Skeletal Muscle Regeneration Research Articles

Clinical, Histopathologic, and Genetic Features of Patients With Myofibrillary and Distal Myopathies: Experience From the Italian Network.

The diagnostic process for myofibrillar myopathies (MFM) and distal myopathies (DM) is particularly complex because of the large number of causative genes, the existence of still molecularly undefined disease entities, and the overlapping features between the 2 categories. This study aimed to characterize a large cohort of patients affected by MFM and DM and identify the most important diagnostic and prognostic aspects of these diseases. Patients with either a myopathological diagnosis of MFM or a clinical diagnosis of DM were included in this retrospective multicentric national study. Demographic, genetic, clinical, and histopathologic data of anonymized patients were collected from the neuromuscular centers of the Italian Association of Myology network. Data regarding 132 patients with MFM (mean age 57.0 ± 15.8 years, 49% female) and 298 patients with DM (mean age 50.7 ± 15.9 years, 40% female) were gathered from 20 neuromuscular centers. 69 patients fulfilled the criteria for both groups (distal myopathies with myofibrillar pathology, DM-MP). Molecular confirmation was achieved in 63% of the patients. Fifty-two percent of the patients with MFM carried pathogenic variants in either DES (n = 30), MYOT (n = 20), or DNAJB6 (n = 18), which were also the most frequent disease-causing genes in DM-MP, while GNE (n = 44) and MYH7 (n = 23) were the genes most commonly carrying pathogenic variants in DM. The mean age at onset varied from <25 years in patients with causative variants in MYH7 and DYSF to 59 years in patients with myotilinopathies. Cardiac involvement was reported in 29% of patients with MFM and 16% of patients with DM, with DES and MYH7 variants significantly associated with the development of cardiomyopathy. Respiratory impairment was more prevalent in patients with TTN and DES variants and rare in other disorders such as GNE myopathy and dysferlinopathies, which were instead associated, together with DNAJB6-related and PLIN4-related myopathies, with the risk of losing ambulation during the disease course. The Italian cohort of patients with MFM and DM recapitulates the phenotypic heterogeneity and the partial overlap between the 2 groups. However, in relative contrast to the encountered phenotypic variability, only 5 genes accounted for most of the molecular diagnoses. Specific genetic entities are associated with significantly increased risk of developing cardiorespiratory complications or loss of ambulation, which has relevant prognostic implications.

Bioactive Hydrogels Inspired by Laminin: An Emerging Biomaterial for Tissue Engineering Applications.

Tissue or organ damage due to severe injuries or chronic diseases can adversely affect the quality of life. Current treatments rely on organ or tissue transplantation which has limitations including unavailability of donors, ethical issues, or immune rejection after transplantations. These limitations can be addressed by tissue regeneration which involves the development of bioactive scaffolds closely mimicking the extracellular matrix (ECM). One of the major components of ECM is the laminin protein which supports several tissues associated with important organs. In this direction, peptide-based hydrogels can effectively mimic the essential characteristics of laminin. While several reports have discussed the structure of laminin, the potential of laminin-derived peptide hydrogels as effective biomaterial for tissue engineering applications is yet to be discussed. In this context, the current review focuses on the structure of laminin and its role as an essential ECM protein. Further, the potential of short peptide hydrogels in mimicking the crucial properties of laminin is proposed. The review further highlights the significance of bioactive hydrogels inspired by laminin - in addressing numerous tissue engineering applications including angiogenesis, neural, skeletal muscle, liver, and adipose tissue regeneration along with a brief outlook on the future applications of these laminin-based hydrogels.

Inhibitor of FTO, Rhein, Restrains the Differentiation of Myoblasts and Delays Skeletal Muscle Regeneration.

N6-methyladenosine (m6A) is a crucial RNA modification affecting skeletal muscle development. Rhein, an anti-inflammatory extract, inhibits FTO, a key demethylase in m6A metabolism. Our study showed that during muscle fiber formation, FTO and ALKBH5 expression increased while m6A levels decreased. After muscle injury, FTO and ALKBH5 expression initially rose but later fell, while m6A levels initially dropped and then recovered. Inhibition of FTO by Rhein reduced MyHC and MyoG expression, indicating myoblast differentiation suppression. In a mouse model, Rhein decreased MyHC expression and muscle fiber cross-sectional area, delaying muscle regeneration. Rhein's ability to increase RNA m6A modification delays skeletal muscle remodeling post-injury, suggesting a new medicinal application for this plant extract.

