Function In Skeletal Muscle Cells Research Articles

Static Strength Training Method for Type 2 Diabetic Mice.

The treatment of type 2 diabetes mellitus (T2DM) is a major difficulty in improving patient health. Exercise is one of the main interventions for T2DM. Static strength training is one of the key forms of traditional sports in China. Research shows that static strength training is an effective clinical method for T2DM intervention, but there is no experimental device suitable for static training in mice. One of the difficulties in moving from clinical to basic research is to design appropriate experimental devices. In order to further study the mechanism of static training intervention in T2DM, a simple method for making a static training device for mice is introduced in this paper. This device has the advantages of simple operation, cheap material, and high feasibility. Previous studies conducted under this protocol have shown that static training can effectively reduce blood glucose levels and improve the mitochondrial function of skeletal muscle cells in T2DM mice. The purpose of introducing this device is to promote research on the mechanism of traditional exercise in the intervention of T2DM and to lay a foundation for the quantitative intervention of exercise.

Therapeutic vs. Suprapharmacological Metformin Concentrations: Different Effects on Energy Metabolism and Mitochondrial Function in Skeletal Muscle Cells in vitro.

Metformin is an oral antidiabetic agent that has been widely used in clinical practice for over 60 years, and is currently the most prescribed antidiabetic drug worldwide. However, the molecular mechanisms of metformin action in different tissues are still not completely understood. Although metformin-induced inhibition of mitochondrial respiratory chain Complex I and activation of AMP-activated protein kinase have been observed in many studies, published data is inconsistent. Furthermore, metformin concentrations used for in vitro studies and their pharmacological relevance are a common point of debate. The aim of this study was to explore the effects of different metformin concentrations on energy metabolism and activity of relevant signaling pathways in C2C12 muscle cells in vitro. In order to determine if therapeutic metformin concentrations have an effect on skeletal muscle cells, we used micromolar metformin concentrations (50 µM), and compared the effects with those of higher, millimolar concentrations (5 mM), that have already been established to affect mitochondrial function and AMPK activity. We conducted all experiments in conditions of high (25 mM) and low glucose (5.5 mM) concentration, in order to discern the role of glucose availability on metformin action. According to our results, micromolar metformin treatment did not cause Complex I inhibition nor AMPK activation. Also, cells cultured in low glucose medium were more sensitive to Complex I inhibition, mitochondrial membrane depolarization and AMPK activation by millimolar metformin, but cells cultured in high glucose medium were more prone to induction of ROS production. In conclusion, even though suprapharmacological metformin concentrations cause Complex I inhibition and AMPK activation in skeletal muscle cells in vitro, therapeutic concentrations cause no such effect. This raises the question if these mechanisms are relevant for therapeutic effects of metformin in skeletal muscle.

TGFβ Induces Changes in Mitochondrial Homeostasis in Skeletal Muscle of Mice with Bone Metastases

