Mouse Muscle Cells Research Articles

Novel Combinatorial MicroRNA-Binding Sites in AAV Vectors Synergistically Diminish Antigen Presentation and Transgene Immunity for Efficient and Stable Transduction.

Recombinant adeno-associated virus (rAAV) platforms hold promise for in vivo gene therapy but are undermined by the undesirable transduction of antigen presenting cells (APCs), which in turn can trigger host immunity towards rAAV-expressed transgene products. In light of recent adverse events in patients receiving high systemic AAV vector doses that were speculated to be related to host immune responses, development of strategies to mute innate and adaptive immunity is imperative. The use of miRNA binding sites (miR-BSs) to confer endogenous miRNA-mediated regulation to detarget transgene expression from APCs has shown promise for reducing transgene immunity. Studies have shown that designing miR-142BSs into rAAV1 vectors were able to repress costimulatory signals in dendritic cells (DCs), blunt the cytotoxic T cell response, and attenuate clearance of transduced muscle cells in mice to allow sustained transgene expression in myofibers with negligible anti-transgene IgG production. In this study, we screened individual and combinatorial miR-BS designs against 26 miRNAs that are abundantly expressed in APCs, but not in skeletal muscle. The highly immunogenic ovalbumin (OVA) transgene was used as a proxy for foreign antigens. In vitro screening in myoblasts, mouse DCs, and macrophages revealed that the combination of miR-142BS and miR-652-5pBS strongly mutes transgene expression in APCs but maintains high myoblast and myocyte expression. Importantly, rAAV1 vectors carrying this novel miR-142/652-5pBS cassette achieve higher transgene levels following intramuscular injections in mice than previous detargeting designs. The cassette strongly inhibits cytotoxic CTL activation and suppresses the Th17 response in vivo. Our approach, thus, advances the efficiency of miRNA-mediated detargeting to achieve synergistic reduction of transgene-specific immune responses and the development of safe and efficient delivery vehicles for gene therapy.

SET knockdown attenuated phenotype modulation and calcium channel associated markers of airway smooth muscle cells in asthmatic mice.

BackgroundDysfunctional phenotype modulation and calcium channels in airway smooth muscle cells (ASMCs) are important characteristics of airway remodeling in chronic asthma. However, the mechanisms underlying these pathological processes remain unclear. SET (I2PP2A, inhibitor-2 of protein phosphatase 2A) has many significant functions and is involved in various physiological and pathological processes. This study aimed to determine the function of SET in chronic asthma.MethodsBALB/c mice were sensitized by ovalbumin injection and repeated inhalation of ovalbumin. The Penh value was measured using the Buxco whole body plethysmography system. A short hairpin RNA of the SET gene was designed and transfected into ASMCs derived from asthmatic mice. Flow cytometry of Annexin-V/propidium iodide staining was used for evaluating cell apoptosis. Western blot was adopted to measure the expression levels of ASMCs phenotype modulation markers and calcium channel-associated proteins.ResultsThe results showed that shRNA targeting SET significantly decreased the expression of SET, and enhanced the apoptosis of ASMCs. SET knockdown promoted the expression of contractile phenotype markers such as α-SMA (alpha smooth muscle Actin), SM-MHC (smooth muscle Myosin heavy chain), and calponin, and inhibited the expression of synthetic phenotype markers including vimentin and CD44. The expression of the calcium channel-related proteins STIM1 (Stromal interaction molecule 1) and Orai1 were also inhibited after SET knockdown.ConclusionsThese data demonstrated that SET participated in the development of airway dysfunction in asthma, suggesting that the silencing of SET may be a new therapeutic target for the treatment of asthma patients.

Periostin Is Required for the Maintenance of Muscle Fibers during Muscle Regeneration.

