Regenerating Muscle Fibers Research Articles

Myoblast-derived exosomes promote the repair and regeneration of injured skeletal muscle in mice.

When skeletal muscle is damaged, satellite cells (SCs) are activated to proliferate rapidly and fuse with the damaged muscle fibers to form new muscle fibers, thereby promoting muscle growth and remodeling and repair of trauma. Exosomes from differentiating human skeletal muscle cells trigger myogenesis of stem cells and provide biochemical cues for skeletal muscle regeneration. Therefore, we hypothesized that, when muscles are injured, myoblast-derived exosomes may regulate muscle repair and regeneration. Here, we investigated the underlying mechanism by applying C2C12-derived exosomes to injured mouse skeletal muscles. The expression levels of skeletal muscle regeneration factors paired box 7 and lipid-promoting factor peroxisome proliferator-activated receptor γ were upregulated, whereas the expression levels of fibrosis factors collagen-1 and α-smooth muscle actin decreased. The expression of proliferating cell nuclear antigen was elevated after applying C2C12-derived exosomes to SCs. Application of C2C12-derived exosomes to fibro-adipogenic progenitors resulted in an increase in peroxisome proliferator-activated receptor γ expression and adipogenesis capacity, whereas α-smooth muscle actin expression and fibrosis capacity decreased. Analysis of the transcriptome and proteome of SCs after treatment with exosomes showed the involvement of multiple biological processes, including proliferation and differentiation of SCs, muscle regeneration, skeletal muscle atrophy, and the inflammatory response after muscle injury. Hence, our data suggest that C2C12-derived exosomes can promote the regeneration of skeletal muscle fibers, accelerate the production of fat from damaged muscles, inhibit the fibrosis of damaged muscles, and accelerate injury repair, which is related to exosome-mediated regulation of the proliferation of SCs, differentiation of fibro-adipogenic progenitors, and modulation of SC mRNA expression and protein formation and decomposition.

Autophagy in non-immune-mediated rhabdomyolysis: Assessment of p62 immunohistochemistry.

p62 immunochemistry (IHC) has been shown to aid diagnosis with distinct patterns of muscle fiber staining observed in some inflammatory, hereditary, and degenerative myopathies, such as immune-mediated necrotizing myopathy (IMNM). The pattern of p62 staining may help narrow the pathological differential diagnosis of rhabdomyolysis. However, there is a lack of information on the pattern of p62 IHC in non-immune-mediated rhabdomyolysis. In this study we aim to describe histopathological findings in non-immune-mediated rhabdomyolysis, with particular emphasis on the pattern of p62 IHC. We retrospectively reviewed the histopathological features of patients with a confirmed diagnoses of non-immune-mediated rhabdomyolysis referred to our center. Five patients were identified. Rhabdomyolysis was determined to be due to statin-associated toxicity in three patients, alcohol overuse in one patient, and intensive exercise in one patient. All patients showed increased numbers of necrotic and regenerating muscle fibers. Diffuse and finely granular sarcoplasmic positive p62 staining was present in scattered non-necrotic muscle fibers in all patients. Disturbance of autophagy appears to be a common mechanism in non-immune-mediated rhabdomyolysis. Our results show p62 IHC is sensitive but lacks specificity. Therefore, the pattern of p62 staining does not distinguish non-immune-mediated rhabdomyolysis from histopathologically similar IMNM.

Muscle magnetic resonance imaging abnormality in neuroleptic malignant syndrome: a case report

