Skeletal Muscle Defects Research Articles

EDMD-Causing Emerin Mutant Myogenic Progenitors Exhibit Impaired Differentiation Using Similar Mechanisms.

Mutations in the gene encoding emerin (EMD) cause Emery–Dreifuss muscular dystrophy (EDMD1), an inherited disorder characterized by progressive skeletal muscle wasting, irregular heart rhythms and contractures of major tendons. The skeletal muscle defects seen in EDMD are caused by failure of muscle stem cells to differentiate and regenerate the damaged muscle. However, the underlying mechanisms remain poorly understood. Most EDMD1 patients harbor nonsense mutations and have no detectable emerin protein. There are three EDMD-causing emerin mutants (S54F, Q133H, and Δ95–99) that localize correctly to the nuclear envelope and are expressed at wildtype levels. We hypothesized these emerin mutants would share in the disruption of key molecular pathways involved in myogenic differentiation. We generated myogenic progenitors expressing wildtype emerin and each EDMD1-causing emerin mutation (S54F, Q133H, Δ95–99) in an emerin-null (EMD−/y) background. S54F, Q133H, and Δ95–99 failed to rescue EMD−/y myogenic differentiation, while wildtype emerin efficiently rescued differentiation. RNA sequencing was done to identify pathways and networks important for emerin regulation of myogenic differentiation. This analysis significantly reduced the number of pathways implicated in EDMD1 muscle pathogenesis.

Parkin deficiency accentuates chronic alcohol intake-induced tissue injury and autophagy defects in brain, liver and skeletal muscle

Alcoholism leads to organ injury including mitochondrial defect and apoptosis with evidence favoring a role for autophagy dysregulation in alcoholic damage. Parkin represents an autosomal recessive inherited gene for Parkinson's disease and an important member of selective autophagy for mitochondria. The association between Parkinson's disease and alcoholic injury remains elusive. This study aimed to examine the effect of parkin deficiency on chronic alcohol intake-induced organ injury in brain, liver and skeletal muscle (rectus femoris muscle). Adult parkin-knockout (PRK-/-) and wild-type mice were placed on Liber-De Carli alcohol liquid diet (4%) for 12weeks prior to assessment of liver enzymes, intraperitoneal glucose tolerance, protein carbonyl content, apoptosis, hematoxylin and eosin morphological staining, and mitochondrial respiration (cytochrome c oxidase, NADH:cytochrome c reductase and succinate:cytochrome c reductase). Autophagy protein markers were monitored by western blot analysis. Our data revealed that chronic alcohol intake imposed liver injury as evidenced by elevated aspartate aminotransferase and alanine transaminase, glucose intolerance, elevated protein carbonyl formation, apoptosis, focal inflammation, necrosis, microvesiculation, autophagy/mitophagy failure and dampened mitochondrial respiration (complex IV, complexes I and III, and complexes II and III) in the brain, liver and rectus femoris skeletal muscle. Although parkin ablation itself did not generate any notable effects on liver enzymes, insulin sensitivity, tissue carbonyl damage, apoptosis, tissue morphology, autophagy or mitochondrial respiration, it accentuated alcohol intake-induced tissue damage, apoptosis, morphological change, autophagy/mitophagy failure and mitochondrial injury without affecting insulin sensitivity. These data suggest that parkin plays an integral role in the preservation against alcohol-induced organ injury, apoptosis and mitochondrial damage.

Systemic therapy in an RNA toxicity mouse model with an antisense oligonucleotide therapy targeting a non-CUG sequence within the DMPK 3'UTR RNA.