Skeletal muscle regeneration is a well-organized process that requires remodeling of the extracellular matrix (ECM). In this study, we revealed the protective role of periostin, a matricellular protein that binds to several ECM proteins during muscle regeneration. In intact muscle, periostin was localized at the neuromuscular junction, muscle spindle, and myotendinous junction, which are connection sites between muscle fibers and nerves or tendons. During muscle regeneration, periostin exhibited robustly increased expression and localization at the interstitial space. Periostin-null mice showed decreased muscle weight due to the loss of muscle fibers during repeated muscle regeneration. Cultured muscle progenitor cells from periostin-null mice showed no deficiencies in their proliferation, differentiation, and the expression of Pax7, MyoD, and myogenin, suggesting that the loss of muscle fibers in periostin-null mice was not due to the impaired function of muscle stem/progenitor cells. Periostin-null mice displayed a decreased number of CD31-positive blood vessels during muscle regeneration, suggesting that the decreased nutritional supply from blood vessels was the cause of muscle fiber loss in periostin-null mice. These results highlight the novel role of periostin in maintaining muscle mass during muscle regeneration.

Chronic exercise training activates histone turnover in mouse skeletal muscle fibers.

Epigenetic regulation of skeletal muscle adaptation to exercise is a recent topic for which there is limited information. This study investigated whether exercise training activates histone turnover in the skeletal muscle fibers of mice. Experiments using a tetracycline-inducible H2B-GFP expression model demonstrated that 4weeks of running training, but not 2weeks of training, significantly promoted the incorporation of H2B-GFP into nucleosomes and the dissociation of histone H3.3 at both transcriptionally upregulated and nonresponsive loci. Muscle-specific PGC-1α-b-overexpressing mice crossed with H2B-GFP mice showed a slight increase in H2B-GFP incorporation at transcriptionally active loci, but not in the dissociation of H3.3 from nucleosomes. Gene expression responses to a single bout of running were significantly enhanced in 4-week trained mice when compared with those in 2-week trained mice. The most drastic increase in the gene response was found in the expression of Hspa1a and Hspa1b, in which the magnitude of upregulation in response to running was significantly enhanced from 8-fold in 2week trained mice to 97- and 121-fold in 4week trained mice, respectively. It was also found that the HSP70 level increased during the training period. In a myonuclear immunohistochemical analysis of chromatin remodelers, we further found that the level of SPT16, an H2A-H2B-specific chaperone, was upregulated after running training. These results revealed that 4weeks of running training activated histone turnover in skeletal muscle fibers. They also suggested that histone turnover led to loosening of the nucleosomes and enhanced gene responses to exercise.

Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression

BackgroundMost single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice over a TFBS called a CArG box in the Tspan2 promoter.ResultsQuantitative RT-PCR showed loss of Tspan2 mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the Tspan2 CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the Tspan2 CArG box displayed similar cell-specific loss of Tspan2 mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of Tspan2 in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders.ConclusionsPE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse.

High extracellular ATP levels released through pannexin-1 channels mediate inflammation and insulin resistance in skeletal muscle fibres of diet-induced obese mice.

