Resveratrol corrects aberrant splicing of RYR1 pre-mRNA and Ca2+ signal in myotonic dystrophy type 1 myotubes

Myotonic dystrophy type 1(DM1) is a spliceopathy related to the mis-splicing of several genes caused by sequestration of nuclear transcriptional RNA-binding factors from non-coding CUG repeats of DMPK pre-mRNAs. Dysregulation of ryanodine receptor 1(RYR1), sarcoplasmatic/endoplasmatic Ca^2+-ATPase(SERCA) and α1 S subunit of voltage-gated Ca^2+ channels(Cav1.1) is related to Ca^2+ homeostasis and excitation-contraction coupling impairment. Though no pharmacological treatment for DM1 exists, aberrant splicing correction represents one major therapeutic target for this disease. Resveratrol(RES, 3,5,4′-trihydroxy-trans-stilbene) is a promising pharmacological tools for DM1 treatment for its ability to directly bind the DNA and RNA influencing gene expression and alternative splicing. Herein, we analyzed the therapeutic effects of RES in DM1 myotubes in a pilot study including cultured myotubes from two DM1 patients and two healthy controls. Our results indicated that RES treatment corrected the aberrant splicing of RYR1, and this event appeared associated with restoring of depolarization-induced Ca^2+ release from RYR1 dependent on the electro-mechanical coupling between RYR1 and Cav1.1. Interestingly, immunoblotting studies showed that RES treatment was associated with a reduction in the levels of CUGBP Elav-like family member 1, while RYR1, Cav1.1 and SERCA1 protein levels were unchanged. Finally, RES treatment did not induce any major changes either in the amount of ribonuclear foci or sequestration of muscleblind-like splicing regulator 1. Overall, the results of this pilot study would support RES as an attractive compound for future clinical trials in DM1. Ethical approval was obtained from the Ethical Committee of IRCCS Fondazione Policlinico Universitario A. Gemelli, Rome, Italy(rs9879/14) on May 20, 2014.

Agaro-Oligosaccharides Prevent Myostatin Hyperexpression and Myosin Heavy Chain Protein Degradation in C2C12 Myotubes Induced by Tumor Necrosis Factor-<i>α</i>

Myostatin is a major factor involved in the regulation of skeletal muscle protein mass. High myostatin levels have been associated with an increase in myotube shrinkage. Enhanced myostatin expression is caused by pro-catabolic reactions involving compounds such as tumor necrosis factor (TNF)-α. The present study investigated the effects of agaro-oligosaccharides (AOSs) on hypercatabolism of myotubes exposed to TNF-α. C2C12 myotubes exposed to TNF-α in the presence or absence of AOSs. Myotube exposure to TNF-α resulted in a reduction in the amount of myosin heavy chain (MyHC) protein and a decrease in myotube diameter, which was associated with increased myostatin mRNA expression. AOSs prevented TNF-α-induced MyHC protein loss and restored normal myostatin mRNA levels, with agarobiose and agarotetraose effectively suppressing the hyperexpression of the mRNA. In addition, expression levels of the known myostatin inhibitors, latent transforming growth factor beta binding protein 3 (Ltbp3) and growth and differentiation factor-associated serum protein 1 (Gasp1) mRNAs, decreased more in TNF-α-induced myotubes than in the TNF-α-free control, possibly resulting in myostatin upregulation. However, AOSs restored nearly normal expression levels of Ltbp3 and Gasp1 mRNA, potentially suppressing myostatin expression. These findings suggest that AOSs could prevent myotube shrinkage induced by TNF-α.