Cancer cachexia is a common neoplastic syndrome of advanced cancers characterized by a loss of skeletal muscle mass and decreased function. Patients with cancer cachexia have higher chances of morbidity and mortality. We have shown that mice inoculated with human MDA‐MB‐231 breast cancer cells, which metastasize to bone, have skeletal muscle weakness, cachexia, and altered skeletal muscle metabolism. Mice with bone metastases have increased serum levels of transforming growth factor beta (TGFβ). Serum TGFβ decreased after treatment with the bone anti‐resorptive agent, zoledronic acid (ZA), suggesting TGFβ is, in part, bone‐derived. Additionally, mice with bone metastases treated with ZA had increased skeletal muscle function. TGFβ has also been shown to decrease the master regulator of mitochondrial biogenesis, peroxisome‐activated receptor γ coactivator 1α (PGC1α), in C2C12 myotubes. Mitochondria provide muscle with energy (ATP) via oxidative phosphorylation, so we measured oxygen consumption and energy production using indirect calorimetry in mice with bone metastases. Oxygen consumption and energy production were both decreased in these mice suggesting a decrease in mitochondrial function. We then examined skeletal muscle mitochondria using transmission electron microscopy and western blotting. We found highly dysmorphic mitochondria and altered protein markers of mitochondrial biogenesis and dynamics. Additionally, LC3B protein was increased in skeletal muscle mitochondria demonstrating increased mitophagy in skeletal muscle of mice with bone metastases. From our previous knowledge and data of TGFβ and its role in mice with bone metastases, we hypothesize that TGFβ causes mitochondrial dysfunction in skeletal muscle of mice with bone metastases. We have shown that a 48‐hour treatment with TGFβ decreases protein expression in markers of mitochondrial biogenesis and dynamics in C2C12 myotubes. This data suggests that TGFβ could be causing the mitochondrial dysfunction and dysmorphia we see in our mouse model, but further experiments are needed to prove this hypothesis. Our data demonstrates the importance of mitochondrial function in skeletal muscle cells and skeletal muscle of mice with bone metastases. Understanding the role TGFβ plays in changes of skeletal muscle mitochondria in mice with bone metastases will help contribute to defining treatments for patients with cancer cachexia.

Subcellular Specialization of Mitochondrial Form and Function in Skeletal Muscle Cells.

Across different cell types and within single cells, mitochondria are heterogeneous in form and function. In skeletal muscle cells, morphologically and functionally distinct subpopulations of mitochondria have been identified, but the mechanisms by which the subcellular specialization of mitochondria contributes to energy homeostasis in working muscles remains unclear. Here, we discuss the current data regarding mitochondrial heterogeneity in skeletal muscle cells and highlight potential new lines of inquiry that have emerged due to advancements in cellular imaging technologies.

Dapagliflozin Promotes Neovascularization by Improving Paracrine Function of Skeletal Muscle Cells in Diabetic Hindlimb Ischemia Mice Through PHD2/HIF-1α Axis.

Diabetes mellitus is associated with a high risk of hindlimb ischemia (HLI) progression and an inevitably poor prognosis, including worse limb salvage and mortality. Skeletal muscle cells can secrete angiogenic factors, which could promote neovascularization and blood perfusion recovery. Thus, paracrine function of skeletal muscle cells, which is aberrant in diabetic conditions, is crucial for therapeutic angiogenesis in diabetic HLI. Dapagliflozin is a well-known anti-hyperglycemia and anti-obesity drug; however, its role in therapeutic angiogenesis is unknown. Herein, we found that dapagliflozin could act as an angiogenesis stimulator in diabetic HLI. We showed that dapagliflozin enhances the viability, proliferation, and migration potentials of skeletal muscle cells and promotes the secretion of multiple angiogenic factors from skeletal muscle cells, most plausibly through PHD2/HIF-1α axis. Furthermore, we demonstrated that conditioned medium from dapagliflozin-treated skeletal muscle cells enhances the proliferation and migration potentials of vascular endothelial and smooth muscle cells, which are two fundamental cells of functional mature vessels. Finally, an in vivo study demonstrated that intramuscular administration of dapagliflozin effectively enhances the formation of mature blood vessels and, subsequently, blood perfusion recovery in diabetic HLI mice. Hence, our results suggest a novel function of dapagliflozin as a potential therapeutic angiogenesis agent for diabetic HLI.

Caveolin-3 deficiency associated with the dystrophy P104L mutation impairs skeletal muscle mitochondrial form and function.