A network pharmacology-based study to investigate the mechanism of curcumin-regulated regenerative repair of quadriceps femoris muscle in KOA rats

BackgroundKnee osteoarthritis (KOA) is a very common musculoskeletal disorder, and patients with KOA often exhibit significant quadriceps femoris muscle atrophy. It is well established that curcumin (CUR) exerts protective effects on skeletal muscle. However, the efficacy of CUR in treating KOA-induced quadriceps femoris muscle atrophy and its underlying mechanisms remain uncertain. In this study, we employed network pharmacology to investigate the mechanism by which CUR promotes regenerative repair of the quadriceps femoris muscle in rats with KOA. MethodsThe potential targets of CUR were obtained from Swiss Target Prediction. The targets of skeletal muscle regeneration were identified from GeneCard and OMIM. A Venn diagram was generated to visualize the intersection of CUR targets and skeletal muscle regeneration targets, and the core targets were identified using STRING. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were conducted using DAVID. Finally, the network pharmacology results were further validated by establishing a KOA rat model using the Hulth method. ResultsNetwork pharmacology analysis and molecular docking results revealed that CUR affects skeletal muscle regeneration through multiple targets and pathways. In vivo experimental results were validated by demonstrating that KOA causes atrophy and induces apoptosis in the quadriceps femoris muscle. Furthermore, CUR was shown to inhibit apoptosis in the quadriceps femoris muscle by regulating STAT3 and FOS, as well as the PI3K/AKT signaling pathway. ConclusionsOur study revealed the apoptosis-inhibiting effects of CUR and its underlying mechanisms. Consequently, CUR has the potential to improve quadriceps femoris muscle atrophy caused by KOA.

Fatty links between multisystem proteinopathy and small VCP-interacting protein.

Multisystem proteinopathy (MSP) is a rare, dominantly inherited disorder that includes a cluster of diseases, including frontotemporal dementia, inclusion body myopathy, and Paget's disease of bone. MSP is caused by mutations in the gene encoding valosin-containing protein (VCP). Patients with the same mutation, even within the same family, can present with a different combination of any or all of the above diseases, along with amyotrophic lateral sclerosis (ALS). The pleiotropic effects may be linked to the greater than 50 VCP co-factors that direct VCP's many roles in the cell. Small VCP-interacting protein (SVIP) is a small protein that directs VCP to autophagosomes and lysosomes. We found that SVIP directs VCP localization to lysosomes in an acylation-dependent manner. We demonstrate that SVIP is myristoylated at Glycine 2 and palmitoylated at Cysteines 4 and 7. Acylation of SVIP is required to mediate cell death in the presence of the MSP-associated VCP variant (R155H-VCP), whereas blocking SVIP myristoylation prevents cytotoxicity. Therefore, SVIP acylation may present a novel target in MSP.

Beyond Bone: A Systematic Review on Bioactive Glass Innovations and Breakthroughs in Skeletal Muscle Regeneration

Beyond Bone: A Systematic Review on Bioactive Glass Innovations and Breakthroughs in Skeletal Muscle Regeneration

The Combined Delivery of the Vegf, Ang, and Gdnf Genes Stimulates Angiogenesis and Improves Post-Ischemic Innervation and Regeneration in Skeletal Muscle.

In this study, the effects of different combinations of the genes Vegf, Ang, and Gdnf injected both using direct virus-mediated injection (adenovirus, Ad5) and umbilical cord blood mononuclear cells (UCBCs) on the processes of stimulation of post-ischemic innervation, angiogenesis, and regeneration in skeletal muscle were investigated in a rat hindlimb chronic ischemia model. It was shown that more pronounced stimulation of angiogenesis and restoration of post-ischemic innervation were achieved both in the early (28 days post-ischemia, dpi) and late (42 dpi) terms of the experiment in the calf muscle when UCBCs delivered the combination of Ad5-Vegf and Ad5-Ang compared to the direct injection of the same vector combination into the area of ischemia. At the same time, the inclusion of Ad5-Gdnf in the combination of Ad5-Vegf and Ad5-Ang directly injected or administered by UCBCs provided a significant increase in the number of centronuclear muscle fibers, indicating stimulation of post-ischemic reparative myogenesis. This study allowed us to determine the most effective gene combinations for angiogenesis and neurogenesis, which, in the future, may serve as the basis for the development of gene and gene cell products for the treatment of chronic lower limb ischemia.