Myotonic dystrophy type 1 (DM1), the most common adult muscular dystrophy, is an autosomal dominant disorder caused by an expansion of a (CTG)n tract within the 3' untranslated region (3'UTR) of the dystrophia myotonica protein kinase (DMPK) gene. Mutant DMPK mRNAs are toxic, present in nuclear RNA foci and correlated with a plethora of RNA splicing defects. Cardinal features of DM1 are myotonia and cardiac conduction abnormalities. Using transgenic mice, we have demonstrated that expression of the mutant DMPK 3'UTR is sufficient to elicit these features of DM1. Here, using these mice, we present a study of systemic treatment with an antisense oligonucleotide (ASO) (ISIS 486178) targeted to a non-CUG sequence within the 3'UTR of DMPK. RNA foci and DMPK 3'UTR mRNA levels were reduced in both the heart and skeletal muscles. This correlated with improvements in several splicing defects in skeletal and cardiac muscles. The treatment reduced myotonia and this correlated with increased Clcn1 expression. Furthermore, functional testing showed improvements in treadmill running. Of note, we demonstrate that the ASO treatment reversed the cardiac conduction abnormalities, and this correlated with restoration of Gja5 (connexin 40) expression in the heart. This is the first time that an ASO targeting a non-CUG sequence within the DMPK 3'UTR has demonstrated benefit on the key DM1 phenotypes of myotonia and cardiac conduction defects. Our data also shows for the first time that ASOs may be a viable option for treating cardiac pathology in DM1.

Effects of SW033291 on the myogenesis of muscle-derived stem cells and muscle regeneration

BackgroundThe unmet medical needs in repairing large muscle defects promote the development of tissue regeneration strategy. The use of bioactive molecules in combination with biomaterial scaffold has become an area of great interest. SW033291, a small-molecule inhibitor targeting 15-hydroxyprostaglandin dehydrogenase (15-PDGH) and subsequently elevating the production of prostaglandin E2 (PGE2), has been proved to accelerate the recovery and potentiate the regeneration of multiple tissues including the bone, liver, and colon. The limited understanding of the potential therapeutic effects on myogenesis motivated us to investigate the role of SW033291 in regulating muscle-derived stem cell (MDSC) myogenic differentiation and MDSC-mediated muscle regeneration.MethodsThe characteristics of rat MDSCs, including cell-specific markers and myogenic differentiation potential, were determined. MDSCs were incubated with SW033291 to evaluate PGE2 production and cytotoxicity. The effects of SW033291 on MDSC myogenic differentiation were assessed by quantitative real-time polymerase chain reaction (qPCR), western blot, and immunocytochemistry. The fibrin gel containing MDSCs and SW033291 was used for muscle regeneration in a tibialis anterior muscle defect model.ResultsOur data demonstrated that MDSCs were well-tolerated to SW033291 and treatment with SW033291 significantly promoted the production of PGE2 by MDSCs. In vitro analysis showed that SW033291 enhanced the myogenic differentiation and myotube formation by upregulating a series of myogenic markers. Additionally, the activation of PI3K/Akt pathway was involved in the mechanism underlying these promotive effects. Then, in situ casting of fibrin gel containing MDSCs and SW033291 was used to repair the tibialis anterior muscle defect; the addition of SW033291 significantly promoted myofiber formation within the defect region with mild immune response, less fibrosis, and sufficient vascularization.ConclusionSW033291 acted as a positive regulator of MDSC myogenic differentiation, and incorporating the compound with MDSCs in fibrin gel could serve as an effective method to repair large skeletal muscle defects.

In Situ Printing of Adhesive Hydrogel Scaffolds for the Treatment of Skeletal Muscle Injuries

Reconstructive surgery remains inadequate for the treatment of volumetric muscle loss (VML). The geometry of skeletal muscle defects in VML injuries varies on a case-by-case basis. Three-dimensional (3D) printing has emerged as one strategy that enables the fabrication of scaffolds that match the geometry of the defect site. However, the time and facilities needed for imaging the defect site, processing to render computer models, and printing a suitable scaffold prevent immediate reconstructive interventions post-traumatic injuries. In addition, the proper implantation of hydrogel-based scaffolds, which have generated promising results in vitro, is a major challenge. To overcome these challenges, a paradigm is proposed in which gelatin-based hydrogels are printed directly into the defect area and cross-linked in situ. The adhesiveness of the bioink hydrogel to the skeletal muscles was assessed ex vivo. The suitability of the in situ printed bioink for the delivery of cells is successfully assessed in vitro. Acellular scaffolds are directly printed into the defect site of mice with VML injury, exhibiting proper adhesion to the surrounding tissue and promoting remnant skeletal muscle hypertrophy. The developed handheld printer capable of 3D in situ printing of adhesive scaffolds is a paradigm shift in the rapid yet precise filling of complex skeletal muscle tissue defects.

Systemic Actions of Breast Cancer Facilitate Functional Limitations.