BackgroundNeuroleptic malignant syndrome (NMS) is a rare and occasionally fatal undesirable reaction to dopamine antagonists, and its phenotype is diverse owing to causative drugs. Classically, elevation of serum creatine kinase is described in NMS. Some reports have described muscular pathological findings; however, muscle magnetic resonance imaging (MRI) has not been reported previously.Case presentationA 63-year-old woman with a history of schizophrenia presented to our hospital with a high fever, excessive sweating, muscle weakness, and elevated serum creatine kinase levels. Muscle MRI revealed T2 high-intensity lesions in several muscles with gadolinium enhancement, and the pathology of the muscle biopsy showed a very mild presence of muscle fiber necrosis and regeneration with type 2c fibers without inflammation. Her symptoms resolved by treatment with levodopa/carbidopa, dantrolene. Finally, the patient was diagnosed with NMS.ConclusionsThis is the first report of muscle MRI abnormalities in a patient with NMS. Muscle MRI abnormalities in NMS may be associated with non-inflammatory myopathic changes. The cause of creatine kinase elevation cannot be explained by abnormal strong muscle contraction nor inflammation.

Early Changes in Structural and Stress-Like Genes after Radiation to the Submental Muscles

INTRODUCTION: Radiation treatments for head and neck cancer can lead to early alterations in swallowing kinematics that ultimately develop into dysphagia and require modifications in oral intake. The submental muscles (ie mylohyoid) are located inferior to the mandible and are crucial for swallowing function. Expression of collagen and heat shock proteins can be altered following muscle injury, which can impair muscle fiber regeneration leading to conditions of muscle atrophy or fibrosis. The aim of this study was to evaluate collagen and heat shock protein (HSP) 72 (HSPA1A/B) expression in a rat model of radiation-induced dysphagia. HSP72 is known to increase in response to stress and is needed to mediate muscle repair after injury. METHODS: We hypothesized that the submental muscles treated with 48 Gy of fractionated radiation would have increased expression of HSPA1A, HSPA1B, COL1, COL3, and COL4 genes at 2 and 4 weeks post treatment. Relative gene expression was measured using RT-PCR. RESULTS: The results showed that there was a sequential increase in expression of HSPA1A, HSPA1B, COL1, and COL3 at both the 2-week and 4-week post irradiation points. There was a greater increase in expression in COL4 at the 2-week compared to the 4-week post irradiation point. The highest change in gene expression was observed with HSPA1A and COL3. CONCLUSION: Results support the idea that irradiation treatment provokes early gene changes that may contribute to modifying the muscle structure and promoting dysphagia which can lead to decreased quality of life for head and neck cancer patients.

Assessment of PABPN1 nuclear inclusions on a large cohort of patients and in a human xenograft model of oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is a rare muscle disease characterized by an onset of weakness in the pharyngeal and eyelid muscles. The disease is caused by the extension of a polyalanine tract in the Poly(A) Binding Protein Nuclear 1 (PABPN1) protein leading to the formation of intranuclear inclusions or aggregates in the muscle of OPMD patients. Despite numerous studies stressing the deleterious role of nuclear inclusions in cellular and animal OPMD models, their exact contribution to human disease is still unclear. In this study, we used a large and unique collection of human muscle biopsy samples to perform an in-depth analysis of PABPN1 aggregates in relation to age, genotype and muscle status with the final aim to improve our understanding of OPMD physiopathology. Here we demonstrate that age and genotype influence PABPN1 aggregates: the percentage of myonuclei containing PABPN1 aggregates increases with age and the chaperone HSP70 co-localize more frequently with PABPN1 aggregates with a larger polyalanine tract. In addition to the previously described PRMT1 and HSP70 co-factors, we identified new components of PABPN1 aggregates including GRP78/BiP, RPL24 and p62. We also observed that myonuclei containing aggregates are larger than myonuclei without. When comparing two muscles from the same patient, a similar amount of aggregates is observed in different muscles, except for the pharyngeal muscle where fewer aggregates are observed. This could be due to the peculiar nature of this muscle which has a low level of PAPBN1 and contains regenerating fibers. To confirm the fate of PABPN1 aggregates in a regenerating muscle, we generated a xenograft model by transplanting human OPMD muscle biopsy samples into the hindlimb of an immunodeficient mouse. Xenografts from subjects with OPMD displayed regeneration of human myofibers and PABPN1 aggregates were rapidly present—although to a lower extent-after muscle fiber regeneration. Our data obtained on human OPMD samples add support to the dual non-exclusive models in OPMD combining toxic PABPN1 intranuclear inclusions together with PABPN1 loss of function which altogether result in this late-onset and muscle selective disease.