Skeletal muscle is a key target organ for insulin's actions and is the main regulator of blood glucose. In obese individuals and animal models, there is a chronic low-grade inflammatory state affecting highly metabolic organs, leading to insulin resistance. We have described that adult skeletal muscle fibres can release ATP to the extracellular medium through pannexin-1 (PANX1) channels. Besides, it is known that high extracellular ATP concentrations can act as an inflammatory signal. Here, we propose that skeletal muscle fibres from obese mice release high levels of ATP, through PANX1 channels, promoting inflammation and insulin resistance in muscle cells. C57BL/6J mice were fed with normal control diet (NCD) or high-fat diet (HFD) for 8weeks. Muscle fibres were isolated from flexor digitorum brevis (FDB) muscle. PANX1-knockdown FDB fibres were obtained by in vivo electroporation of a short hairpin RNA Panx1 plasmid. We analysed extracellular ATP levels in a luciferin/luciferase assay. Gene expression was studied with quantitative real-time PCR (qPCR). Protein levels were evaluated by immunoblots, ELISA and immunofluorescence. Insulin sensitivity was analysed in a 2-NBDG (fluorescent glucose analogue) uptake assay, immunoblots and IPGTT. HFD-fed mice showed significant weight gain and insulin resistance compared with NCD-fed mice. IL-6, IL-1β and TNF-α protein levels were increased in FDB muscle from obese mice. We observed high levels of extracellular ATP in muscle fibres from obese mice (197 ± 55pmol ATP/μg RNA) compared with controls (32 ± 10pmol ATP/μg RNA). ATP release in obese mice fibres was reduced by application of 100μmol/l oleamide (OLE) and 5μmol/l carbenoxolone (CBX), both PANX1 blockers. mRNA levels of genes linked to inflammation were reduced using OLE, CBX or 2U/ml ATPase apyrase in muscle fibres from HFD-fed mice. In fibres from mice with pannexin-1 knockdown, we observed diminished extracellular ATP levels (78 ± 10pmol ATP/μg RNA vs 252 ± 37pmol ATP/μg RNA in control mice) and a lower expression of inflammatory markers. Moreover, a single pulse of 300μmol/l ATP to fibres from control mice reduced insulin-mediated 2-NBDG uptake and promoted an elevation in mRNA levels of inflammatory markers. PANX-1 protein levels were increased two- to threefold in skeletal muscle from obese mice compared with control mice. Incubation with CBX increased Akt activation and 2-NBDG uptake in HFD fibres after insulin stimulation, rescuing the insulin resistance condition. Finally, in vivo treatment of HFD-fed mice with CBX (i.p. injection of 10mg/kg each day) for 14days, compared with PBS, reduced extracellular ATP levels in skeletal muscle fibres (51 ± 10pmol ATP/μg RNA vs 222 ± 28pmol ATP/μg RNA in PBS-treated mice), diminished inflammation and improved glycaemic management. In this work, we propose a novel mechanism for the development of inflammation and insulin resistance in the skeletal muscle of obese mice. We found that high extracellular ATP levels, released by overexpressed PANX1 channels, lead to an inflammatory state and insulin resistance in skeletal muscle fibres of obese mice.

Effect of miR-506-3p on Proliferation and Apoptosis of Airway Smooth Muscle Cells in Asthmatic Mice by Regulating CCL2 Gene Expression and Mediating TLR4/NF-κB Signaling Pathway Activation.

We aimed to investigate the effect of miR-506-3p on the proliferation and apoptosis of airway smooth muscle cells (ASMCS) in asthmatic mice by regulating the activation of TLR4/NF-κB signaling pathway through targeted regulation of C-C Motif Chemokine Ligand 2 (CCL2) expression. Twenty-four BALB/c mice of specific pathogen-free grade were selected to establish asthmatic mouse model, which were randomly divided into normal control group and asthma model group (n = 12 for each group). HE and IHC staining, bioinformatics and dual luciferase reporter assay, RT-PCR MTT, flow cytometry and Western blot were used in this research. HE staining showed airway epithelium thickening, submucosal inflammatory cell infiltration and airway smooth muscle thickening, and the positive expression rate of CCL2 was significantly increased in asthma model group (all P < 0.05). CCL2 was the target gene of miR-506-3p. Moreover, the expression of miR-506-3p in asthma model group was significantly decreased, the mRNA and protein expression levels of CCL2, TLR4, NF-κB (p65) and Bcl-2 were significantly increased, while those of Bax were decreased (all P < 0.05). In miR-506-3p mimic group or siRNA-CCL2 group, the expression of CCL2, TLR4, NF-κB (p65) and Bcl-2 decreased obviously, while that of Bax increased, cell proliferation decreased, G1 phase prolonged, G2 & S phases shortened, and apoptosis rate increased significantly (all P < 0.05), whereas the opposite trends were found in miR-506-3p inhibitor group (all P < 0.05). However, there was no statistical difference in the above-mentioned indexes in miR-506-3p inhibitor + siRNA-CCL2 group (all P > 0.05). Overexpression of miR-506-3p can inhibit ASMCS proliferation and promote apoptosis via inhibiting CCL2 expression and suppressing the activation of TLR4/NF-κB signaling pathway. Inhibited expression of miR-506-3p can reverse the positive role of CCL2 gene silencing. Our study is the first to prove the beneficial role of miR-506-3p-CCL2-TLR4/NF-κB regulatory axis in the development of asthma.