Abstract We032: FOXK1-Gli2 network regulates cardiomyocyte proliferation and heart regeneration

Unlike adult mammals, the neonatal mouse has an exceptional regenerative capacity following myocardial injury. Our goal was to decipher and define novel factors and mechanisms that have a role in regeneration of the injured adult mouse heart. FOXK1 is a transcription factor that regulates cell cycle kinetics, myogenic stem cell proliferation, and skeletal muscle regeneration. During development, its expression is restricted to progenitors, somites and the heart. In these studies, we identified a novel role for FOXK1 as a regulator of cardiomyocyte (CM) proliferation and regeneration through the regulation of Gli2 . Analysis of single nucleus RNAseq datasets of neonatal hearts revealed that FOXK1 was expressed in neonatal CMs. qPCR analysis from P1 hearts of control and Foxk1 null mice confirmed this finding and demonstrated dysregulation of the cell cycle regulators, Ccne1 and p21 , in the absence of Foxk1 . Control and Foxk1 null mice were pulsed with EdU from P3 to P7 which demonstrated that the absence of Foxk1 led to a decrease in CM proliferation in-vivo. Then, using a transgenic mouse model that we engineered to overexpress FOXK1 in muscle ( MCK-Foxk1 ), we observed CM proliferation in adult MCK-Foxk1 mice, which showed increased incorporation of EdU and increased pH3 expression compared to control. Bulk RNA-seq analysis of P1, P7, and P14 hearts indicated increased CM proliferation in the MCK-Foxk1 mouse and revealed Gli2 as a potential downstream target of FOXK1. A gel shift assay was performed, which demonstrated that FOXK1 was able to bind to a FOXK1 motif upstream of the Gli2 gene. ChIP-PCR further verified that Gli2 was a direct downstream target of FOXK1 in the embryonic stem cell-embryoid body (ES-EB) system. We then performed ligation of the left anterior descending coronary artery (LAD) in adult control and MCK-Foxk1 mice. Echocardiography following the LAD-ligation injuries showed improved cardiac function in MCK-Foxk1 transgenic mice compared with controls. These results identify FOXK1 as a regulator of CM proliferation and heart regeneration and suggest that this function is mediated through the regulation of Gli2 gene expression.

Taurine Alleviates Ferroptosis-Induced Metabolic Impairments in C2C12 Myoblasts by Stabilizing the Labile Iron Pool and Improving Redox Homeostasis.

Ferroptosis adversely affects the viability, differentiation, and metabolic integrity of C2C12 myoblasts, contributing to the decline in skeletal muscle health. The intricate mechanisms behind this process are not fully understood. In this study, we induced ferroptosis in myoblasts using targeted inducers and found a marked decrease in specific redox metabolites, particularly taurine. Taurine supplementation effectively reversed the deleterious effects of ferroptosis, significantly increased cellular glutathione levels, reduced MDA and ROS levels, and rejuvenated impaired myogenic differentiation. Furthermore, taurine downregulated HO-1 expression and decreased intracellular Fe2+ levels, thereby stabilizing the labile iron pool. Using NMR metabolomic analysis, we observed that taurine profoundly promoted glycerophospholipid metabolism, which is critical for cell membrane repair, and enhanced mitochondrial bioenergetics, thereby increasing the energy reserves essential for muscle satellite cell regeneration. These results suggest that taurine is a potent ferroptosis inhibitor that attenuates key drivers of this process, strengthens oxidative defenses, and improves redox homeostasis. This combined effect protects cells from ferroptosis-induced damage. This study highlights the potential of taurine as a valuable ferroptosis inhibitor that protects skeletal muscle from ferroptosis-induced damage and provides a basis for therapeutic strategies to rejuvenate and facilitate the regeneration of aging skeletal muscle.

A novel variant in the GNE gene in a Malian patient presenting with distal myopathy.

GNE-myopathy (GNE-M) is a rare autosomal recessive disorder caused by variants in the GNE gene. We report a novel variant in GNE causing GNE-M in a Malian family. A 19-year-old male patient from consanguineous marriage was seen for progressive walking difficulty. Neurological examination found predominant distal muscle weakness and atrophy, decreased tendon reflexes, predominating in lower limbs. Electroneuromyography showed an axonal neuropathy pattern. However, whole exome sequencing (WES) revealed a novel biallelic variant in GNE c.1838G > A:p.Gly613Glu, segregating with the phenotype within the family. This study highlights its diagnosis challenges in sub-Saharan Africa and broadens the genetic spectrum of this rare disease.