Antidiabetic effect of Chrysophyllum albidum is mediated by enzyme inhibition and enhancement of glucose uptake via 3T3-L1 adipocytes and C2C12 myotubes

Objective:To investigate the in vivo and in vitro antidiabetic potential of Chrysophyllum albidum.Methods:The effects of oral treatment with hydro-ethanolic extract(125,250 and 500 mg/kg)of the stem bark of Chrysophyllum albidum and glibenclamide for 21 d on glucose level,serum enzyme markers for liver function,lipid profile,total protein,serum urea,serum creatinine,and body weight were evaluated in experimental diabetic rats administered with 45 mg/kg of streptozotocin.In vitro assays including glucose uptake in C2 C12 cells and 3 T3-L1 adipose tissues,α-glucosidase andα-amylase inhibition were employed to evaluate the possible mechanism of hypoglycemic action of the extract.DPPH and nitric oxide radical antioxidant activity of the extract was also measured.Results:The increased levels of blood glucose,triglycerides,lowdensity lipoprotein,total cholesterol,serum aspartate,and alanine transaminases,creatinine,and urea in the diabetic animals were reduced significantly(P<0.01)after treatment with Chrysophyllum albidum extract.The decreased total protein and high-density lipoprotein concentrations were normalized after treatment.In addition,the extract significantly(P<0.01)increased the transport of glucose in 3 T3-L1 cells and C2 C12 myotubes and exhibited considerable potential to inhibitα-amylase andα-glucosidase.It also demonstrated potent antioxidant action by scavenging considerably DPPH and nitric oxide radicals.Conclusions:Chrysophyllum albidum stem bark extract exhibits considerable antidiabetic effect by stimulating glucose uptake and utilization in C2 C12 myotubes and 3 T3-L1 adipocytes as well as inhibiting the activities ofα-amylase andα-glucosidase.

Methanolic extract of Momordica cymbalaria enhances glucose uptake in L6 myotubes in vitro by up-regulating PPAR-γ and GLUT-4

The present study was undertaken to evaluate the influence of the methanolic fruit extract of Momordica cymbalaria （MFMC） on PPARγ, （Peroxisome Proliferator Activated Receptor gamma） and GLUT-4 （Glucose transporter-4） with respect to glucose transport. Various concentrations of MFMC ranging from 62.5 to 500 μg·mL^-1 were evaluated for glucose uptake activity in vitro using L6 myotubes, rosiglitazone was used as a reference standard. The MFMC showed significant and dose-dependent increase in glucose uptake at the tested concentrations, further, the glucose uptake activity of MFMC （500 μg·mL^-1） was comparable with rosigilitazone. Furthermore, MFMC has shown up-regulation of GLUT-4 and PPARy gene expressions in L6 myotubes. In addition, the MFMC when incubated along with cycloheximide （CHX）, which is a protein synthesis inhibitor, has shown complete blockade of glucose uptake. This indicates that new protein synthesis is required for increased GLUT-4 translocation. In conclusion, these findings suggest that MFMC is enhancing the glucose uptake significantly and dose dependently through the enhanced expression of PPARμ and GLUT-4 in vitro.

Crosstalk among canonical Wnt and Hippo pathway members in skeletal muscle and at the neuromuscular junction

Skeletal muscles are essential for locomotion,posture,and metabolic regulation.To understand physiological processes,exercise adaptation,and muscle-related disorders,it is critical to understand the molecular pathways that underlie skeletal muscle function.The process of muscle contra ction,orchestrated by a complex interplay of molecular events,is at the core of skeletal muscle function.Muscle contraction is initiated by an action potential and neuromuscular transmission requiring a neuromuscular junction.Within muscle fibers,calcium ions play a critical role in mediating the interaction between actin and myosin filaments that generate force.Regulation of calcium release from the sarcoplasmic reticulum plays a key role in excitation-contraction coupling.The development and growth of skeletal muscle are regulated by a network of molecular pathways collectively known as myogenesis.Myogenic regulators coordinate the diffe rentiation of myoblasts into mature muscle fibers.Signaling pathways regulate muscle protein synthesis and hypertrophy in response to mechanical stimuli and nutrient availability.Seve ral muscle-related diseases,including congenital myasthenic disorders,sarcopenia,muscular dystrophies,and metabolic myopathies,are underpinned by dys regulated molecular pathways in skeletal muscle.Therapeutic interventions aimed at preserving muscle mass and function,enhancing regeneration,and improving metabolic health hold promise by targeting specific molecular pathways.Other molecular signaling pathways in skeletal muscle include the canonical Wnt signaling pathway,a critical regulator of myogenesis,muscle regeneration,and metabolic function,and the Hippo signaling pathway.In recent years,more details have been uncovered about the role of these two pathways during myogenesis and in developing and adult skeletal muscle fibers,and at the neuromuscular junction.In fact,research in the last few years now suggests that these two signaling pathways are interconnected and that they jointly control physiological and pathophysiological processes in muscle fibers.In this review,we will summarize and discuss the data on these two pathways,focusing on their concerted action next to their contribution to skeletal muscle biology.However,an in-depth discussion of the noncanonical Wnt pathway,the fibro/a dipogenic precursors,or the mechanosensory aspects of these pathways is not the focus of this review.