SEDT2/METTL14-mediated m6A methylation awakening contributes to hypoxia-induced pulmonary arterial hypertension in mice.

Pulmonary arterial hypertension (PAH) is a fatal disease whose molecular mechanism is unknown. The trimethylation of lysine 36 on histone 3 (H3K36me3) catalyzed by SETD2 and the modification of N6-methyladenine (m6A) mRNA mediated by METTL14 play important roles in a variety of normal and pathological biological processes. However, the role of these epigenetic controls in the pathogenesis of PAH remains unclear. In this study, the expression of SETD2 and METTL14 was elevated in pulmonary artery smooth muscle cells (PASMCs) of hypoxia-induced PAH mice. We further constructed a mouse model with SETD2 specific knockout in smooth muscle cells (SETD2SM22α Cre). Our results suggest that the lack of SETD2 in SMCs protected mice from hypoxia-induced PAH and significantly reduced right ventricular systolic pressure (RVSP), right ventricular/left ventricular plus septum [RV/(LV+S)] weight ratio, and pulmonary median width. In addition, the absence of SETD2 in SMCs alleviates the level of METTL14 expression and the m6A RNA methylation level in PAH SMCs. These results obtained from mice suggest that strategies that target the inhibition of SETD2/METTL14 activity may be a viable treatment for PAH in a clinical setting.

Inhibition of Lipid Accumulation in Skeletal Muscle and Liver Cells: A Protective Mechanism of Bilirubin Against Diabetes Mellitus Type 2.

Ectopic lipid accumulation in skeletal muscle and liver drives the pathogenesis of diabetes mellitus type 2 (DMT2). Mild hyperbilirubinaemia has been repeatedly suggested to play a role in the prevention of DMT2 and is known for its capacity to shape an improved lipid phenotype in humans and in animals. To date, the effect of bilirubin on lipid accumulation in tissues that are prone to ectopic lipid deposition is unclear. Therefore, we analyzed the effect of bilirubin on lipid accumulation in skeletal muscle and liver cell lines. C2C12 skeletal mouse muscle and HepG2 human liver cells were treated with physiological concentrations of free fatty acids (FFA) (0.5 mM and 1 mM) and unconjugated bilirubin (UCB) (17.1 and 55 µM). The intracellular presence of UCB upon exogenous UCB administration was confirmed by HPLC and the lipid accumulation was assessed by using Nile red. Exposure of both cell lines to UCB significantly reduced lipid accumulation by up to 23% (p ≤ 0.001) in HepG2 and by up to 17% (p ≤ 0.01) in C2C12 cells at 0.5 and 5 h under hypoglycaemic conditions. Simultaneously, UCB slightly increased FFA uptake in HepG2 cells after 0.5 and 5 h and in C2C12 cells after 12 h as confirmed by gas chromatographic analyses of the remaining FFA content in the incubation media. The effects of UCB on lipid accumulation and uptake were abolished in the presence of higher glucose concentrations. Monitoring the uptake of a radiolabeled glucose analogue [18F]FDG: (2-deoxy-2-[18F]fluoro-D-glucose) into both cell types further indicated higher glucose consumption in the presence of UCB. In conclusion, our findings show that UCB considerably decreases lipid accumulation in skeletal muscle and liver cells within a short incubation time of max. 5 h which suggests that mildly elevated bilirubin levels could lower ectopic lipid deposition, a major key element in the pathogenesis of DMT2.

Sterol-resistant SCAP Overexpression in Vascular Smooth Muscle Cells Accelerates Atherosclerosis by Increasing Local Vascular Inflammation through Activation of the NLRP3 Inflammasome in Mice.