Breast cancer is a disease of a specific organ, but its effects are felt throughout the body. The systemic effects of breast cancer can lead to functional limitations in patients who suffer from muscle weakness, fatigue, pain, fibromyalgia, or many other dysfunctions, which hasten cancer-associated death. Mechanistic studies have identified quite a few molecular defects in skeletal muscles that are associated with functional limitations in breast cancer. These include circulating cytokines such as TNF-α, IL-1, IL-6, and TGF-β altering the levels or function of myogenic molecules including PAX7, MyoD, and microRNAs through transcriptional regulators such as NF-κB, STAT3, and SMADs. Molecular defects in breast cancer may also include reduced muscle mitochondrial content and increased extracellular matrix deposition leading to energy imbalance and skeletal muscle fibrosis. This review highlights recent evidence that breast cancer-associated molecular defects mechanistically contribute to functional limitations and further provides insights into therapeutic interventions in managing functional limitations, which in turn may help to improve quality of life in breast cancer patients.

Glass-activated regeneration of volumetric muscle loss

Volumetric muscle loss (VML) resulting from injuries to skeletal muscles has profound consequences in healthcare. Current VML treatment based on the use of soft materials including biopolymers and decellularized extracellular matrix (dECM) is challenging due to their incapability of stimulating the formation of satellite cells (SCs), muscle stem cells, which are required for muscle regeneration. Additional stem cells and/or growth factors have to be incorporated in these constructs for improved efficacy. Here we report an approach by using bioactive glasses capable of regenerating VML without growth factors or stem cells. One silicate and two borate compositions with different degradation rates (2.4% for silicate 45S5; 5.3% and 30.4% for borate 8A3B and 13-93B3, respectively, in simulated body fluid (SBF) at 37 °C for 30 days) were used for this study. Our in vitro models demonstrate the ability of ions released from bioactive glasses in promoting angiogenesis and stimulating cells to secrete critical muscle-related growth factors. We further show the activation of SCs and the regeneration of skeletal muscles in a rat VML model. Considering these promising results, this work reveals a potentially simple and safe approach to regenerating skeletal muscle defects. Statement of significance(1) This is the first report on an inorganic material used in skeletal muscle regeneration through in vitro and in vivo models.(2) Bioactive glass is found to activate the production of satellite cells (SCs), muscle stem cells, without the incorporation of extra stem cells or growth factors.(3) The work represents a simple, safe, low-cost yet efficient means for healing muscle defects.

THE ROLE OF PLATELET-RICH FIBRIN FACILITATES THE HEALING OF GASTROCNEMIUS MUSCLE DEFECT: A PRELIMINARY STUDY ON ANIMAL MODEL

Background: Scar formation has been a classical issue in the healing of skeletal muscle defect which will results in decrease of strength and function. Platelet-rich plasma (PRP) has been studied to enhance the healing of muscle defect. The disadvantages of it include the risk of immunologic reaction, time-consuming preparation, and lack of scaffold element. Platelet-rich fibrin (PRF) is a new generation of platelet concentrate that provides good source of growth factors and scaffold element necessary for the healing. This study is to provide a fondation of PRF preparation and implantation for the healing of experimental defect of gastrocnemius muscle in an animal model.Purpose: The goal is to provide a fondation of PRF preparation and implantation for the healing of experimental defect of gastrocnemius muscle in an animal model.Methods: Animal Care and Use Committee, Airlangga University approved the study. Twenty New Zealand white rabbits were divided into two groups. In control group, a defect was created in the right gastrocnemius and no implantation was done. In study group, similar defect was created with implantation of PRF. Two and four weeks after surgery, the defect was examined immunohistochemically for the expression of Pax7 protein.Results: The implanted group showed higher IRS score in two and four weeks compared to the control group.Conclusion: The use of PRF facilitates the healing of muscle injury. This study will be used as a fondation for further study on PRF.