Identification of transition factors in myotube formation from proteome and transcriptome analyses

Muscle fibers are the main component of skeletal muscle and undergo maturation through the formation of myotubes.During early development,a population of skeletal muscle satellite cells(SSCs)proliferate into myoblasts.The myoblasts then undergo further differentiation and fusion events,leading to the development of myotubes.However,the mechanisms involved in the transition from SSCs to myotube formation remain unclear.In this study,we characterized changes in the proteomic and transcriptomic expression profiles of SSCs,myoblasts(differentiation for 2 d)and myotubes(differentiation for 10 d).Proteomic analysis identified SLMAP and STOM as potentially associated with myotube formation.In addition,some different changes in MyoD,MyoG,Myosin7 and Desmin occurred after silencing SLMAP and STOM,suggesting that they may affect changes in the myogenic marker.GO analysis indicated that the differentiation and migration factors SVIL,ENSCHIG00000026624(AQP1)and SERPINE1 enhanced the transition from SSCs to myoblasts,accompanied by changes in the apoptotic balance.In the myoblast vs.myotube group,candidates related to cell adhesion and signal transduction were highly expressed in the myotubes.Additionally,CCN2,TGFB1,MYL2 and MYL4 were identified as hub-candidates in this group.These data enhance our existing understanding of myotube formation during early development and repair.

World’s First Myoblast Treatment of Human Cancer Found Safe and Efficacious

Evolution of placental mammals over the past 160 million years witnesses the relative sparing of muscles from cancer attacks. In 1) nude mice with human gastrointestinal or lung tumors, and 2) human subjects with liver, lung or gastrointestinal tumors, intra-tumor implantation of allogeneic human myoblasts induced cancer apoptosis, inhibiting metastasis and tumor growth. We postulate four mechanisms of cancer apoptosis: a) myoblasts releasing tumor necrosis factor-α (TNF-α);b) deprivation of nutrients and oxygen;c) local inflammatory and immunologic attacks;and d) prevention from metastasis. These basic and clinical studies demonstrated preliminary safety and efficacy of intra-tumor myoblast implantation in the development of prevention and treatment for cancer, now the number one disease killer of mankind.

GSK3β inhibitor promotes myelination and mitigates muscle atrophy after peripheral nerve injury