Atherosclerosis is a serious age-related pathology, and one of its hallmarks is the presence of chronic inflammation. Sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) is a cholesterol sensor that plays an essential role in regulating intracellular cholesterol homeostasis. Accordingly, dysregulation of the SCAP-SREBP pathway has been reported to be closely associated with an increased risk of obesity, hypercholesterolemia, and cardiovascular disease. In this study, we explored whether sterol-resistant SCAP (D443N mutation) in vascular smooth muscle cells (VSMCs) of mice promotes vascular inflammation and accelerates the occurrence and progression of atherosclerosis. We established a transgenic knock-in mouse model of atherosclerosis with an activating D443N mutation at the sterol-sensing domain of SCAP (SCAPD443N) by microinjection. Next, SCAPD443N/ApoE-/- mice were generated by crossing SCAPD443N mice with apolipoprotein E-/- (ApoE-/-) background mice. We found that sterol-resistant SCAP markedly amplified and accelerated the progression of atherosclerotic plaques in SCAPD443N/ApoE-/- mice compared with that in control ApoE-/- mice. Similarly, in SCAPD443N mice, aortic atherosclerotic plaques both appeared earlier and were greater in number than that in control SCAP+/+ mice, both of which were fed a Western diet for 12 or 24 weeks. Moreover, we observed that sterol-resistant SCAP significantly increased local inflammation and induced endothelial dysfunction in the aortas of SCAPD443N mice and SCAPD443N/ApoE-/- mice. In vitro, we also found that sterol-resistant SCAP overexpression in VSMCs increased the release of inflammatory cytokines and induced endothelial cell injury when both cell types were cocultured. Furthermore, we demonstrated that sterol-resistant SCAP overexpression in VSMCs promoted SCAP and NLRP3 inflammasome cotranslocation to the Golgi and increased the activation of the NLRP3 inflammasome pathway. These findings suggested that sterol-resistant SCAP in VSMCs of mice induced vascular inflammation and endothelial dysfunction, consequently accelerating atherosclerosis by activating the NLRP3 inflammasome pathway.

The effects of low-frequncy high-volume electrical stimulation on satellite cell activation and anabolic signaling pathway in single muscle fibers of old mice

Purpose This is the first study to examine whether age impacts the response of single muscle fibers to high/low frequency and high/low volume electrical pulse stimulation. We performed in vitro experiments to evaluate the effect of low-frequency high-volume electrical pulse stimulation (EPS) on mechanistic target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK) signaling pathway, and satellite cell activation in singles fibers of young and aged muscles. Methods Isolated single fibers from gastocnemius in 12-wk (n=21) and 72-wk (n=21) old male C57BL/6 mice were divided into four groups: 1) control (Con) received no EPS, 2) low-frequency low-volume EPS (LL), 3) low-frequency high-volume EPS (LH), and 4) high-frequency low-volume EPS (HL) were made to contract using independent EPS protocols. Satellite cell activation and anabolic pathway (mTOR and MAPK signaling) were measured before and after EPS. Results The number of quiescent (Pax7+/Ki67-) and active (Pax7+/Ki67+) satellite cells, myonuclear content and the phosphorylation of 4E-BP1 and ERK were higher in young when compared with old. However, regardless of age, LH and HL EPS significantly increased the number of activate satellite cells (142%, both) and phosphorylation of mTOR (129% and 133%, respectively), p70S6K (133% and 136%, respectively) and 4E-BP1 (140% and 129%, respectively) compared with Con. The protein expression of ERK phosphorylation only increased by LH EPS in both the young and old groups (123% and 125%, respectively). Conclusion Low-frequency high-volume EPS stimulated satellite cell activation and the mTOR signaling pathway in older similar to young muscle.