6-Bromoindirubin-3\u2032-oxime intercepts GSK3 signaling to promote and enhance skeletal muscle differentiation affecting miR-206 expression in mice

Dystrophies are characterized by progressive skeletal muscle degeneration and weakness as consequence of their molecular abnormalities. Thus, new drugs for restoring skeletal muscle deterioration are critically needed. To identify new and alternative compounds with a functional role in skeletal muscle myogenesis, we screened a library of pharmacologically active compounds and selected the small molecule 6-bromoindirubin-3′-oxime (BIO) as an inhibitor of myoblast proliferation. Using C2C12 cells, we examined BIO’s effect during myoblast proliferation and differentiation showing that BIO treatment promotes transition from cell proliferation to myogenic differentiation through the arrest of cell cycle. Here, we show that BIO is able to promote myogenic differentiation in damaged myotubes in-vitro by enriching the population of newly formed skeletal muscle myotubes. Moreover, in-vivo experiments in CTX-damaged TA muscle confirmed the pro-differentiation capability of BIO as shown by the increasing of the percentage of myofibers with centralized nuclei as well as by the increasing of myofibers number. Additionally, we have identified a strong correlation of miR-206 with BIO treatment both in-vitro and in-vivo: the enhanced expression of miR-206 was observed in-vitro in BIO-treated proliferating myoblasts, miR-206 restored expression was observed in a forced miR-206 silencing conditions antagomiR-mediated upon BIO treatment, and in-vivo in CTX-injured muscles miR-206 enhanced expression was observed upon BIO treatment. Taken together, our results highlight the capacity of BIO to act as a positive modulator of skeletal muscle differentiation in-vitro and in-vivo opening up a new perspective for novel therapeutic targets to correct skeletal muscle defects.

Postnatal β2 adrenergic treatment improves insulin sensitivity in lambs with IUGR but not persistent defects in pancreatic islets or skeletal muscle.

Key points Previous studies in fetuses with intrauterine growth restriction (IUGR) have shown that adrenergic dysregulation was associated with low insulin concentrations and greater insulin sensitivity.Although whole‐body glucose clearance is normal, 1‐month‐old lambs with IUGR at birth have higher rates of hindlimb glucose uptake, which may compensate for myocyte deficiencies in glucose oxidation.Impaired glucose‐stimulated insulin secretion in IUGR lambs is due to lower intra‐islet insulin availability and not from glucose sensing.We investigated adrenergic receptor (ADR) β2 desensitization by administering oral ADRβ modifiers for the first month after birth to activate ADRβ2 and antagonize ADRβ1/3. In IUGR lambs ADRβ2 activation increased whole‐body glucose utilization rates and insulin sensitivity but had no effect on isolated islet or myocyte deficiencies.IUGR establishes risk for developing diabetes. In IUGR lambs we identified disparities in key aspects of glucose‐stimulated insulin secretion and insulin‐stimulated glucose oxidation, providing new insights into potential mechanisms for this risk. Placental insufficiency causes intrauterine growth restriction (IUGR) and disturbances in glucose homeostasis with associated β adrenergic receptor (ADRβ) desensitization. Our objectives were to measure insulin‐sensitive glucose metabolism in neonatal lambs with IUGR and to determine whether daily treatment with ADRβ2 agonist and ADRβ1/β3 antagonists for 1 month normalizes their glucose metabolism. Growth, glucose‐stimulated insulin secretion (GSIS) and glucose utilization rates (GURs) were measured in control lambs, IUGR lambs and IUGR lambs treated with adrenergic receptor modifiers: clenbuterol atenolol and SR59230A (IUGR‐AR). In IUGR lambs, islet insulin content and GSIS were less than in controls; however, insulin sensitivity and whole‐body GUR were not different from controls. Of importance, ADRβ2 stimulation with β1/β3 inhibition increases both insulin sensitivity and whole‐body glucose utilization in IUGR lambs. In IUGR and IUGR‐AR lambs, hindlimb GURs were greater but fractional glucose oxidation rates and ex vivo skeletal muscle glucose oxidation rates were lower than controls. Glucose transporter 4 (GLUT4) was lower in IUGR and IUGR‐AR skeletal muscle than in controls but GLUT1 was greater in IUGR‐AR. ADRβ2, insulin receptor, glycogen content and citrate synthase activity were similar among groups. In IUGR and IUGR‐AR lambs heart rates were greater, which was independent of cardiac ADRβ1 activation. We conclude that targeted ADRβ2 stimulation improved whole‐body insulin sensitivity but minimally affected defects in GSIS and skeletal muscle glucose oxidation. We show that risk factors for developing diabetes are independent of postnatal catch‐up growth in IUGR lambs as early as 1 month of age and are inherent to the islets and myocytes.