P.61 Imaging Mass Cytometry reveals new clues to understand the pathogenesis of Becker muscular dystrophy

Muscular dystrophies are genetic diseases characterised by muscle fiber loss and its replacement by fibrotic and adipose tissue. Several cell types, including macrophages and fibroadipogenic precursor cells (FAPS) interact with degenerative muscle fibers locally in what is known as the degenerative niche. Recent technological advances known as spatial biology allow dissecting cell-to-cell interactions in vivo using samples of tissue, although this has not been applied to human muscle tissue yet. We analysed muscle biopsies of controls and patients with Becker muscular dystrophy (BMD) at early, moderate and advanced stages of degeneration using Hyperion Imaging Mass Cytometry (IMC). We labelled single sections of the biopsies with 40 markers labelling different components of the muscle structure. We developed a deep learning algorithm to analyse the images and studied changes in the composition and spatial correlations of the different markers across disease progression. We included 2 control and 8 muscle biopsies of BMD. We observed a progressive increase in the amount of collagen-I and III, that significantly correlated with the amount of CD68+ macrophages (R=0.93 and R=0.94), most of them positive for the M2 marker CD206. Fat and collagen I surrounded vessels during the first stages of disease progression, substituting muscle fibres in later stages. We observed a negative correlation between regenerative muscle fibers and collagen-I (R=-0.83) and with CD68 cells (R:-0.90). We also observed a modification in the number of satellite cells in the biopsies across disease progression. IMC enables the study of changes in muscle structure along disease progression in patients with muscular dystrophies establishing spatio-temporal correlations opening the door to confirm and understand new potential pathogenic pathways in vivo in human samples.

The chemokine receptor CXCR4 regulates satellite cell activation, early expansion, and self-renewal, in response to skeletal muscle injury.

Acute skeletal muscle injury is followed by satellite cell activation, proliferation, and differentiation to replace damaged fibers with newly regenerated muscle fibers, processes that involve satellite cell interactions with various niche signals. Here we show that satellite cell specific deletion of the chemokine receptor CXCR4, followed by suppression of recombination escapers, leads to defects in regeneration and satellite cell pool repopulation in both the transplantation and in situ injury contexts. Mechanistically, we show that endothelial cells and FAPs express the gene for the ligand, SDF1α, and that CXCR4 is principally required for proper activation and for transit through the first cell division, and to a lesser extent the later cell divisions. In the absence of CXCR4, gene expression in quiescent satellite cells is not severely disrupted, but in activated satellite cells a subset of genes normally induced by activation fail to upregulate normally. These data demonstrate that CXCR4 signaling is essential to normal early activation, proliferation, and self-renewal of satellite cells.

Dystrophin Restoration after Adeno-Associated Virus U7–Mediated Dmd Exon Skipping Is Modulated by Muscular Exercise in the Severe D2-Mdx Duchenne Muscular Dystrophy Murine Model

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease caused by Dmd mutations, resulting in the absence of dystrophin in skeletal muscle, and a greater susceptibility to damage during contraction (exercise). The current study evaluated whether voluntary exercise impacts a Dmd exon skipping and muscle physiology in a severe DMD murine model. D2-mdx mice were intramuscularly injected with an adeno-associated virus (AAV) U7 snRNA to correct Dmd reading frame, and allowed to voluntary run on a wheel for 1 month. Voluntary running did not induce muscle fiber regeneration, as indicated by the percentage of centronucleated fibers, Myh3 and Myh4 expression, and maximal force production, and thus possibly did not compromise the gene therapy approach. Voluntary running did not impact the number of viral genomes and the expression of U7 and Dmd 1 month after injection of AAV-U7 injected just before exercise initiation, but reduced the amount of dystrophin in dystrophin-expressing fibers from 80% to 65% of the muscle cross-sectional area. In conclusion, voluntary running did not induce muscle damage and had no drastic detrimental effect on the AAV gene therapy exon skipping approach in a severe murine DMD model. Moreover, these results suggest considering exercise as an additional element in the design and conception of future therapeutic approaches for DMD.

