the transdifferentiation process of human fibroblasts (tHFs), in addition to the differentiation of murine skeletal myoblasts (C2C12), and the differentiation of primary human skeletal myoblasts (HSkM). Differentiating or transdifferentiating cells were exposed to single or combinations of biological ligands, including Follistatin, GDF8, FGF2, GDF11, GDF15, hGH, TMSB4X, BMP4, BMP7, IL6, and TNF-α. These were selected for their critical roles in myogenesis and regeneration. C2C12 and tHFs displayed significant differentiation deficits when exposed to FGF2, BMP4, BMP7, and TNF-α, while proliferation was significantly enhanced by FGF2. When exposed to combinations of ligands, we observed consistent deficit differentiation when TNF-α was included. Finally, our direct reprogramming technique allowed for the assembly of elongated, cross-striated, and aligned tHFs within tissue-engineered 3D skeletal muscle constructs. In conclusion, we describe an efficient system to transdifferentiate human fibroblasts into myogenic cells and a platform for the generation of tissue-engineered constructs. Future directions will involve the evaluation of the functional characteristics of these engineered tissues.

Fermented Antler Improves Endurance during Exercise Performance by Increasing Mitochondrial Biogenesis and Muscle Strength in Mice

In this study, we investigated whether antler fermented with lactic acid bacteria (LAB) increases mitochondrial biogenesis and muscle strength in vitro and in vivo. LAB from a strain library were grown in antler extract agar at the Yakult Central Research Institute of Korea. Isolated LAB, named Lactobacillus curvatus HY7602, were used to ferment antlers. Analysis of the effects of fermented antler (FA) revealed that it enhanced the insulin-like growth factor 1 (IGF-I), signaling pathway and mitochondrial metabolic activity in mouse skeletal myotube (C2C12) cells. Next, we evaluated the effect of non-fermented antler (NFA) and FA on exercise performance in C57BL/6J mice. The results showed that HY7602-FA increased treadmill exercise capacity and forced swimming endurance. Furthermore, blood markers associated with muscle fatigue, endurance, and energy supply (e.g., alanine aminotransferase, lactate dehydrogenase, creatinine, creatine kinase, and lactate) in the FA-intake group were lower than in the NFA-intake group. In addition, the expression index of genes associated with muscle protein synthesis, and with mitochondrial energy production and supply, in muscle tissue was remarkably higher in the FA group than in the control and NFA groups. Taken together, these results suggested that HY7602-FA may be an effective functional food and health supplement.

The Golgi apparatus is the main microtubule-organizing center in differentiating skeletal muscle cells.

Studies in differentiating skeletal muscle cells in vitro have revealed that the microtubule-organizing center shifts from the centrosome to the perinuclear sites. As the Golgi apparatus surrounds the nucleus in a myotube, it is unclear whether microtubules are nucleated at the nuclear envelope or at the surrounding Golgi apparatus. In this study, we investigated the positional relationship between the microtubule nucleating sites and the Golgi apparatus in C2C12 myotubes and in primary cultured mouse skeletal myotubes. We focused on gaps in the perinuclear Golgi apparatus where the nuclear envelope was not covered with the Golgi apparatus. In microtubule regrowth assay, microtubule regrowth after cold-nocodazole depolymerization of preexisting microtubules was not found at the gap of the perinuclear Golgi apparatus. Most of the microtubule regrowth was detected at the CDK5RAP2 (CDK5 regulatory subunit-associated protein 2)-rich spots on the perinuclear Golgi apparatus. Disruption of the perinuclear Golgi apparatus with brefeldin A treatment eliminated the perinuclear microtubule regrowth. The Golgi apparatus of undifferentiated myoblasts and those at the cytoplasm of myotubes were also the microtubule nucleating sites. From these observations, we concluded that most of the perinuclear microtubule nucleation occurs on the Golgi apparatus surrounding the nucleus.

A muscle fatigue-like contractile decline was recapitulated using skeletal myotubes from Duchenne muscular dystrophy patient-derived iPSCs

SummaryDuchenne muscular dystrophy (DMD) is a muscle degenerating disease caused by dystrophin deficiency, for which therapeutic options are limited. To facilitate drug development, it is desirable to develop in vitro disease models that enable the evaluation of DMD declines in contractile performance. Here, we show MYOD1-induced differentiation of hiPSCs into functional skeletal myotubes in vitro with collagen gel and electrical field stimulation (EFS). Long-term EFS training (0.5 Hz, 20 V, 2 ms, continuous for 2 weeks) mimicking muscle overuse recapitulates declines in contractile performance in dystrophic myotubes. A screening of clinically relevant drugs using this model detects three compounds that ameliorate this decline. Furthermore, we validate the feasibility of adapting the model to a 96-well culture system using optogenetic technology for large-scale screening. Our results support a disease model using patient-derived iPSCs that allows for the recapitulation of the contractile pathogenesis of DMD and a screening strategy for drug development.