Roux-en-Y gastric bypass surgery restores insulin-mediated glucose partitioning and mitochondrial dynamics in primary myotubes from severely obese humans.

Background/Objectives:Impaired insulin-mediated glucose partitioning is an intrinsic metabolic defect in skeletal muscle from severely obese humans (BMI ≥ 40 kg/m2). Roux-en-Y gastric bypass (RYGB) surgery has been shown to improve glucose metabolism in severely obese humans. The purpose of the study was to determine the effects of RYGB surgery on glucose partitioning, mitochondrial network morphology, and markers of mitochondrial dynamics skeletal muscle from severely obese humans.Subject/Methods:Human skeletal muscle cells were isolated from muscle biopsies obtained from RYGB patients (BMI = 48.0 ± 2.1, n=7) prior to, 1-month and 7-months following surgery and lean control subjects (BMI = 22.4 ± 1.1, n=7). Complete glucose oxidation, non-oxidized glycolysis rates, mitochondrial respiratory capacity, mitochondrial network morphology and regulatory proteins of mitochondrial dynamics were determined in differentiated human myotubes.Results:Myotubes derived from severely obese humans exhibited enhanced glucose oxidation (13.5%; 95%CI [7.6, 19.4], P = 0.043) and reduced non-oxidized glycolysis (−1.3%; 95%CI [−11.1, 8.6]) in response to insulin stimulation at 7-months after RYGB when compared to the pre-surgery state (−0.6%; 95%CI [−5.2, 4.0] and 19.5%; 95%CI [4.0, 35.0], P =0.006), and were not different from the lean controls (16.7%; 95%CI [11.8, 21.5] and 1.9%; 95%CI [−1.6, 5.4], respectively). Further, number of fragmented mitochondria and Drp1(Ser616) phosphorylation and were trended to reduced/reduced (0.0104, 95%CI [0.0085, 0.0126], P = 0.091 and 0.0085, 95%CI [0.0068, 0.0102], P = 0.05) in myotubes derived from severely obese humans at 7-months after RYGB surgery in comparison to the pre-surgery state. Finally, Drp1(Ser616) phosphorylation was negatively correlated with insulin-stimulated glucose oxidation (r = −0.49, P = 0.037).Conclusion/Interpretation:These data indicate that an intrinsic metabolic defect of glucose partitioning in skeletal muscle from severely obese humans is restored by RYGB surgery. The restoration of glucose partitioning may be regulated through reduced mitochondrial fission protein Drp1 phosphorylation.

P.104Skeletal muscle reduction of Dnm2 with antisense oligonucleotides in myotubular myopathy

Centronuclear myopathies (CNM) are a group of severe muscle diseases for which no effective therapy is currently available. The most severe and neonatal X-linked form is known as myotubular myopathy and caused by loss-of-function mutations in Myotubularin (MTM1), while the main autosomal dominant form is due to mutations in Dynamin 2 (DNM2). We previously showed that genetic reduction of DNM2 expression in Mtm1 knockout (Mtm1KO) mice prevents development of muscle pathology. We are now investigating skeletal muscle targeting of Dnm2 reduction in mice. Indeed specific muscle isoforms are expressed during muscle maturation and in adulthood, through alternative splicing. A single intraperitoneal injection of 25mg/kg of antisense oligonucleotides targeting the total pool of Dnm2 increased the lifespan, whole body strength, and reduced disease severity in Mtm1KO mice, however a single injection alone was not sufficient to rescue the disease for the lifespan of the mice. Repeated injections of ASO was able to revert the disease phenotype in mice within 2 weeks, and the effects lasted several months after the final injection. ASO was still detected in skeletal muscles at this time point, but not in key tissues such as the liver, which may explain in part the longterm improvement in the myopathic phenotype. New ligand-conjugated antisense (LICA) oligonucleotides targeting Dnm2 are now being tested in Mtm1KO mice for potency and efficacy. ASO-mediated Dnm2 knockdown can efficiently correct skeletal muscle defects due to loss of MTM1, providing an attractive therapeutic strategy for this disease. Effective muscle targeting will be an important step in translation of this approach to the clinic for patients.