Pregabalin synchronizes the regeneration of nerve and muscle fibers optimizing the gait recovery of MDX dystrophic mice.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder induced by mutations in the dystrophin gene, leading to a degeneration of muscle fibers, triggering retrograde immunomodulatory, and degenerative events in the central nervous system. Thus, neuroprotective drugs such as pregabalin (PGB) can improve motor function by modulating plasticity, together with anti-inflammatory effects. The present work aimed to study the effects of PGB on axonal regeneration after axotomy in dystrophic and non-dystrophic mice. For that, MDX and C57BL/10 mouse strains were subjected to peripheral nerve damage and were treated with PGB (30 mg/kg/day, i.p.) for 28 consecutive days. The treatment was carried out in mice as soon as they completed 5 weeks of life, 1 week before the lesion, corresponding to the peak period of muscle degeneration in the MDX strain. Six-week-old mice were submitted to unilateral sciatic nerve crush and were sacrificed in the 9th week of age. The ipsi and contralateral sciatic nerves were processed for immunohistochemistry and qRT-PCR, evaluating the expression of proteins and gene transcripts related to neuronal and Schwann cell activity. Cranial tibial muscles were dissected for evaluation of neuromuscular junctions using α-bungarotoxin, and the myelinated axons of the sciatic nerve were analyzed by morphometry. The recovery of motor function was monitored throughout the treatment through tests of forced locomotion (rotarod) and spontaneous walking track test (Catwalk system). The results show that treatment with PGB reduced the retrograde cyclic effects of muscle degeneration/regeneration on the nervous system. This fact was confirmed after peripheral nerve injury, showing better adaptation and response of neurons and glia for rapid axonal regeneration, with efficient muscle targeting and regain of function. No side effects of PGB treatment were observed, and the expression of pro-regenerative proteins in neurons and Schwann cells was upregulated. Morphometry of the axons was in line with the preservation of motor endplates, resulting in enhanced performance of dystrophic animals. Overall, the present data indicate that pregabalin is protective and enhances regeneration of the SNP during the development of DMD, improving motor function, which can, in turn, be translated to the clinic.

Caveolin-3 regulates the activity of Ca2+/calmodulin-dependent protein kinase II in C2C12 cells.

Caveolins, encoded by the Cav gene family, are the main components of caveolae. Caveolin-3 (Cav3) is specifically expressed in muscle cells. Mutations in Cav3 are responsible for a group of muscle diseases called caveolinopathies, and Cav3 deficiency is associated with sarcolemmal membrane alterations, disorganization of T-tubules, and disruption of specific cell-signaling pathways. However, Cav3 overexpression increases the number of sarcolemmal caveolae and muscular dystrophy-like regenerating muscle fibers with central nuclei, suggesting that the alteration of Cav3 expression levels or localization influences muscle cell functions. Here, we used mouse C2C12 myoblasts in which Cav3 expression was suppressed with short hairpin RNA and found that Cav3 suppression impaired myotube differentiation without affecting the expression of MyoD and Myog. We also observed an increase of intracellular Ca2+ levels, total calpain activity, and Ca2+-dependent calmodulin kinase II (CaMKII) levels in Cav3-depleted myoblasts. Importantly, those phenotypes due to Cav3 suppression were caused by the ryanodine receptor activation. Furthermore, pharmacological inhibition of CaMKII rescued the impairment of myoblast differentiation due to Cav3 knockdown. Our results suggest that Cav3 regulates intracellular Ca2+ concentrations by modulating ryanodine receptor activity in muscle cells and that CaMKII suppression in muscle could be a novel therapy for caveolinopathies.