A novel PAI-1 inhibitor prevents ageing-related muscle fiber atrophy

Sarcopenia is among the most common medical problems of the aging population worldwide and a major social concern. Here, we explored the therapeutic potential of TM5484, a novel orally available PAI-1 inhibitor, to prevent sarcopenia. The sarcopenic phenotypes of the calf muscle of 12- and 6-month-old middle-aged mice were compared. Although significant decline of isometric gastrocnemius muscle force was detected in the older untreated mice, those administered TM5484 had significantly greater calf muscle force, as determined using isometric measurements by electrical stimulation. Histological analysis indicated that cross-sectional gastrocnemius muscle fibers in untreated older mice were thinner than those in younger mice; however, TM5484-treated group showed thicker fibers than younger mice. Treatment with TM5484 for 6 months enhanced Igf1, Atrogin-1, Mt-Co1, and Chrna1 mRNA expression in the mice gastrocnemius muscle, with increased serum IGF-1 concentration. TM5484 induced dose-dependent Igf1, Atrogin-1, and Chrna1 expression in C2C12 myoblastic cells, confirming cell autonomous effect. Further, the presence of plasmin for 72 h caused significantly increased Igf1 expression in C2C12 cells. These findings suggest that oral PAI-1 inhibitors represent a promising therapeutic candidate for preventing sarcopenia progression in humans.

A Novel HGF/SF Receptor (MET) Agonist Transiently Delays the Disease Progression in an Amyotrophic Lateral Sclerosis Mouse Model by Promoting Neuronal Survival and Dampening the Immune Dysregulation.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease with no effective treatment. The Hepatocyte Growth Factor/Scatter Factor (HGF/SF), through its receptor MET, is one of the most potent survival-promoting factors for motor neurons (MN) and is known as a modulator of immune cell function. We recently developed a novel recombinant MET agonist optimized for therapy, designated K1K1. K1K1 was ten times more potent than HGF/SF in preventing MN loss in an in vitro model of ALS. Treatments with K1K1 delayed the onset of muscular impairment and reduced MN loss and skeletal muscle denervation of superoxide dismutase 1 G93A (SOD1G93A) mice. This effect was associated with increased levels of phospho-extracellular signal-related kinase (pERK) in the spinal cord and sciatic nerves and the activation of non-myelinating Schwann cells. Moreover, reduced activated microglia and astroglia, lower T cells infiltration and increased interleukin 4 (IL4) levels were found in the lumbar spinal cord of K1K1 treated mice. K1K1 treatment also prevented the infiltration of T cells in skeletal muscle of SOD1G93A mice. All these protective effects were lost on long-term treatment suggesting a mechanism of drug tolerance. These data provide a rational justification for further exploring the long-term loss of K1K1 efficacy in the perspective of providing a potential treatment for ALS.

Decreasing HepG2 Cytotoxicity by Lowering the Lipophilicity of Benzo[d]oxazolephosphinate Ester Utrophin Modulators.

Utrophin modulation is a disease-modifying therapeutic strategy for Duchenne muscular dystrophy that would be applicable to all patient populations. To improve the suboptimal profile of ezutromid, the first-in-class clinical candidate, a second generation of utrophin modulators bearing a phosphinate ester moiety was developed. This modification significantly improved the physicochemical and ADME properties, but one of the main lead molecules was found to have dose-limiting hepatotoxicity. In this work we describe how less lipophilic analogues retained utrophin modulatory activity in a reporter gene assay, upregulated utrophin protein in dystrophic mouse muscle cells, but also had improved physicochemical and ADME properties. Notably, ClogP was found to directly correlate with pIC50 in HepG2 cells, hence leading to a potentially safer toxicological profiles in this series. Compound 21 showed a balanced profile (H2K EC50: 4.17 μM, solubility: 477 μM, mouse hepatocyte T 1/2 > 240 min) and increased utrophin protein 1.6-fold in a Western blot assay.