2267-PUB: Skeletal Muscle mRNA Transcriptional Signature of Response to Exercise of Patients in Different Stages of Glucose Tolerance

Physical activity is an effective method to prevent and treat type 2 diabetes (T2D); however, the mechanisms of its beneficial effects have not been precisely elucidated. Skeletal muscle is a major contributor to overall glucose homeostasis in the body and is responsible for the efficacy of exercise intervention in the prevention and treatment of T2D. Currently, there is a lack of studies investigating the transcriptional signature of exercise training on skeletal muscles of patients with different stages of glucose tolerance. Thus, the aim of this study was to identify the mRNA signature of skeletal muscle adaptation to three-month exercise intervention in patients with different dysregulations of glucose metabolism. For this project we included 35 sedentary males, aged 24-65 years old, with BMI 26-33 kg/m2, divided into three groups: 15 normoglycemics, 12 prediabetics and 8 subjects with type 2 diabetes, treated with metformin only. RNA samples were obtained from biopsied Vastus lateralis muscles, before and after a 3-month supervised exercise intervention that occurred thrice weekly and consisted of mixed aerobic (60-70% of individual’s VO2max) and strength (60-70% of the repetition maximum for each exercise) activities. Selected groups of patients did not differ in age, BMI, number of performed trainings or diet during the intervention. mRNA sequencing was performed on an Illumina HiSeq 4000. We found highly significant differential mRNA expression in normoglycemics vs. T2D and prediabetic subjects, mostly related to mitochondrial processes, including the oxidative phosphorylation and thermogenesis pathways. Furthermore, we found that exercise intervention mostly affected these pathways, suggesting that a major defect of skeletal muscle in type 2 diabetes development is related to mitochondrial function and can be diminished by exercise. Disclosure L. Szczerbinski: None. M. Niemira: None. K. Szczerbinski: None. U. Puchta: None. A. Citko: None. E. Siewiec: None. J. Zapolska: None. M. Taylor: None. S. Larsen: None. M. Gorska: None. A. Kretowski: None. Funding Polish Diabetes Association; Polish Ministry of Science and Higher Education

Quantitative RyR1 reduction and loss of calcium sensitivity of RyR1Q1970fsX16+A4329D cause cores and loss of muscle strength.

Recessive ryanodine receptor 1 (RYR1) mutations cause congenital myopathies including multiminicore disease (MmD), congenital fiber-type disproportion and centronuclear myopathy. We created a mouse model knocked-in for the Q1970fsX16+A4329D RYR1 mutations, which are isogenic with those identified in a severely affected child with MmD. During the first 20weeks after birth the body weight and the spontaneous running distance of the mutant mice were 20% and 50% lower compared to wild-type littermates. Skeletal muscles from mutant mice contained 'cores' characterized by severe myofibrillar disorganization associated with misplacement of mitochondria. Furthermore, their muscles developed less force and had smaller electrically evoked calcium transients. Mutant RyR1 channels incorporated into lipid bilayers were less sensitive to calcium and caffeine, but no change in single-channel conductance was observed. Our results demonstrate that the phenotype of the RyR1Q1970fsX16+A4329D compound heterozygous mice recapitulates the clinical picture of multiminicore patients and provide evidence of the molecular mechanisms responsible for skeletal muscle defects.

Probiotics Ameliorate Gut‐Microbial Dysbiosis, Intestinal Permeability, Systemic Inflammation, and Skeletal Muscle Dysfunction in Cystathionine‐β‐synthase‐Deficient Mice