Impact of Food on the Oral Absorption of N-Acetyl-D-Mannosamine in Healthy Men and Women.

N-Acetyl-D-mannosamine (ManNAc) is an endogenous monosaccharide and precursor of N-acetylneuraminic acid (Neu5Ac), a critical sialic acid. ManNAc is currently under clinical development to treat GNE myopathy, a rare muscle-wasting disease. In this randomized, open-label, 2-sequence, crossover study, 16 healthy women and men were administered a single oral dose of ManNAc under fasting and fed conditions. Blood samples were collected for 48hours after dosing for quantification of plasma ManNAc and Neu5Ac concentrations. Noncompartmental pharmacokinetic and deconvolution analyses were performed using baseline-corrected plasma concentration data. Administration of ManNAc in the fed state resulted in a 1.6-fold increase in ManNAc exposure, compared to fasting conditions. A concurrent increase in Neu5Ac exposure was observed in the presence of food. Deconvolution analysis indicated that the findings were attributed to prolonged absorption rather than an enhanced rate of absorption. The impact of food on ManNAc pharmacokinetics was greater in women than men (fed/fasted area under the concentration-time curve from time 0 to infinity mean ratio: 198% compared to 121%). It is hypothesized that the presence of food slows gastric emptying, allowing a gradual release of ManNAc into the small intestine, translating into improved ManNAc absorption. The results suggest that taking ManNAc with food may enhance its therapeutic activity and/or reduce the daily dosage requirement.

Design, development, and benchmarking of a bioreactor integrated with 3D bioprinting: Application to skeletal muscle regeneration

In recent years, great efforts have been spent to create engineered muscle constructs recapitulating the 3D architecture and applying external stimulations. In this regard, tissue engineering approaches could be very promising in regenerating skeletal muscle, in which bioprinting techniques have produced encouraging results especially regarding 3D architecture. Tensile stimuli showed a fundamental role in regulating the behavior of muscle cells both in terms of 3D organizations and protein expression. Despite this promising premise, the combination of 3D bioprinting and mechanical stimulation has been poorly investigated, calling for novel approaches dealing with the mechanical stimulation of the 3D bioprinted construct and the integration of the bioprinting phase into the stimulation device. To this aim, the present work proposes the design, manufacturing, and benchmarking of a bioprinting-integrated mechanical platform conceived for mechanically stimulating a 3D muscle model directly printed into the bioreactor to foster the integration of printing and stimulation. The study consists of three main steps: 1) the design, fabrication, and mechanical characterization of stretchable supports suitable for bioprinting and long-term cell culture; 2) the design, assisted by computational tools, and the fabrication of the smart Petri dish containing the stimulation mechanism and of the final cyclic mechanical platform; 3) the in-vitro validation of the proposed platform in terms of transmission of the mechanical stimulation to the 3D construct and the biological effect of dynamic culture on 3D bioprinted muscle cells. The results highlighted excellent viability and demonstrated that the external stimulus influences the murine myoblasts behavior already after 7 days of culture. In conclusion, prototypes are now available of a mechanical platform that integrates the 3D bioprinting and is capable of stimulating 3D biological constructs for applications in the field of muscle tissue engineering.

Serotonin reuptake inhibitors improve muscle stem cell function and muscle regeneration in male mice

Serotonin reuptake inhibitor antidepressants such as fluoxetine are widely used to treat mood disorders. The mechanisms of action include an increase in extracellular level of serotonin, neurogenesis, and growth of vessels in the brain. We investigated whether fluoxetine could have broader peripheral regenerative properties. Following prolonged administration of fluoxetine in male mice, we showed that fluoxetine increases the number of muscle stem cells and muscle angiogenesis, associated with positive changes in skeletal muscle function. Fluoxetine also improved skeletal muscle regeneration after single and multiples injuries with an increased muscle stem cells pool and vessel density associated with reduced fibrotic lesions and inflammation. Mice devoid of peripheral serotonin treated with fluoxetine did not exhibit beneficial effects during muscle regeneration. Specifically, pharmacological, and genetic inactivation of the 5-HT1B subtype serotonin receptor also abolished the enhanced regenerative process induced by fluoxetine. We highlight here a regenerative property of serotonin on skeletal muscle.