DHA/EPA Improves GLUT4 Translocation, Glycogen Synthesis and Aerobic Glycolysis in Skeletal Muscle of db/db Mice

Abstract Objectives The aim of this study was to investigate the effects of DHA and EPA on glucose metabolism including glucose uptake and disposal in skeletal muscle and C2C12 myotubes. Methods Four-week-old db/db diabetic mice were fed with control diet enriched with DHA/EPA (purity > 99%, 1% wt/wt) for 10 weeks. To further explore the underlying mechanism, C2C12 myotubes were induced insulin resistance by palmitate and treated with 25 and 50 μM DHA/EPA for 24 h after differentiation. Results The untargeted metabolome of skeletal muscle showed BCAAs and other metabolites associated with glycolysis and TCA cycle were altered by DHA/EPA treatment. Further detections revealed DHA/EPA treatment promoted the translocation of GLUT4 via increasing Rab8a and SNAP23 expression, and enhanced the activity of GS and PDH. In vitro, the glucose consumption was improved coupled with promoted Rab8a or SNAP23, and GS and PDH were also activated under DHA/EPA intervention increased glucose consumption via promoted Rab8a and SNAP23. The GS and PDH were also activated, which were in line with the results in vivo. Conclusions Long-term intake of DHA and EPA may have a protective effect on diabetes through promoted GLUT4 translocation, glycogen synthesis and aerobic glycolysis in skeletal muscle. Funding Sources This work was supported by the National Natural Science Foundation of China (grant number 81773419 and 81300309), Chinese Institute of Nutrition DSM Research Fund (grant number CNS-DSM-2017–035), China National Program for Support of Top-notch Young Professionals and China Postdoctoral Science Foundation (grant number 2020M681869).

When multiple caveolins make the difference: Cav1 partly compensates Cav3 alterations and rescues ion channels expression

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Fondazione Cariplo Caveolae are small-membrane invagination that contribute both to buffering excessive contraction-dependent membrane strain and to initiation of membrane repair. Moreover, they constitute micro-domains where receptors and ion channels are clustered, favouring their functional interaction. Caveolin-3 (Cav3) is the key structural component of muscular caveolae. Mutations in Cav3 gene are associated with alterations of the skeletal muscle architecture leading to some rare forms of hereditary skeletal myopathies and/or cardiomyopathies called caveolinopathies. Notably, skeletal muscle dysfunctions usually precede cardiac dysfunctions, even though the mutated Cav3 is expressed in both cell types. An important difference between skeletal fibers and cardiomyocytes is that in the latter, caveolin-1 (Cav1) participates with Cav3 to form caveolae; skeletal myotubes instead do not express Cav1.The delay or lack of onset of cardiac alterations in caveolinopathies may depend on a preserved micro-domains organization in the heart compared to skeletal muscle, due to Cav1 expression. We decided to focus on a specific mutation T78K found in heterozygous in a patient with Ripple muscle disease and hyperCKemia. We have characterized human cardiomyocytes (CM) differentiated from induced pluripotent stem cells (iPSC) derived from this patient and one healthy control. In particular, we have investigated which caveolin isoforms are expressed at day 30 of differentiation, finding both Cav1 and Cav3, with a significant decrease of Cav3 isoform in T78K-CM. Their different expressions significantly increase T78K membrane resistance (3.27 ± 0.6 GΩ versus 1.64 ± 0.4 GΩ in the CTRL-CM), and consequently membrane excitability. The T78K_CM showed an increase spontaneous beating rate compared to CTRL (1,75± 0,08 Hz and 0,89 ± 0,4 Hz, respectively). Previous laboratory analysis conducted in caveolin-free MEF cells, co-transfected with WT and T78K Cav3 mutation, revealed that the T78K mutant is dominant, inducing the retention of WT Cav3 in the perinuclear areas and causes significant reduction in current density of three ion channels (HCN4, Kv1.5 and Kir2.1) known to interact with caveolins. The dominant decreased in cav3 expression is in line with previous data in skeletal muscle biopsy, however, electrophysiological data would be likely incompatible with life. For this reason, we decided to compare the impact of this mutation in CHO cells that exhibit high levels of Cav1, and in cav-1 expressing MEF line. In these systems, the membrane localization of Cav3 T78K is rescued both in heterozygous and homozygous conditions. In line with caveolin membrane expression, HCN4, Kv1.5 and Kir2.1 density is also rescued to normal levels. These results constitute the first evidence of a possible role of Cav1 in compensating membrane disorganization and disfunction due to Cav3 mutations in the heart, making this organ less susceptible to caveolinopathies.