Recent studies have reported that dietary supplementation of probiotics is a novel approach for enhancing skeletal development and metabolism in both humans and animals. However, the precise role of probiotics in regulating skeletal muscle mass at the cellular and molecular levels in mice still needs to be clarified. Here we show that altered gut‐microbiota eubiosis, systemic inflammation and skeletal muscle dysfunction caused by hydrogen sulfide deficiency and whether the intragastric administration of probiotics (GSL#5) could retard H2S deficiency‐related skeletal muscle defects. To test this hypothesis, we employed 8‐week‐old female wild‐type (WT), cystathionine‐beta‐synthase heterozygote knockout (CBS+/−), and CBS+/− mice supplemented with probiotics (GSL#5) at the dosage of 1×109 CFU every other day through oral gavage for 6 weeks. In the murine model, H2S deficiency (CBS+/− mice) increased gut permeability, and upregulated the inflammatory cytokines TNFα (TNF), and IL‐17 in the blood and the skeletal muscle. Further, the gut microbiota is significantly altered in CBS‐deficient mice as assessed by the Denaturing gradient gel electrophoresis (DGGE) analysis. We documented that long non‐coding RNA‐PVT1 (lncRNA‐PVT1) is, indeed, upregulated in the soleus muscles of CBS‐deficient mice (4.2 fold), as assessed by RT2 lncRNA qPCR Assay. The mechanistic study shows that depressed H2S signaling stimulates the transcription factor C/EBP‐α binding to the promoter of lncRNA‐PVT1 and promotes the expression of lncRNA‐PVT1. Using an electrophoretic mobility shift assay and chromatin immunoprecipitations, we identified that lncRNA‐PVT1 interacts with TNFAIP3 interacting protein 1, TNIP1, leading to the acetylation of NF‐κB (P65 subunit) activity in the soleus muscle of CBS‐deficient mice. Further, activation of P65 region of NF‐κB transcriptionally upregulates atrogin‐1, MuRF‐1 expression. Overexpression of lncRNA‐PVT1 in cultured C2C12 muscle cell lines stimulates an increase in MuRF‐1 (2.3‐fold) and atrogin‐1 (3.7‐fold) expression. In addition, systemic delivery of synthetic lncRNA‐PVT1 in control mice causes the myofibers to undergo atrophy. Gene knockout of lncRNA‐PVT1 in CBS‐deficient mice attenuates atrophy of the myofibers and muscle wasting. Overall, administration of probiotics in CBS‐deficient mice reverses the gut‐microbiota dysbiosis and preserves skeletal muscle mass. In conclusion, our findings provide novel evidence that the administration of probiotics could balance the gut‐microbiota homeostasis as well as skeletal muscle function by modulating systemic inflammation in H2S‐deficient miceSupport or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Assessment of skeletal muscle early myopathy with contrast-enhanced ultrasound in rabbit model of diabetes mellitus

Objective To evaluate early skeletal muscle myopathy with contrast-enhanced ultrasound (CEUS) in diabetic rabbits. Methods Alloxan (100 mg/kg) was given intravenously to 11 New Zealand white rabbits. Another 5 age-matched normal rabbits were used as controls.CEUS was performed at baseline and 1, 2, 3 months after the establishment of diabetes, respectively. Then, skeletal muscle samples were obtained for pathological observation. Additionally, the diabetic rabbits were divided into 3 groups according to their pathologic findings: mild, moderate, and severe myopathy group. Micro-perfusion parameters, including artery-to-vein transit time (A-VTT), muscle tissue mean transit time (M-MTT) and muscle tissue peak intensity (M-PI) were calculated. Results The diabetic rabbits exhibited a lower body weight increase and a decrease of muscle thickness.Plasma levels of triglyceride, cholesterol and creatinine were significantly higher in diabetic group than those in control group (P<0.05). The A-VTT and M-MTT values of the diabetic rabbits significantly increased over time (all P<0.05), whereas M-PI value significantly decreased compared with that in control group (P=0.013). Besides, the A-VTT value was significantly higher in severe myopathy group than that in mild group (P=0.001). The M-MTT values in both moderate and severe groups were significantly higher when compared to that in mild group (all P<0.05). The A-VTT and M-MTT values were correlated with the severity of diabetic myopathy(ρ=0.898, P<0.001; ρ=0.527, P=0.01). Conclusions Diabetic rabbits have defective skeletal muscle micro-perfusion in the early stage. CEUS can quantify impaire muscle microcirculation, and is a valuable tool for assessment of diabetic myopathy. Key words: Contrast-enhanced ultrasound; Diabetes mellitus; Myopathy; Microcirculation

Defective sarcomere assembly in smyd1a and smyd1b zebrafish mutants.