BackgroundCaveolin‐3 (Cav3) is the principal structural component of caveolae in skeletal muscle. Dominant pathogenic mutations in the Cav3 gene, such as the Limb Girdle Muscular Dystrophy‐1C (LGMD1C) P104L mutation, result in substantial loss of Cav3 and myopathic changes characterized by muscle weakness and wasting. We hypothesize such myopathy may also be associated with disturbances in mitochondrial biology. Herein, we report studies assessing the effects of Cav3 deficiency on mitochondrial form and function in skeletal muscle cells.MethodsL6 myoblasts were stably transfected with Cav3P104L or expression of native Cav3 repressed by shRNA or CRISPR/Cas9 genome editing prior to performing fixed/live cell imaging of mitochondrial morphology, subcellular fractionation and immunoblotting, or analysis of real time mitochondrial respiration. Skeletal muscle from wild‐type and Cav3−/− mice was processed for analysis of mitochondrial proteins by immunoblotting.ResultsCaveolin‐3 was detected in mitochondrial‐enriched membranes isolated from mouse gastrocnemius muscle and L6 myoblasts. Expression of Cav3P104L in L6 myoblasts led to its targeting to the Golgi and loss of native Cav3 (>95%), including that associated with mitochondrial membranes. Cav3P104L reduced mitochondrial mass and induced fragmentation of the mitochondrial network that was associated with significant loss of proteins involved in mitochondrial biogenesis, respiration, morphology, and redox function [i.e. PGC1α, succinate dehyrdogenase (SDHA), ANT1, MFN2, OPA1, and MnSOD). Furthermore, Cav3P104L myoblasts exhibited increased mitochondrial cholesterol and loss of cardiolipin. Consistent with these changes, Cav3P104L expression reduced mitochondrial respiratory capacity and increased myocellular superoxide production. These morphological, biochemical, and functional mitochondrial changes were phenocopied in myoblasts in which Cav3 had been silenced/knocked‐out using shRNA or CRISPR. Reduced mitochondrial mass, PGC1α, SDHA, ANT1, and MnSOD were also demonstrable in Cav3−/− mouse gastrocnemius. Strikingly, Cav3 re‐expression in Cav3KO myoblasts restored its mitochondrial association and facilitated reformation of a tubular mitochondrial network. Significantly, re‐expression also mitigated changes in mitochondrial superoxide, cholesterol, and cardiolipin content and recovered cellular respiratory capacity.ConclusionsOur results identify Cav3 as an important regulator of mitochondrial homeostasis and reveal that Cav3 deficiency in muscle cells associated with the Cav3P104L mutation invokes major disturbances in mitochondrial respiration and energy status that may contribute to the pathology of LGMD1C.

Tyrosol Facilitates Neovascularization by Enhancing Skeletal Muscle Cells Viability and Paracrine Function in Diabetic Hindlimb Ischemia Mice.

As one of the most severe manifestations of diabetes, vascular complications are the main causes of diabetes-related morbidity and mortality. Hyperglycemia induces systemic abnormalities, including impaired angiogenesis, causing diabetic patients to be highly susceptible in suffering hindlimb ischemia (HLI). Despite its severe prognosis, there is currently no effective treatment for diabetic HLI. Skeletal muscle cells secrete multiple angiogenic factors, hence, recently are reported to be critical for angiogenesis; however, hyperglycemia disrupted the paracrine function in skeletal muscle cells, leading to the impaired angiogenesis potential observed in diabetic patients. The present study showed that tyrosol, a phenylethanoid compound, suppresses accumulation of intracellular reactive oxygen species (ROS) caused by hyperglycemia, most plausibly by promoting heme oxygenase-1 (HO-1) expression in skeletal muscle cells. Consequently, tyrosol exerts cytoprotective function against hyperglycemia-induced oxidative stress in skeletal muscle cells, increases their proliferation vigorously, and simultaneously suppresses apoptosis. Furthermore, tyrosol grossly increases the secretion of vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) from skeletal muscle cells. This leads to enhanced proliferation and migration capabilities of vascular endothelial and smooth muscle cells, two types of cells that are responsible in forming blood vessels, through cell-cell communication. Finally, in vivo experiment using the diabetic HLI mouse model showed that tyrosol injection into the gastrocnemius muscle of the ischemic hindlimb significantly enhances the formation of functional blood vessels and subsequently leads to significant recovery of blood perfusion. Overall, our findings highlight the potential of the pharmacological application of tyrosol as a small molecule drug for therapeutic angiogenesis in diabetic HLI patients.