Bioenergetic defects in muscle fibers of RYR1 mutant knock-in mice associated with malignant hyperthermia

Mutations in the skeletal muscle ryanodine receptor gene (RYR1) can cause susceptibility to malignant hyperthermia (MH), a potentially lethal genetic condition triggered by volatile anesthetics. MH is associated with hypermetabolism, which has directed research interest into oxidative phosphorylation and muscle bioenergetics. The most common cause of MH in the United Kingdom is the c.7300G>A RYR1 variant, which is present in ∼16% of MH families. Our study focuses on the MH susceptible G2435R-RYR1 knock-in mouse model, which is the murine equivalent of the human c.7300G>A genotype. Using a combination of transcriptomics, protein expression, and functional analysis, we investigated adult muscle fiber bioenergetics in this mouse model. RNA-Seq data showed reduced expression of genes associated with mitochondria and fatty acid oxidation in RYR1 mutants when compared with WT controls. Mitochondrial function was assessed by measuring oxygen consumption rates in permeabilized muscle fibers. Comparisons between WT and homozygous G2435R-RYR1 mitochondria showed a significant increase in complex I-facilitated oxidative phosphorylation in mutant muscle. Furthermore, we observed a gene-dose-specific increase in reactive oxygen species production in G2435R-RYR1 muscle fibers. Collectively, these findings provide evidence of metabolic defects in G2435R-RYR1 knock-in mouse muscle under basal conditions. Differences in metabolic profile could be the result of differential gene expression in metabolic pathways, in conjunction with mitochondrial damage accumulated from chronic exposure to increased oxidative stress.

High-throughput screening identifies modulators of sarcospan that stabilize muscle cells and exhibit activity in the mouse model of Duchenne muscular dystrophy

BackgroundDuchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by mutations in the dystrophin gene. Loss of dystrophin prevents the formation of a critical connection between the muscle cell membrane and the extracellular matrix. Overexpression of sarcospan (SSPN) in the mouse model of DMD restores the membrane connection and reduces disease severity, making SSPN a promising therapeutic target for pharmacological upregulation.MethodsUsing a previously described cell-based promoter reporter assay of SSPN gene expression (hSSPN-EGFP), we conducted high-throughput screening on libraries of over 200,000 curated small molecules to identify SSPN modulators. The hits were validated in both hSSPN-EGFP and hSSPN-luciferase reporter cells. Hit selection was conducted on dystrophin-deficient mouse and human myotubes with assessments of (1) SSPN gene expression using quantitative PCR and (2) SSPN protein expression using immunoblotting and an ELISA. A membrane stability assay using osmotic shock was used to validate the functional effects of treatment followed by cell surface biotinylation to label cell surface proteins. Dystrophin-deficient mdx mice were treated with compound, and muscle was subjected to quantitative PCR to assess SSPN gene expression.ResultsWe identified and validated lead compounds that increased SSPN gene and protein expression in dystrophin-deficient mouse and human muscle cells. The lead compound OT-9 increased cell membrane localization of compensatory laminin-binding adhesion complexes and improved membrane stability in DMD myotubes. We demonstrated that the membrane stabilizing benefit is dependent on SSPN. Intramuscular injection of OT-9 in the mouse model of DMD increased SSPN gene expression.ConclusionsThis study identifies a pharmacological approach to treat DMD and sets the path for the development of SSPN-based therapies.

Golgi Outposts Nucleate Microtubules in Cells with Specialized Shapes.

Classically, animal cells nucleate or form new microtubules off the perinuclear centrosome. In recent years, the Golgi outpost has emerged as a satellite organelle that can function as an acentrosomal microtubule-organizing center (MTOC), nucleating new microtubules at distances far from the nucleus or cell body. Golgi outposts can nucleate new microtubules in specialized cells with unique cytoarchitectures, including Drosophila neurons, mouse muscle cells, and rodent oligodendrocytes. This review compares and contrasts topics of functional relevance, including Golgi outpost heterogeneity, formation and transport, as well as regulation of microtubule polarity and branching. Golgi outposts have also been implicated in the pathology of diseases including muscular dystrophy, and neurodegenerative diseases, such as Parkinson's disease (PD). Since Golgi outposts are relatively understudied, many outstanding questions regarding their function and roles in disease remain.