Two smyd1 paralogues, smyd1a and smyd1b, have been identified in zebrafish. Although Smyd1b function has been reported in fast muscle, its function in slow muscle and the function of Smyd1a, in general, are uncertain. In this study, we generated 2 smyd1a mutant alleles and analyzed the muscle defects in smyd1a and smyd1b single and double mutants in zebrafish. We demonstrated that knockout of smyd1a alone had no visible effect on muscle development and fish survival. This was in contrast to the smyd1b mutant, which exhibited skeletal and cardiac muscle defects, leading to early embryonic lethality. The smyd1a and smyd1b double mutants, however, showed a stronger muscle defect compared with smyd1a or smyd1b mutation alone, namely, the complete disruption of sarcomere organization in slow and fast muscles. Immunostaining revealed that smyd1a; smyd1b double mutations had no effect on myosin gene expression but resulted in a dramatic reduction of myosin protein levels in muscle cells of zebrafish embryos. This was accompanied by the up-regulation of hsp40 and hsp90-α1 gene expression. Together, our studies indicate that both Smyd1a and Smyd1b partake in slow and fast muscle development although Smyd1b plays a dominant role compared with Smyd1a.-Cai, M., Han, L., Liu, L., He, F., Chu, W., Zhang, J., Tian, Z., Du, S. Defective sarcomere assembly in smyd1a and smyd1b zebrafish mutants.

Differential lethal and sublethal effects in embryonic zebrafish exposed to different sizes of silver nanoparticles

Various parameters can influence the toxic response to silver nanoparticles (Ag NPs), including the size and surface properties, as well as the exposure environment and the biological site of action. Herein, we assess the intestinal toxicity of three different sizes (10, 40, and 100 nm) of Ag NPs in embryonic zebrafish, and describe the relationship between the properties and behavior of Ag NPs in the exposure medium, and induction of lethal and sublethal effects. We find that the composition of the medium and the size contribute to differential NPs agglomeration, release of Ag ions, and subsequent effects during exposure. The exposure medium causes dramatic reduction in silver dissolution due to the presence of salts and divalent cations, which limits the lethal potential of silver ions. Lethality is observed primarily for embryos exposed to medium sized Ag NPs (40 nm), but not to the supernatant originated from particles, which suggests that the exposure to particulate silver is the main cause of mortality. On the other hand, the exposure to 10 nm and 100 nm NPs, as well as Ag ions, only causes sublethal developmental defects in skeletal muscles and intestine, and induces a nitric oxide imbalance.

Skeletal muscle differentiation potential of cells derived from adipose tissue

Autologous adipose tissue is an ideal donor for muscle transformation and repair of skeletal muscle defects. Adipose derived stem cells, dedifferentiated adipocytes, and brown adipose cells can differentiate into skeletal muscle cells under certain conditions. Adipose derived stem cells can improve muscle function after transplantation in vivo. Adipose tissue derived cells may be the promising candidates for the therapy of skeletal muscle defects. This paper summarizes the skeletal muscle differentiation potential of cells derived from adipose tissue to provide references for further studies. Key words: Adipose tissue; Adipose derived stem cells; Dedifferentiated; Transdifferentiation; Skeletal muscle

Nuclear defects in skeletal muscle from a Dynamin 2-linked centronuclear myopathy mouse model

Dynamin 2 (DNM2) is a key protein of the endocytosis and intracellular membrane trafficking machinery. Mutations in the DNM2 gene cause autosomal dominant centronuclear myopathy (CNM) and a knock-in mouse model expressing the most frequent human DNM2 mutation in CNM (Knock In-Dnm2R465W/+) develops a myopathy sharing similarities with human disease. Using isolated muscle fibres from Knock In-Dnm2R465W/+ mice, we investigated number, spatial distribution and morphology of myonuclei. We showed a reduction of nuclear number from 20 weeks of age in Tibialis anterior muscle from heterozygous mice. This reduction is associated with a decrease in the satellite cell content in heterozygous muscles. The concomitant reduction of myonuclei number and cross-section area in the heterozygous fibres contributes to largely maintain myonuclear density and volume of myonuclear domain. Moreover, we identified signs of impaired spatial nuclear distribution including alteration of distance from myonuclei to their nearest neighbours and change in orientation of the nuclei. This study highlights reduction of number of myonuclei, a key regulator of the myofiber size, as a new pathomechanism underlying muscle atrophy in the dominant centronuclear myopathy. In addition, this study opens a new line of investigation which could prove particularly important on satellite cells in dominant centronuclear myopathy.