Analysis of Ca(2+) signaling mechanisms - our experience on the intercellular communication in muscle remodeling.

The aim of this study was to evaluate cell diversity by considering how Ca(2+) signaling has been adapted in skeletal muscle cell function. We characterized single C2C12 myoblasts through intracellular Ca(2+) signaling kinetics after exposure to specific drugs and calcium blockers using fast fluorescence microspectrofluorimetry followed by ATP effect analysis, which confirmed the expression of functional purinergic adenosine and P2 receptors. Further, we found that glutamate sensitivity of C2C12 cells was mediated by ionotropic glutamate receptors; on the other hand, most cells were responsive to cyclopiazonic acid, which inhibits the sarco-endoplasmic reticulum Ca(2+)-ATPase pump. These results suggest that C2C12 cells possess functional L- and P/Q-type voltage-operated Ca(2+) channels, ryanodine receptors and functional sarcoplasmic reticulumCa(2+) stores (typical for muscle cells), adenosine and P2 purinergic receptors, as well as ionotropic glutamate receptors. The evaluation of intracellular Ca(2+) signaling is a promising approach towards a better understanding and control of the physiopathological properties of myogenic cells that could be used as a predictive factor in the selection of optimal cells for scaffold recellularization or for tissue engineered constructs used in stem cell therapy.

Salidroside-Pretreated Mesenchymal Stem Cells Enhance Diabetic Wound Healing by Promoting Paracrine Function and Survival of Mesenchymal Stem Cells Under Hyperglycemia.

Systemic abnormalities cause several complications in diabetes patients. Impaired wound healing is a serious complication that leads to severe foot ulcer and amputation. Mesenchymal stem cells (MSCs) have been considered a promising strategy for promoting wound healing due to their paracrine function. However, their poor survival after transplantation limits their therapeutic effect and applications. Salidroside, a glucopyranoside, has been reported to exert cytoprotective effects. Our previous study revealed that salidroside could promote the paracrine function of skeletal muscle cells. However, whether salidroside could improve MSCs survival under hyperglycemic condition and, subsequently, promote wound healing in diabetic model mice remains unknown. Here, we found that salidroside pretreatment effectively reversed the hyperglycemia‐induced suppression of the expression of crucial wound healing factors in MSCs, such as heme oxygenase‐1 (HO‐1), fibroblast growth factor 2 (FGF2), and hepatocyte growth factor (HGF). Salidroside pretreatment also suppressed the hyperglycemia‐induced intracellular reactive oxygen species (ROS) levels in MSCs, thereby lowering the apoptosis rate and enhancing MSCs survival rate. Furthermore, salidroside improved the MSCs migration potential that was impaired under hyperglycemia. in vivo experiments revealed that salidroside pretreatment prior to transplantation significantly enhanced the effect of MSCs in promoting wound closure in diabetic mice. Collectively, our results suggest that pretreatment with salidroside could be an effective strategy to enhance the survival rate and the therapeutic effect of MSCs. Thus, our article suggested a novel, potential MSC‐based strategy for diabetic wound healing. stem cells translational medicine 2019;8:404–414

Specific effects of neuregulin-1β on the communication between DRG neurons and skeletal muscle cells in vitro.