Calcium entry units (CEUs): perspectives in skeletal muscle function and disease

In the last decades the term Store-operated Ca2+ entry (SOCE) has been used in the scientific literature to describe an ubiquitous cellular mechanism that allows recovery of calcium (Ca2+) from the extracellular space. SOCE is triggered by a reduction of Ca2+ content (i.e. depletion) in intracellular stores, i.e. endoplasmic or sarcoplasmic reticulum (ER and SR). In skeletal muscle the mechanism is primarily mediated by a physical interaction between stromal interaction molecule-1 (STIM1), a Ca2+ sensor located in the SR membrane, and ORAI1, a Ca2+-permeable channel of external membranes, located in transverse tubules (TTs), the invaginations of the plasma membrane (PM) deputed to propagation of action potentials. It is generally accepted that in skeletal muscle SOCE is important to limit muscle fatigue during repetitive stimulation. We recently discovered that exercise promotes the assembly of new intracellular junctions that contains colocalized STIM1 and ORAI1, and that the presence of these new junctions increases Ca2+ entry via ORAI1, while improving fatigue resistance during repetitive stimulation. Based on these findings we named these new junctions Ca2+ Entry Units (CEUs). CEUs are dynamic organelles that assemble during muscle activity and disassemble during recovery thanks to the plasticity of the SR (containing STIM1) and the elongation/retraction of TTs (bearing ORAI1). Interestingly, similar structures described as SR stacks were previously reported in different mouse models carrying mutations in proteins involved in Ca2+ handling (calsequestrin-null mice; triadin and junctin null mice, etc.) or associated to microtubules (MAP6 knockout mice). Mutations in Stim1 and Orai1 (and calsequestrin-1) genes have been associated to tubular aggregate myopathy (TAM), a muscular disease characterized by: (a) muscle pain, cramping, or weakness that begins in childhood and worsens over time, and (b) the presence of large accumulations of ordered SR tubes (tubular aggregates, TAs) that do not contain myofibrils, mitochondria, nor TTs. Interestingly, TAs are also present in fast twitch muscle fibers of ageing mice. Several important issues remain un-answered: (a) the molecular mechanisms and signals that trigger the remodeling of membranes and the functional activation of SOCE during exercise are unclear; and (b) how dysfunctional SOCE and/or mutations in Stim1, Orai1 and calsequestrin (Casq1) genes lead to the formation of tubular aggregates (TAs) in aging and disease deserve investigation.

Decreased smooth muscle function, peristaltic activity, and gastrointestinal transit in dystrophic (mdx) mice.

Duchenne muscular dystrophy (DMD) is characterized by the lack of dystrophin in skeletal, cardiac, and smooth muscle. Slow colonic transit and constipation are common in DMD patients and animal models of DMD. However, the cause of this hypocontractility and the expression of contractile proteins in smooth muscle are unknown. The aim of the study was to investigate the expression of contractile proteins in the colonic smooth muscle and the function of the colon in control and mdx mice. Muscle contraction was measured in muscle strips and isolated muscle cells. Peristaltic activity was measured in ex vivo preparations by spatiotemporal mapping, and gastrointestinal (GI) transit in vivo was measured by the distribution of fluorescent marker along the intestine and colon. mRNA expression of contractile proteins smoothelin, caldesmon, calponin, and tropomyosin was measured by qRT-PCR. Expression of mRNA for contractile proteins was decreased in colonic smooth muscle of mdx mice compared with control. Contraction in response to acetylcholine and KCl was decreased in colonic muscle strips and in isolated muscle cells of mdx mice. Distension of ex vivo colons with Krebs buffer induced peristalsis in both control and mdx mice; however, significantly fewer full peristaltic waves were recorded in the colons of mdx mice. GI transit was also inhibited in mdx mice. The data indicate that the lack of dystrophin causes decrease in colonic smooth muscle contractility, peristalsis, and GI transit and provides the basis for analysis of mechanisms involved in smooth muscle dysfunction in DMD.

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.