Extrusion-Based 3D Bioprinting of Bioactive and Piezoelectric Scaffolds as Potential Therapy for Treating Critical Soft Tissue Wounds.

Objective: This study focuses on developing bioactive piezoelectric scaffolds that could deliver bioelectrical cues to potentially treat injuries to soft tissues such as skeletal muscles and promote active regeneration. Approach: To address the underexplored aspect of bioelectrical cues in skeletal muscle tissue engineering (SMTE), we developed piezoelectric bioink based on natural bioactive materials such as sodium alginate, gelatin, and chitosan. Extrusion-based 3D bioprinting was utilized to develop scaffolds that mimic muscle stiffness and generate electrical stimulation (E-stim) when subjected to forces. The biocompatibility of these scaffolds was tested with the C2C12 muscle cell line. Results: The bioink demonstrated suitable rheological properties for 3D bioprinting, resulting in high-resolution composite sodium alginate-gelatin-chitosan scaffolds with good structural fidelity. The scaffolds exhibited a 42-60 kPa stiffness, similar to muscle. When a controlled force of 5N was applied to the scaffolds at a constant frequency of 4 Hz, they generated electrical fields and impulses (charge), indicating their suitability as a stand-alone scaffold to generate E-stim and instill bioelectrical cues in the wound region. The cell viability and proliferation test results confirm the scaffold's biocompatibility with C2C12s and the benefit of piezoelectricity in promoting muscle cell growth kinetics. Our study indicates that our piezoelectric bioink and scaffolds offer promise as autonomous E-stim-generating regenerative therapy for SMTE. Innovation: A novel approach for treating skeletal muscle wounds was introduced by developing a bioactive electroactive scaffold capable of autonomously generating E-stim without stimulators and electrodes. This scaffold offers a unique approach to enhancing skeletal muscle regeneration through bioelectric cues, addressing a major gap in the SMTE, that is, fibrotic tissue formation due to delayed muscle regeneration. Conclusion: A piezoelectric scaffold was developed, providing a promising solution for promoting skeletal muscle regeneration. This development can potentially address skeletal muscle injuries and offers a unique approach to facilitating skeletal muscle wound healing.

Muscle cell-derived Ccl8 is a negative regulator of skeletal muscle regeneration.

Skeletal muscles undergo robust regeneration upon injury, and infiltrating immune cells play a major role in not only clearing damaged tissues but also regulating the myogenic process through secreted cytokines. Chemokine C-C motif ligand 8 (Ccl8), along with Ccl2 and Ccl7, has been reported to mediate inflammatory responses to suppress muscle regeneration. Ccl8 is also expressed by muscle cells, but a role of the muscle cell-derived Ccl8 in myogenesis has not been reported. In this study, we found that knockdown of Ccl8, but not Ccl2 or Ccl7, led to increased differentiation of C2C12 myoblasts. Analysis of existing single-cell transcriptomic datasets revealed that both immune cells and muscle stem cells (MuSCs) in regenerating muscles express Ccl8, with the expression by MuSCs at a much lower level, and that the temporal patterns of Ccl8 expression were different in MuSCs and macrophages. To probe a function of muscle cell-derived Ccl8 invivo, we utilized a mouse system in which Cas9 was expressed in Pax7+ myogenic progenitor cells (MPCs) and Ccl8 gene editing was induced by AAV9-delivered sgRNA. Depletion of Ccl8 in Pax7+ MPCs resulted in accelerated muscle regeneration after barium chloride-induced injury in both young and middle-aged mice, and intramuscular administration of a recombinant Ccl8 reversed the phenotype. Accelerated regeneration was also observed when Ccl8 was depleted in Myf5+ or MyoD+ MPCs by similar approaches. Our results suggest that muscle cell-derived Ccl8 plays a unique role in regulating the initiation of myogenic differentiation during injury-induced muscle regeneration.

Enhanced secretion of promyogenic exosomes by quiescent muscle cells.