The communication between primary afferent neuron and skeletal muscle (SKM) is one of the important factors on maintaining the structure and function of SKM cells. Neuregulin-1β (NRG-1β) signaling is essential for regulating synaptic neurotransmission. Here, we established a neuromuscular coculture model of dorsal root ganglion (DRG) sensory neurons and SKM cells to explore the nerve-muscle communication in the presence of exogenous NRG-1β. The expression of three distinct subtypes (TrkA, TrkB, and TrkC) of tyrosine kinase receptors was monitored for the phenotypical alterations of the neurons. The aggregation extent of acetylcholine receptor (AChR) represents the specific changes of SKM cells after NRG-1β incubation in this neuromuscular coculture model. The results showed that NRG-1β not only enhanced neurite outgrowth of DRG neurons but also increased the length and branches of SKM cells. NRG-1β treatment not only induced expression of all the three subtypes of Trk receptors in neurons but also promoted AChR aggregation on the surface of SKM cells. The effects of NRG-1β could be blocked by administration of ERK1/2 inhibitor PD98059, PI3K inhibitor LY294002, and JAK2 inhibitor AG490, respectively. These data imply that NRG-1β is essential for the nerve-muscle communication by enhancing growth and modifying phenotypes of the two different kinds of cells. The specific effects produced by NRG-1β add novel interpretation for nerve-muscle communication between sensory neurons and SKM cells.

Protein arginine methyltransferase expression and activity during myogenesis.

Despite the emerging importance of protein arginine methyltransferases (PRMTs) in regulating skeletal muscle plasticity, PRMT biology during muscle development is complex and not completely understood. Therefore, our purpose was to investigate PRMT1, -4, and -5 expression and function in skeletal muscle cells during the phenotypic remodeling elicited by myogenesis. C2C12 muscle cell maturation, assessed during the myoblast (MB) stage, and during days 1, 3, 5, and 7 of differentiation, was employed as an in vitro model of myogenesis. We observed PRMT-specific patterns of expression and activity during myogenesis. PRMT4 and -5 gene expression was unchanged, while PRMT1 mRNA and protein content were significantly induced. Cellular monomethylarginines (MMAs) and symmetric dimethylarginines (SDMAs), indicative of global and type II PRMT activities, respectively, remained steady during development, while type I PRMT activity indicator asymmetric dimethylarginines (ADMAs) increased through myogenesis. Histone 4 arginine 3 (H4R3) and H3R17 contents were elevated coincident with the myonuclear accumulation of PRMT1 and -4. Collectively, this suggests that PRMTs are methyl donors throughout myogenesis and demonstrate specificity for their protein targets. Cells were then treated with TC-E 5003 (TC-E), a selective inhibitor of PRMT1 in order to specifically examine the enzymes role during myogenic differentiation. TC-E treated cells exhibited decrements in muscle differentiation, which were consistent with attenuated mitochondrial biogenesis and respiratory function. In summary, the present study increases our understanding of PRMT1, -4, and -5 biology during the plasticity of skeletal muscle development. Our results provide evidence for a role of PRMT1, via a mitochondrially mediated mechanism, in driving the muscle differentiation program.

Differentiation-related glycan epitopes identify discrete domains of the muscle glycocalyx.

The neuromuscular junction (NMJ) is enriched with glycoproteins modified with N-acetylgalactosamine (GalNAc) residues, and four nominally GalNAc-specific plant lectins have historically been used to identify the NMJ and the utrophin-glycoprotein complex. However, little is known about the specific glycan epitopes on skeletal muscle that are bound by these lectins, the glycoproteins that bear these epitopes or how creation of these glycan epitopes is regulated. Here, we profile changes in cell surface glycosylation during muscle cell differentiation and identify distinct differences in the binding preferences of GalNAc-specific lectins, Wisteria floribunda agglutinin (WFA), Vicia villosa agglutinin (VVA), soybean agglutinin (SBA) and Dolichos biflorus agglutinin (DBA). While we find that all four GalNAc binding lectins specifically label the NMJ, each of the four lectins binds distinct sets of muscle glycoproteins; furthermore, none of the major adhesion complexes are required for binding of any of the four GalNAc-specific lectins. Analysis of glycosylation-related transcripts identified target glycosyltransferases and glycosidases that could potentially create GalNAc-containing epitopes; reducing expression of these transcripts by siRNA highlighted differences in lectin binding specificities. In addition, we found that complex N-glycans are required for binding of WFA and SBA to murine C2C12 myotubes and for WFA binding to wild-type skeletal muscle, but not for binding of VVA or DBA. These results demonstrate that muscle cell surface glycosylation is finely regulated during muscle differentiation in a domain- and acceptor-substrate-specific manner, suggesting that temporal- and site-specific glycosylation are important for skeletal muscle cell function.