N-CAM Expression: The Study of Muscle Disease in a Tertiary Center of Thailand

Background: Neural cell adhesion molecule (N-CAM), or CD56, is a cell-surface glycoprotein that plays a critical role in mediating intercellular adhesion in the central nervous system. It is also found to be expressed on embryonic muscle but disappears in healthy adult muscle. However, denervated or regenerating muscles can express N-CAM. Objective: To evaluate the value of N-CAM expressions in diagnosing muscle diseases in the Thai population. Materials and Methods: The present study used immunohistochemistry to interpret N-CAM expression in 75 muscle biopsy specimens diagnosed with myopathies and non-myopathies from a 3-year retrospective cohort. Results: Using the chi-squared test, there was statistically significant associativity between N-CAM expression and the diagnosis of inflammatory myopathy, but not in muscular dystrophy, neurogenic change, or non-specific myopathy even though they also showed N-CAM expression. The inflammatory myopathy showed positive N-CAM in 15 out of 17 cases with statistical significance (p<0.001, OR 14.250, 95% CI 2.960 to 68.606). The authors did not find any N-CAM expression in the present study mitochondrial myopathy or non-myopathy cases, whose biopsies were defined as a control group due to lack of myopathic change and non-specific clinical symptoms. Conclusion: The authors suggest the evaluation of N-CAM expression as a complementary tool in the diagnosis of muscle diseases that have denervated or regenerating muscle fibers, especially in inflammatory myopathy. Keywords: Neural cell adhesion molecule; N-CAM; CD56; Muscle disease; Myopathy

The role of the aging microenvironment on the fate of PDGFRβ lineage cells in skeletal muscle repair

BackgroundDuring aging, perturbation of muscle progenitor cell (MPC) constituents leads to progressive loss of muscle mass and accumulation of adipose and fibrotic tissue. Mesenchymal stem cells (MSCs) give rise to adipocytes and fibroblasts that accumulate in injured and pathological skeletal muscle through constitutive activation of platelet-derived growth factor receptors (PDGFRs). Although the role of the PDGFRα has been widely explored, there is a paucity of evidence demonstrating the role of PDGFRβ in aged skeletal muscle.MethodsIn this study, we investigated the role of PDGFRβ lineage cells in skeletal muscle during aging by using Cre/loxP lineage tracing technology. The PDGFR-Cre mice were crossed with global double-fluorescent Cre reporter mice (mTmG) that indelibly marks PDGFRβ lineage cells. Those cells were analyzed and compared at different ages in the skeletal muscle of the mice.ResultsOur results demonstrated that PDGFRβ lineage cells isolated from the muscles of young mice are MPC-like cells that exhibited satellite cell morphology, expressed Pax7, and undergo myogenic differentiation producing myosin heavy chain expressing myotubes. Conversely, the PDGFRβ lineage cells isolated from muscles of old mice displayed MSC morphology with a reduced myogenic differentiation potential while expressing adipogenic and fibrotic differentiation markers. PDGFRβ lineage cells also gave rise to newly regenerated muscle fibers in young mice after muscle injury, but their muscle regenerative process is reduced in old mice.ConclusionsOur data suggest that PDGFRβ lineage cells function as MPCs in young mice, while the same PDGFRβ lineage cells from old mice undergo a fate switch participating in adipose and fibrotic tissue infiltration in aged muscle. The inhibition of fate-switching in PDGFRβ lineage cells may represent a potential approach to prevent fibrosis and fatty infiltration in skeletal muscle during the aging process.

Inducible Heat Shock Protein 70 Levels in Patients and the mdx Mouse Affirm Regulation during Skeletal Muscle Regeneration in Muscular Dystrophy.