Delay of axon regeneration after peripheral nerve injury usually leads to progressive muscle atrophy and poor functional recovery. The Wnt/β-catenin signaling pathway is considered to be one of the main molecular mechanisms that lead to skeletal muscle atrophy in the elderly. We hold the hypothesis that the innervation of target muscle can be promoted by accelerating axon regeneration and decelerating muscle cell degeneration so as to improve functional recovery of skeletal muscle following peripheral nerve injury. This process may be associated with the Wnt/β-catenin signaling pathway. Our study designed in vitro cell models to simulate myelin regeneration and muscle atrophy. We investigated the effects of SB216763, a glycogen synthase kinase 3 beta inhibitor, on the two major murine cell lines RSC96 and C2C12 derived from Schwann cells and muscle satellite cells. The results showed that SB216763 stimulated the Schwann cell migra- tion and myotube contraction. Quantitative polymerase chain reaction results demonstrated that myelin related genes, myelin associated glycoprotein and cyclin-D1, muscle related gene myogenin and endplate-associated gene nicotinic acetylcholine receptors levels were stimulated by SB216763. Immunocytochemical staining revealed that the expressions of ^-catenin in the RSC96 and C2C12 cytosolic and nuclear compartments were increased in the SB216763-treated cells. These findings confirm that the glycogen synthase kinase 3 beta in- hibitor, SB216763, promoted the myelination and myotube differentiation through the Wnt/β-catenin signaling pathway and contributed to nerve remyelination and reduced denervated muscle atrophy after peripheral nerve injury.

Porcine skeletal muscle tissue fabrication for cultured meat production using three-dimensional bioprinting technology

Cultured meat produced through in vitro cell culture technology is regarded as a technical revolution.In this study,three-dimensional(3D)bioprinting technology was used to mimic the growth environment in vivo and construct a 3D culture system in vilro.Hydroge1 bioinks,namely,sodium alginate-gelatin and gelatin-methacrylate(GelMA)-silk fbroin,produced using two different curing processes were blended,and their rheological properties,mechanical properties,and biocompatibilities were compared.The 4%GelMA-20%silk fibroin hydroge1(GS2)demonstrated good performance and was hybridized with porcine skeleta1 muscle satellite cells for 3D printing to construct network structures of size 15 mm×15 mm and porosity 1000 um in 4-,6-,and 8-1ayer struchures.After 16 days of culture,4-and 6-1ayer grid structures formed compact muscle fbers organized by multinucleated myotubes.These results suggested that 3D bioprinting and GeIMA-silk fbroin hydrogels have great potential in fabricating porcine skeleta1 muscle tissue for use as cultured meat.

Arginine promotes myogenic differentiation and myotube formation through the elevation of cytoplasmic calcium concentration

This study aimed to explore the mechanism underlying arginine-promoted myogenesis of myoblasts.C2C12 cells were cultured with a medium containing 0.1,0.4,0.8,or 1.2 mmol/L arginine,respectively.Cell proliferation,viability,differentiation indexes,cytoplasmic Ca^(2+)concentration,and relative mRNA expression levels of myogenic regulatory factors(MRF)and key Ca2+channels were measured in the absence or presence of 2 chemical inhibitors,dantrolene(DAN,10μmol/L)and nisoldipine(NIS,10μmol/L),respectively.Results demonstrated that arginine promoted myogenic differentiation and myotube formation.Compared with the control(0.4 mmol/L arginine),1.2 mmol/L arginine upregulated the relative mRNA expression levels of myogenin(MyoG)and Myomaker at d 2 during myogenic induction(P<0.05).Cytoplasmic Ca^(2+)concentrations were significantly elevated by arginine supplementation at d 2 and 4(P<0.05).Relative mRNA expression levels of Ca^(2+)channels including the type 1 ryanodine recepto r(RyR1)and voltage-gated Ca^(2+)channel(Cav1.1)were upregulated by 1.2 mmol/L arginine during2-d myogenic induction(P<0.01).However,arginine-promoted myogenic potential of myoblasts was remarkably compromised by DAN and NIS,respectively(P<0.05).These findings evidenced that the supplementation of arginine promoted myogenic differentiation and myotube formation through increasing cytoplasmic Ca^(2+)concentration from both extracellular and sarcoplasmic reticulum Ca^(2+).

IS4 peptide forms ion channel in rat skeletal muscle cell membrane

A 22-mer peptide, identical to the primary sequence of domain I segment 4 (IS4) of rat brain sodium channel I, has been synthesized. IS4 peptide can incorporate into cultured rat skeletal myotube membranes and form ion channels. With patch clamp cell-attached technique single channel currents through IS4 channels can be recorded. The single channel conduc-