Sarcolipin-Mediated Regulation of SERCA by Computer Simulations

The sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) is a transmembrane pump that, upon ATP hydrolysis, pumps Ca2+ against a concentration gradient from the cytosol to the sarco- or endoplasmic reticulum, thus terminating muscle contraction and priming the cell for the next excitation-contraction stimulus1. SERCA function in skeletal muscle cells is regulated by sarcolipin2,3 (SLN), a 31 amino acid transmembrane peptide that inhibits SERCA by lowering its apparent Ca2+ affinity. By combining solid-state NMR and cross-linking experiments, we characterized the structure of the SERCA/SLN complex in its natural membrane environment in both Ca2-E1:ATP and Hn-E2:ATP states of the catalytic cycle. In this work, we employ molecular dynamics computer simulations to investigate SERCA's mechanism of regulation by mapping the effects of SLN binding on SERCA's free energy landscape, sub-microsecond structural dynamics, and allosteric coupling between the transmembrane and cytoplasmic domains. Our calculations indicate that upon binding to SERCA, SLN increases its average tilt angle with respect the unbound state. The interaction between the two proteins results in a shift of the free energy basin of SERCA and in an altered coupling between its transmembrane and cytoplasmic domains, which may be responsible for the reduced activity of SERCA in its SLN-bound state.

Milk-derived ribonuclease 5 preparations induce myogenic differentiation in vitro and muscle growth in vivo

Ribonuclease 5, also known as angiogenin, is a stable and abundant ribonuclease in milk whey protein, which is able to regulate several cellular functions, including capillary formation, neuron survival, and epithelial cell growth. Ribonuclease 5 is important for protein synthesis directly stimulating rRNA synthesis in the nucleolus. Here, we show that biologically active RNase5 can be purified from bovine milk. Furthermore, we show that milk-derived RNase5 directly stimulates muscle cell differentiation in vitro, inducing C2C12 cell differentiation and myogenesis. When supplemented into the diet of healthy adult mice, milk-derived RNase5 preparations promoted muscle weight gain and grip strength. Collectively, these data indicate that milk-derived RNase5 preparations exhibit a novel role in skeletal muscle cell function.

Anoctamin 6 Regulates C2C12 Myoblast Proliferation

Anoctamin 6 (Ano6) belongs to a conserved gene family (TMEM16) predicted to code for eight transmembrane proteins with putative Ca2+-activated chloride channel (CaCC) activity. Recent work revealed that disruption of ANO6 leads to a blood coagulation defect and impaired skeletal development. However, its function in skeletal muscle cells remains to be determined. By using a RNA interference mediated (RNAi) loss-of-function approach, we show that Ano6 regulates C2C12 myoblast proliferation. Ano6 is highly expressed in C2C12 myoblasts and its expression decreases upon differentiation. Knocking down Ano6 significantly reduces C2C12 myoblast proliferation but has minimal effect on differentiation. Ano6 deficiency significantly reduces ERK/AKT phosphorylation, which has been shown to be involved in regulation of cancer cell proliferation by another Anoctamin member. Taken together, our data demonstrate for the first time that Ano6 plays an essential role in C2C12 myoblast proliferation, likely via regulating the ERK/AKT signaling pathway.