Stress-inducible heat shock protein 70 (HSP70) is both a protective chaperone involved in protein homeostasis and an immune regulator. In both capacities, HSP70 has been implicated in muscle disorders, yet with fragmented and differing results. In this study we aimed to compare results obtained in the mouse model for the severest form of muscular dystrophy (MD) equivalent to Duchenne MD, termed the mdx mouse, with results obtained in human MD. Skeletal muscle and serum samples were obtained from 11 healthy controls, 11 fully characterized patients diagnosed with Becker MD and limb girdle MD (LGMD), and six muscle disease controls. In addition, muscle extracts were prepared from tibialis anterior of mdx and control mice at ages 4, 8 and 12 weeks. The HSP70 levels were quantified using RT-PCR, western blotting and protein arrays, and localized in muscle tissue sections using double immunofluorescence. We found selective and significant 2.2-fold upregulation of HSP70 protein in mdx tibialis muscle at the earliest disease phase only. In LGMD and Becker MD patients, HSP70 protein levels were not significantly different from those of healthy muscle and serum. HSP70 was localized to regenerating muscle fibers both in mouse and human MD skeletal muscle tissues. Toll-like receptor (TLR) 2 and TLR4 expression was moderately increased on the sarcolemma in MD muscle, yet protein levels were not significantly different from normal controls. HSP70 upregulation in MD appears disease stage-dependent, marking the phase of most active muscle regeneration in the mdx mouse. We postulate that well-timed supportive therapeutic interventions with HSP70 agonists could potentially improve muscle tissue's regenerative capacities in MD, attenuating loss of muscle mass while we await gene therapies to become more widely available.

Tuning Myogenesis by Controlling Gelatin Hydrogel Properties through Hydrogen Peroxide-Mediated Cross-Linking and Degradation.

Engineering skeletal muscle tissue in vitro is important to study the mechanism of myogenesis, which is crucial for regenerating muscle cells. The physicochemical properties of the cellular microenvironment are known to govern various cell behaviours. Yet, most studies utilised synthetic materials to model the extracellular matrix that suffers from cytotoxicity to the cells. We have previously reported that the physicochemical property of hydrogels obtained from horseradish peroxidase (HRP)-catalysed cross-linking could be controlled by a simple adjustment to the exposure time to air containing H2O2. In this study, we evaluated the influence of physicochemical properties dynamics in the gelatin possessing phenol groups (Gelatin-Ph) hydrogel to regulate the myogenesis in vitro. We controlled the Young’s modulus of the Gelatin-Ph hydrogel by tuning the air containing 16 ppm H2O2 exposure time for 15–60 min. Additionally, prolonged exposure to air containing H2O2 also induced Gelatin-Ph degradation. Myoblasts showed higher adhesion and myotube formation on stiff hydrogel (3.53 kPa) fabricated through 30 min of exposure to air containing H2O2 compared to those on softer hydrogel (0.77–2.79 kPa) fabricated through 15, 45, and 60 min of the exposure. These results demonstrate that the myogenesis can be tuned by changes in the physicochemical properties of Gelatin-Ph hydrogel mediated by H2O2.

Regeneration of Skeletal Muscle Fibers and Regulation of Myosatellitocytes Metabolism

Regeneration of Skeletal Muscle Fibers and Regulation of Myosatellitocytes Metabolism

Natural antisense RNA Foxk1-AS promotes myogenic differentiation by inhibiting Foxk1 activity

BackgroundNatural antisense RNAs are RNA molecules that are transcribed from the opposite strand of either protein-coding or non-protein coding genes and have the ability to regulate the expression of their sense gene or several related genes. However, the roles of natural antisense RNAs in the maintenance and myogenesis of muscle stem cells remain largely unexamined.MethodsWe analysed myoblast differentiation and regeneration by overexpression and knockdown of Foxk1-AS using lentivirus and adeno-associated virus infection in C2C12 cells and damaged muscle tissues. Muscle injury was induced by BaCl2 and the regeneration and repair of damaged muscle tissues was assessed by haematoxylin–eosin staining and quantitative real-time PCR. The expression of myogenic differentiation-related genes was verified via quantitative real-time PCR, Western blotting and immunofluorescence staining.ResultsWe identified a novel natural antisense RNA, Foxk1-AS, which is transcribed from the opposite strand of Foxk1 DNA and completely incorporated in the 3′ UTR of Foxk1. Foxk1-AS targets Foxk1 and functions as a regulator of myogenesis. Overexpression of Foxk1-AS strongly inhibited the expression of Foxk1 in C2C12 cells and in tibialis anterior muscle tissue and promoted myoblast differentiation and the regeneration of muscle fibres damaged by BaCl2. Furthermore, overexpression of Foxk1-AS promoted the expression of Mef2c, which is an important transcription factor in the control of muscle gene expression and is negatively regulated by Foxk1.ConclusionThe results indicated that Foxk1-AS represses Foxk1, thereby rescuing Mef2c activity and promoting myogenic differentiation of C2C12 cells and regeneration of damaged muscle fibres.DfdN9uwC-a513-TBDoKHuCVideo