Associations Among Adipose Tissue Immunology, Inflammation, Exosomes and Insulin Sensitivity in People With Obesity and Nonalcoholic Fatty Liver Disease

Insulin resistance is a key factor in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). We evaluated the importance of subcutaneous abdominal adipose tissue (SAAT) inflammation and both plasma and SAAT-derived exosomes in regulating insulin sensitivity in people with obesity and NAFLD. Adipose tissue inflammation (macrophage and T-cell content and expression of proinflammatory cytokines), liver and whole-body insulin sensitivity (assessed using a hyperinsulinemic-euglycemic clamp and glucose tracer infusion), and 24-hour serial plasma cytokine concentrations were evaluated in 3 groups stratified by adiposity and intrahepatic triglyceride (IHTG) content: (1) lean with normal IHTG content (LEAN; N= 14); (2) obese with normal IHTG content (OB-NL; N= 28); and (3) obese with NAFLD (OB-NAFLD; N= 28). The effect of plasma and SAAT-derived exosomes on insulin-stimulated Akt phosphorylation in human skeletal muscle myotubes and mouse primary hepatocytes was assessed in a subset of participants. Proinflammatory macrophages, proinflammatory CD4 and CD8 T-cell populations, and gene expression of several cytokines in SAAT were greater in the OB-NAFLD than the OB-NL and LEAN groups. However, with the exception of PAI-1, which was greater in the OB-NAFLD than the LEAN and OB-NL groups, 24-hour plasma cytokine concentration areas-under-the-curve were not different between groups. The percentage of proinflammatory macrophages and plasma PAI-1 concentration areas-under-the-curve were inversely correlated with both hepatic and whole-body insulin sensitivity. Compared with exosomes from OB-NL participants, plasma and SAAT-derived exosomes from the OB-NAFLD group decreased insulin signaling in myotubes and hepatocytes. Systemic insulin resistance in people with obesity and NAFLD is associated with increased plasma PAI-1 concentrations and both plasma and SAAT-derived exosomes. ClinicalTrials.gov number: NCT02706262 (https://clinicaltrials.gov/ct2/show/NCT02706262).

Diversity-oriented synthesis through gamma radiolysis: Preparation of unusual ecdysteroid derivatives activating Akt and AMPK in skeletal muscle cells

Gamma-ray radiation is a unique way to induce chemical transformations of bioactive compounds. In the present study, we pursued this approach to the diversity-oriented synthesis of analogs of 20-hydroxyecdysone (20E), an abundant ecdysteroid with a range of beneficial, non-hormonal bioactivities in mammals including humans. Gamma irradiations of aqueous solutions of 20E were conducted either in N2- or N2O-saturated solutions. Centrifugal partition chromatography was used to fractionate crude resulting irradiated materials using a biphasic solvent system composed of tert-butyl alcohol - ethyl acetate - water (0.45:0.9:1, v/v/v) in ascending mode. Subsequently, the products were purified by RP-HPLC. Fourteen ecdysteroids, including five new compounds, were isolated, and their structure were elucidated by 1D and 2D NMR and HRMS. Compounds 2–4, 7, 9, 12 and 15 were tested for their capacity to increase the Akt- and AMPK-phosphorylation of C2C12 murine skeletal myotubes in vitro. The compounds were similarly active on Akt as their parent compound. Stachysterone B (7) and a new ring-rearranged compound (12) were more potent than 20E in activating AMPK, indicating a stronger cytoprotective effect. Our results demonstrate the use of gamma irradiation in expanding the chemical diversity of ecdysteroids to obtain new, unusual bioactive metabolites.

Weak Electromagnetic Fields Accelerate Fusion of Myoblasts.

Weak electromagnetic fields (WEF) alter Ca2+ handling in skeletal muscle myotubes. Owing to the involvement of Ca2+ in muscle development, we investigated whether WEF affects fusion of myoblasts in culture. Rat primary myoblast cultures were exposed to WEF (1.75 µT, 16 Hz) for up to six days. Under control conditions, cell fusion and creatine kinase (CK) activity increased in parallel and peaked at 4–6 days. WEF enhanced the extent of fusion after one and two days (by ~40%) vs. control, but not thereafter. Exposure to WEF also enhanced CK activity after two days (almost four-fold), but not afterwards. Incorporation of 3H-thymidine into DNA was enhanced by one-day exposure to WEF (~40%), indicating increased cell replication. Using the potentiometric fluorescent dye di-8-ANEPPS, we found that exposure of cells to 150 mM KCl resulted in depolarization of the cell membrane. However, prior exposure of cells to WEF for one day followed by addition of KCl resulted in hyperpolarization of the cell membrane. Acute exposure of cells to WEF also resulted in hyperpolarization of the cell membrane. Twenty-four hour incubation of myoblasts with gambogic acid, an inhibitor of the inward rectifying K+ channel 2.1 (Kir2.1), did not affect cell fusion, WEF-mediated acceleration of fusion or hyperpolarization. These data demonstrate that WEF accelerates fusion of myoblasts, resulting in myotube formation. The WEF effect is associated with hyperpolarization but WEF does not appear to mediate its effects on fusion by activating Kir2.1 channels.