GPR43 regulates metabolic function in skeletal muscle cells

Introduction: Preliminary data suggests a putative role for GPR43 in regulating systemic health via processes including adipogenesis, inflammation and secretion of gastrointestinal peptides. This suggests GPR43 may have a role in weight control. Given muscle is a major determinant of systemic metabolic health and energy balance, we sought to characterise the effect of activating GPR43 in skeletal and cardiac muscle cells. Methods: C2C12 skeletal muscle myotubes and H9c2 cardiac muscle myoblasts were exposed to a GPR43 specific phenylacetamide (PA) agonist (70 nM to 7 M) for 15min (n = 5—8/group). Protein expression was quantified by Western blotting. Metabolic activity of C2C12 and H9c2 cells treated with PA was determined via MTT assay (n = 10—12/group). Results: In C2C12 myotubes 70 nM PA significantly up-regulated total JNK and AMPK proteins compared to control (p≤ 0.05). There was no change in the phosphorylation of these markers or the abundance of total or phosphorylated ERK1/2 protein at any dose used. Metabolic activity in C2C12 myotubes was decreased at 70 nM and 7 M PA compared to control (p≤ 0.05). Interestingly, PA treatment did not alter protein expression or metabolic activity in H9c2 myoblasts. Conclusion: These data suggest GPR43 may have a specific role in mediating skeletal muscle metabolic function. Decrements in metabolic activity in C2C12 myotubes imply GPR43 agonism may impinge upon nutrient metabolism in skeletal muscle. This has the potential to promote aberrations of systemic metabolic health. Thus, targeting GPR43 may form a novel means of treating obesity and type 2 diabetes mellitus in which skeletal muscle fatty acid and glucose metabolism is perturbed. Further work is needed to clarify the signaling pathways mediating decreased metabolic activity and how this relates to functional changes in nutrient metabolism. LMC was supported by a scholarship (PB 10M 5472) from the National Heart Foundation of Australia.

Mitochondrial and performance adaptations to exercise training in mice lacking skeletal muscle LKB1

LKB1 and its downstream targets of the AMP-activated protein kinase family are important regulators of many aspects of skeletal muscle cell function, including control of mitochondrial content and capillarity. LKB1 deficiency in skeletal and cardiac muscle (mLKB1-KO) greatly impairs exercise capacity. However, cardiac dysfunction in that genetic model prevents a clear assessment of the role of skeletal muscle LKB1 in the observed effects. Our purposes here were to determine whether skeletal muscle-specific knockout of LKB1 (skmLKB1-KO) decreases exercise capacity and mitochondrial protein content, impairs accretion of mitochondrial proteins after exercise training, and attenuates improvement in running performance after exercise training. We found that treadmill and voluntary wheel running capacity was reduced in skmLKB1-KO vs. control (CON) mice. Citrate synthase activity, succinate dehydrogenase activity, and pyruvate dehydrogenase kinase content were lower in KO vs. CON muscles. Three weeks of treadmill training resulted in significantly increased treadmill running performance in both CON and skmLKB1-KO mice. Citrate synthase activity increased significantly with training in both genotypes, but protein content and activity for components of the mitochondrial electron transport chain increased only in CON mice. Capillarity and VEGF protein was lower in skmLKB1-KO vs. CON muscles, but VEGF increased with training only in skmLKB1-KO. Three hours after an acute bout of muscle contractions, PGC-1α, cytochrome c, and VEGF gene expression all increased in CON but not skmLKB1-KO muscles. Our findings indicate that skeletal muscle LKB1 is required for accretion of some mitochondrial proteins but not for early exercise capacity improvements with exercise training.

The miR‐696 contributes to insulin resistance by reducing mitochondrial function and antioxidant capacity in skeletal muscle cells