Morphological evaluation of the effect of continuous direct electrostimulation with the help of the bioelectric system on the denervated circular musculature of the eye in case of experimental single facial nerve injury

The use of direct electrical stimulation to produce shortening of the denervated circular oculus in experimental facial nerve palsy (FNP) allows for complete closure of the ocular slit. The aim of this study is to study morphological changes in the nerves under the influence of direct electrical stimulation with biphasic pulses over a long period of time. Materials and methods: A model of a single facial nerve injury with consequent paralysis of orbicularis oculi muscle was created in the experiment on 12 adult animals. The animals were divided into 2 groups: main and control. In the main group, the stimulating part of the bioelectric device for eye closure was implanted, after which the ability to work in real-time mode to perform long-duration stimulation of orbicularis oculi muscle was tested. The control group did not receive any effect effects on orbicularis oculi muscle. After 30 days, all the animals underwent orbicularis oculi muscle elimination on the side of the facial nerve palsy with the following morphological examination. Results: An intergroup comparative analysis using the nonparametric U-Mann-Whitney criterion for changes in the Ki-67 proliferation index revealed a statistically significant difference between the comparison group and the experimental group, that was affected by the electrical stimulation with the increase of the parameter for the group with electrical stimulation according to the control group from 28,3 (25,0; 30.8) to 30.9 (28.8; 33.3), or 1.1 times (p=0.04); Analysis of variation in mitochondrial photocurrent density revealed statistically significant differences between the control group and the experimental group, with the increase of this parameter for the group with electrical stimulation compared with the control group from 10.5 (6.3; 14.4) to 15.8 (12.8 17,6), i.e. by 1.5 times (p=0.0004); a statistically significant increase of the parameter for the group with electrical stimulation versus the control group from 48,9 (40,6; 63.0) to 64.4 (60.0; 72.4), i.e. by a factor of 1.3 (p = 0.00005). Conclusions: The assessment of the effects of electrostimulation on the musculoskeletal apparatus after neuroraphy (in the experiment) revealed structural and ultrastructural changes, indicating the increased morphofunctional activity of renewed regeneration of muscle fibers in the study under the influence of direct electrical stimulation. In addition, no specific pathological changes were detected under the influence of direct electrostimulation for 30 days.

Optimized lentiviral vector to restore full-length dystrophin via a cell-mediated approach in a mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the DMD gene. Restoration of full-length dystrophin protein in skeletal muscle would have therapeutic benefit, but lentivirally mediated delivery of such a large gene in vivo has been hindered by lack of tissue specificity, limited transduction, and insufficient transgene expression. To address these problems, we developed a lentiviral vector, which contains a muscle-specific promoter and sequence-optimized full-length dystrophin, to constrain dystrophin expression to differentiated myotubes/myofibers and enhance the transgene expression. We further explored the efficiency of restoration of full-length dystrophin in vivo, by grafting DMD myoblasts that had been corrected by this optimized lentiviral vector intramuscularly into an immunodeficient DMD mouse model. We show that these lentivirally corrected DMD myoblasts effectively reconstituted full-length dystrophin expression in 93.58% ± 2.17% of the myotubes in vitro. Moreover, dystrophin was restored in 64.4% ± 2.87% of the donor-derived regenerated muscle fibers in vivo, which were able to recruit members of the dystrophin-glycoprotein complex at the sarcolemma. This study represents a significant advance over existing cell-mediated gene therapy strategies for DMD that aim to restore full-length dystrophin expression in skeletal muscle.