Signaling interactions are important during skeletal muscle regeneration, where muscle cells in distinct states (quiescent, reactivated, proliferating and differentiated) must coordinate their response to injury. Here, we probed the role of secreted small extracellular vesicles (sEV/exosomes) using a culture model of physiologically relevant cell states seen in muscle regeneration. Unexpectedly, G0 myoblasts exhibited enhanced secretion of sEV (∼150nm) displaying exosome markers (Alix, TSG101, flotillin-1, and CD9), and increased expression of Kibra, a regulator of exosome biogenesis. Perturbation of Kibra levels confirmed a role in controlling sEV secretion rates. Purified sEVs displayed a common exosome marker-enriched proteome in all muscle cell states, as well as state-specific proteins. Exosomes derived from G0 cells showed an antioxidant signature, and were most strongly internalized by differentiated myotubes. Functionally, donor exosomes from all muscle cell states could activate an integrated Wnt reporter in target cells, but only G0-derived exosomes could induce myogenic differentiation in proliferating cells. Taken together, we provide evidence that quiescence in muscle cells is accompanied by enhanced secretion of exosomes with distinct uptake, cargo and signal activating features. Our study suggests the novel possibility that quiescent muscle stem cells in vivo may play a previously under-appreciated signaling role during muscle homeostasis.

Biodegradable conductive IPN in situ cryogels with anisotropic microchannels and sequential delivery of dual-growth factors for skeletal muscle regeneration

Biodegradable and anisotropic cryogels that simulate conductivity and ECM orientation structure of skeletal muscle, and release multiple growth factors, are expected for in situ skeletal muscle tissue engineering. Herein, biodegradable, conductive and anisotropic interpenetrating network (IPN) in situ cryogels are fabricated through Schiff base/acylhydrazone crosslinking via combining unidirectional freezing and cyclic freeze-thaw processes. The cryogels have good anisotropic mechanical properties and oriented microchannel structure, and induce the oriented alignment of myoblasts. The introduction of aniline tetramer enhances the mechanical properties and conductivity of the cryogels. The conductive cryogels significantly improve the proliferation and myogenic differentiation of C2C12 cells during 3D culture. The sequential delivery of insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF) can induce migration of human umbilical vein endothelial cells (HUVECs), proliferation of human skin myofibroblasts (HMFB), and myogenic differentiation of C2C12 cells in vitro. In particular, conductive and anisotropic cryogels with dual-growth factors can significantly improve the repair efficiency of volumetric muscle loss (VML) in vivo. This study provides a new strategy to fabricate anisotropic and conductive IPN in situ cryogel biomimetic scaffolds that can encapsulate dual-growth factors and deliver them in time sequence, which significantly promote the efficient repair of VML via an in situ tissue engineering approach.

Interactions between myoblasts and macrophages under high glucose milieus result in inflammatory response and impaired insulin sensitivity.

Skeletal muscle handles about 80% of insulin-stimulated glucose uptake and become the major organ occurring insulin resistance (IR). Many studies have confirmed the interactions between macrophages and skeletal muscle regulated the inflammation and regeneration of skeletal muscle. However, despite of the decades of research, whether macrophages infiltration and polarization in skeletal muscle under high glucose (HG) milieus results in the development of IR is yet to be elucidated. C2C12 myoblasts are well-established and excellent model to study myogenic regulation and its responses to stimulation. Further exploration of macrophages' role in myoblasts IR and the dynamics of their infiltration and polarization is warranted. To evaluate interactions between myoblasts and macrophages under HG, and its effects on inflammation and IR in skeletal muscle. We detected the polarization status of macrophages infiltrated to skeletal muscles of IR mice by hematoxylin and eosin and immunohistochemical staining. Then, we developed an in vitro co-culture system to study the interactions between myoblasts and macrophages under HG milieus. The effects of myoblasts on macrophages were explored through morphological observation, CCK-8 assay, Flow Cytometry, and enzyme-linked immunosorbent assay. The mediation of macrophages to myogenesis and insulin sensitivity were detected by morphological observation, CCK-8 assay, Immunofluorescence, and 2-NBDG assay. The F4/80 and co-localization of F4/80 and CD86 increased, and the myofiber size decreased in IR group (P < 0.01, g = 6.26). Compared to Mc group, F4/80+CD86+CD206- cells, tumor necrosis factor-α (TNFα), inerleukin-1β (IL-1β) and IL-6 decreased, and IL-10 increased in McM group (P < 0.01, g > 0.8). In McM + HG group, F4/80+CD86+CD206- cells, monocyte chemoattractant protein 1, TNFα, IL-1β and IL-6 were increased, and F4/80+CD206+CD86- cells and IL-10 were decreased compared with Mc + HG group and McM group (P < 0.01, g > 0.8). Compered to M group, myotube area, myotube number and E-MHC were increased in MMc group (P < 0.01, g > 0.8). In MMc + HG group, myotube area, myotube number, E-MHC, GLUT4 and glucose uptake were decreased compared with M + HG group and MMc group (P < 0.01, g > 0.8). Interactions between myoblasts and macrophages under HG milieus results in inflammation and IR, which support that the macrophage may serve as a promising therapeutic target for skeletal muscle atrophy and IR.