Insulin resistance is associated to type II diabetes but its mechanism is not fully understood. Micro‐RNAs have been described as non‐coding molecules associated with post‐transcriptional regulation of several physiological processes including diabetes. We have observed that miR‐696 is regulated in an opposite manner compared to PGC‐1α leading to a proposition that the miR‐696 may be involved to insulin resistance. Mice mioblast cells (C2C12) were treated with palmitic acid (500μM) for 24 h. The insulin resistance was confirmed by evaluation of Akt phosphorylation after insulin incubation by western blot. Expression of mature miR‐696 was evaluated by real‐time RT‐PCR using TaqMan® MicroRNA Assay. The expression of microRNAs was normalized with the SnoR‐202. Gene expression of PGC1α was evaluated by real time RT‐PCR using Sybr Green. Analysis of oxygen consumption was performed by oxygraph (Hansatech) using CCCP and oligomycin as controls. We observed that PGC1α transcription was markedly reduced in insulin resistant cells, an effect that was reflected by a low mitochondrial respiration, reduced antioxidant capacity and elevated miR‐696 level. In contrast, the PGC1α transcription was markedly elevated after AICAR treatment as reflected by elevated mitochondrial respiration and antioxidant capacity and low miR‐696 level. Interestingly, the inhibition of miR‐696 transcription in insulin resistant cells re‐established the insulin response, PGC1α transcription and mitochondrial function indicating an association of miR‐696 and mitochondrial biogenesis. Therefore, we are suggesting that miR‐696 contributes to insulin resistance reducing mitochondrial biogenesis and mitochondrial function in skeletal muscle cells.

METTL21C: From GWAS to in vitro function in skeletal muscle cells

Sarcopenia and osteoporosis are among the most serious and costly public health problems and often occur concurrently. Bivariate genome‐wide association studies (GWAS) identified METTL21C as a candidate gene for bone‐muscle pleotropy. Intriguingly, METTL21C is highly expressed in skeletal muscles, but the roles of this gene are largely unknown. We used siRNAs to silence METTL21C in C2C12 myoblasts to study its function on myogenesis. After 24h of transfection, real‐time PCR revealed that METTL21C expression was reduced by 30–40% in the siRNA‐treated cells. Two days after the onset of differentiation, a significantly reduced number of fusing myocytes was observed. At day 4, early myotubes were clearly observed in the control group but not in the siRNA‐treated cells. By day 7, fully matured and developed myotubes were observed in the control but not in the siRNA‐treated cells. Silencing of this relatively unknown gene led to a striking phenotype of fewer, shorter and smaller myotubes. These results suggest that METTL21C plays an important role in skeletal muscle cell differentiation. We are now performing similar studies in MLO‐Y4 osteocytes and plan to expand these studies to include biochemical and functional analyses to determine and validate its pleiotropic effects in in‐vitro models of bone and muscle research. Support: NIH‐NIA P01 AG039355 and Missouri Life Sci. Res. Board (MB) and R01 AR057118 (DK)

Proline Isomerase Pin1 Represses Terminal Differentiation and Myocyte Enhancer Factor 2C Function in Skeletal Muscle Cells

Reversible proline-directed phosphorylation at Ser/Thr-Pro motifs has an essential role in myogenesis, a multistep process strictly regulated by several signaling pathways that impinge on two families of myogenic effectors, the basic helix-loop-helix myogenic transcription factors and the MEF2 (myocyte enhancer factor 2) proteins. The question of how these signals are deciphered by the myogenic effectors remains largely unaddressed. In this study, we show that the peptidyl-prolyl isomerase Pin1, which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds to induce conformational changes of its target proteins, acts as an inhibitor of muscle differentiation because its knockdown in myoblasts promotes myotube formation. With the aim of clarifying the mechanism of Pin1 function in skeletal myogenesis, we investigated whether MEF2C, a critical regulator of the myogenic program that is the end point of several signaling pathways, might serve as a/the target for the inhibitory effects of Pin1 on muscle differentiation. We show that Pin1 interacts selectively with phosphorylated MEF2C in skeletal muscle cells, both in vitro and in vivo. The interaction with Pin1 requires two novel critical phospho-Ser/Thr-Pro motifs in MEF2C, Ser98 and Ser110, which are phosphorylated in vivo. Overexpression of Pin1 decreases MEF2C stability and activity and its ability to cooperate with MyoD to activate myogenic conversion. Collectively, these findings reveal a novel role for Pin1 as a regulator of muscle terminal differentiation and suggest that Pin1-mediated repression of MEF2C function could contribute to this function.