A myogenic niche with a proper mechanical stress environment improves abdominal wall muscle repair by modulating immunity and preventing fibrosis

Volumetric muscle loss (VML) healing is often complicated by fibrosis, which impairs muscle regeneration and function. Adjusting mechanical stress in the repair environment may modulate immunity and reduce fibrosis. In this study, we aimed to create a biomaterial with suitable tension conditions and bidirectional tissue-inducing abilities to prevent fibrosis thus promote muscle regeneration and induce aponeurosis-like structures to restore muscle force transmission. A protocol was developed to manufacture decellularized muscle aponeurosis (D-MA) patches with an intact extracellular matrix (ECM) and low cytotoxicity. D-MA optimized the mechanical stress distribution in muscle injury sites and decreased the number of proinflammatory macrophages and myofibroblasts, thereby attenuating muscle fibrosis. Muscle and aponeurosis ECM environments had different microstructures and mechanical properties, which specifically enhanced stem cell differentiation into muscle-like cells on muscle ECM and tenocyte-like cells on aponeurosis ECM in vitro. Four weeks after orthotopic implantation, the biphasic muscle-aponeurosis-like tissue was successfully regenerated by the D-MA scaffold. The regenerated muscle fibers in D-MA were more abundant than those in the fibrotic decellularized muscle (D-M) scaffold. D-MA can be used to repair abdominal defects, which significantly improves the repair outcomes. Our results suggest D-MA as a promising material for VML repair.

P043 Challenges in the diagnosis and management of immune-mediated necrotizing myopathy in a patient on long-term statins

Abstract Background/Aims Statins are very common medications prescribed for cardiovascular disease prevention and with their ever-increasing use; we expect a significant increase in even the rarest side-effects. Immune-mediated necrotising myopathy (IMNM) is a rare type of autoimmune myopathy characterised by relatively severe proximal weakness and is most associated with the development of anti-HMGCoAR (3-hydroxy-3-methylglutaryl CoA Reductase) autoantibodies in statin users. The objective of this clinical case report is to highlight this rare condition to avoid incorrect diagnosis, particularly in primary care, to ensure prompt and specific management. Methods A 51-year-old female presented to her GP with gradual onset muscle weakness particularly around her shoulders and legs. She had a history of hypercholesterolemia, for which she had been on atorvastatin 20mg daily for 4 years. Results Following a blood test by her GP, a raised CK of 16,670 iU/L prompted a hospital admission due to suspected skeletal muscle damage. MRI scan of thighs showed features of myositis and muscle biopsy showed necrotic regenerating muscle fibres with lymphohistiocytic inflammation consistent with necrotizing myopathy. Blood tests revealed positive anti-HMGCoAR antibodies (negative ANA, DNA, ENA & other myositis-specific antibodies) confirming a diagnosis of anti-HMGCoAR IMNM. Atorvastatin was stopped and treatment was initiated with high-dose IV methylprednisolone followed by oral prednisolone tapering course. She showed only minor improvement in symptoms over the initial 6 weeks. Methotrexate was then added, with gradual improvement in her symptoms and normalization of CK over the next 6 months. Conclusion IMNM is unique in that it does not improve with statin discontinuation alone, and usually requires a combination of steroid and steroid-sparing immunosuppressant. Early diagnosis is crucial as commencing treatment early leads to better outcomes in patients. Anti-HMGCoAR antibodies are highly specific for this condition and not associated per se with hyperlipidaemia, self-limiting statin side effects or genetic muscle disease. In patients presenting with muscle weakness who are taking statins, it is beneficial to perform anti-HMGCoAR antibody test (especially if persistent symptoms despite statin cessation) to promptly differentiate this rare condition from more common but less severe statin related myotoxicities. Disclosure F. Khan: None. A. Kuttikat: None.