Curcumin-activated Wnt5a pathway mediates Ca2+ channel opening to affect myoblast differentiation and skeletal muscle regeneration.

Skeletal muscle injury is one of the most common sports injuries; if not properly treated or not effective rehabilitation treatment after injury, it can be transformed into chronic cumulative injury. Curcumin, an herbal ingredient, has been found to promote skeletal muscle injury repair and regeneration. The Wnt5a pathway is related to the expression of myogenic regulatory factors, and Ca2+ promotes the differentiation and fusion process of myoblasts. This study explored the effect and mechanism of curcumin on myoblast differentiation during the repair and regeneration of injured skeletal muscle and its relationship with the Wnt5a pathway and Ca2+ channel. Myogenic differentiation of C2C12 cells was induced with 2% horse serum, and a mouse (male, 10weeks old) model of acute skeletal muscle injury was established using cardiotoxin (20μL). In addition, we constructed a Wnt5a knockdown C2C12 cell model and a Wnt5a knockout mouse model. Besides, curcumin was added to the cell culture solution (80mg/L) and fed to the mice (50mg/kg). Fluorescence microscopy was used to determine the concentration of Ca2+. Western blot and RT-qPCR were used to detect the protein and mRNA levels of Wnt5a, CaN, NFAT2, MyoD, Myf5, Pax7, and Myogenin. The expression levels of MyoD, Myf5, Myogenin, MHC, Desmin, and NFAT2 were detected using immunofluorescence techniques. In addition, MyoD expression was observed using immunohistochemistry, and morphological changes in mouse muscle tissue were observed using HE staining. During myoblast differentiation and muscle regeneration, Wnt5a expression was upregulated (P<0.001) and the Wnt5a signalling pathway was activated. Wnt5a overexpression promoted the expression of MyoD, Myf5, Myogenin, MHC, and Desmin (P<0.05), and conversely, knockdown of Wnt5a inhibited their expression (P<0.001). The Wnt5a pathway mediated the opening of Ca2+ channels, regulated the expression levels of CaN, NFAT2, MyoD, Myf5, Myogenin, MHC, and Desmin (P<0.01) and promoted the differentiation of C2C12 myoblasts and the repair and regeneration of injured skeletal muscle. The expression of Wnt5a, CaN, NFAT2, MyoD, Myogenin, Myf5, and MHC in C2C12 myoblast was significantly increased after curcumin intervention (P<0.05); however, their expression decreased significantly after knocking down Wnt5a on the basis of curcumin intervention (P<0.05). Similarly, in Wnt5a knockout mice, the promotion of muscle regeneration by curcumin was significantly attenuated. Curcumin can activate the Wnt5a signalling pathway and mediate the opening of Ca2+ channels to accelerate the myogenic differentiation of C2C12 cells and the repair and regeneration of injured skeletal muscle.

The IRE1α/XBP1 signaling axis drives myoblast fusion in adult skeletal muscle

Skeletal muscle regeneration involves a signaling network that regulates the proliferation, differentiation, and fusion of muscle precursor cells to injured myofibers. IRE1α, one of the arms of the unfolded protein response, regulates cellular proteostasis in response to ER stress. Here, we demonstrate that inducible deletion of IRE1α in satellite cells of mice impairs skeletal muscle regeneration through inhibiting myoblast fusion. Knockdown of IRE1α or its downstream target, X-box protein 1 (XBP1), also inhibits myoblast fusion during myogenesis. Transcriptome analysis revealed that knockdown of IRE1α or XBP1 dysregulates the gene expression of molecules involved in myoblast fusion. The IRE1α-XBP1 axis mediates the gene expression of multiple profusion molecules, including myomaker (Mymk). Spliced XBP1 (sXBP1) transcription factor binds to the promoter of Mymk gene during myogenesis. Overexpression of myomaker in IRE1α-knockdown cultures rescues fusion defects. Inducible deletion of IRE1α in satellite cells also inhibits myoblast fusion and myofiber hypertrophy in response to functional overload. Collectively, our study demonstrates that IRE1α promotes myoblast fusion through sXBP1-mediated up-regulation of the gene expression of multiple profusion molecules, including myomaker.