Muscle Regeneration Process Research Articles

Bioactive MXene hydrogel promotes structural and functional regeneration of skeletal muscle through improving autophagy and muscle innervation

Complete skeletal muscle regeneration after traumatic injuries remains a challenge due to impaired regenerative capability and dysregulated microenvironments. Autophagy plays a crucial role in the muscle regeneration process by regulating myogenic and non-myogenic cells. Herein, we report a bioactive MXene hydrogel (FPGM) capable of upregulating autophagy and increasing muscle innervation to restore skeletal muscle structure and function. FPGM possessed excellent electrical conductivity, tissue adhesive ability and antioxidation, which could eliminate excess reactive oxygen species to reduce oxidative stress and decrease the secretion of pro-inflammatory cytokine. FPGM upregulated the autophagy level of myoblasts and promoted the migration and tube formation of endothelial cells as well as myogenic differentiation with negligible toxicity. FPGM accelerated muscle fiber formation and skeletal muscle regeneration by improving autophagy, which could regulate microenvironment through raising M2 macrophages to alleviate excessive inflammation, facilitating angiogenesis and decreasing fibrous scar tissue formation in vivo. Importantly, FPGM could efficiently restore muscle function by improving muscle innervation, tibialis anterior compound muscle action potential amplitude and neuromuscular conduction. This work demonstrates that bioactive MXene hydrogel should be a promising candidate for complete skeletal muscle regeneration.

Diarylpropionitrile-stimulated ERβ nuclear accumulation promotes MyoD-induced muscle regeneration in mdx mice by interacting with FOXO3A

Duchenne muscular dystrophy (DMD) is an X-linked recessive progressive degenerative disease of skeletal muscle, characterized by intramuscular inflammation, muscle regeneration disorder and replacement of muscle with fibroadipose tissue. DMD is caused by the absence of normal dystrophy. Impaired self-renew ability and limited differentiation capacity of satellite cells are proved as main reasons for muscle regeneration failure. The deficiency of estrogen impedes the process of muscle regeneration. However, the role of estrogen receptor β (ERβ) in muscle regeneration is still unclear. This study aims to investigate the role and the pharmacological effect of ERβ activation on muscle regeneration in mdx mice. This study showed that mRNA levels of ERβ and myogenic-related genes both witnessed increasing trends in dystrophic context. Our results revealed that treatment with selective ERβ agonist (DPN, diarylpropionitrile) significantly increased myogenic differentiation 1 (MyoD-1) level and promoted muscle regeneration in mdx mice. Similarly, in mdx mice with muscle-specific estrogen receptor α (ERα) ablation, DPN treatment still promoted muscle regeneration. Moreover, we demonstrated that myoblasts differentiation was accompanied by raised nuclear accumulation of ERβ. DPN treatment augmented the nuclear accumulation of ERβ and, thus, contributed to myotubes formation. One important finding was that forkhead box O3A (FOXO3A), as a pivotal transcription factor in Myod-1 transcription, participated in the ERβ-promoted muscle regeneration. Overall, we offered an interesting explanation about the crucial role of ERβ during myogenesis.

IL-33 Facilitates Fibro-Adipogenic Progenitors to Establish the Pro-Regenerative Niche after Muscle Injury.

During the process of muscle regeneration post-injury in adults, muscle stem cells (MuSCs) function is facilitated by neighboring cells within the pro-regenerative niche. However, the precise mechanism triggering the initiation of signaling in the pro-regenerative niche remains unknown. Using single-cell RNA sequencing, 14 different muscle cells are comprehensively mapped during the initial stage following injury. Among these, macrophages and fibro-adipogenic progenitor cells (FAPs) exhibit the most pronounced intercellular communication with other cells. In the FAP subclusters, the study identifies an activated FAP phenotype that secretes chemokines, such as CXCL1, CXCL5, CCL2, and CCL7, to recruit macrophages after injury. Il1rl1, encoding the protein of the interleukin-33 (IL-33) receptor, is identified as a highly expressed signature surface marker of the FAP phenotype. Following muscle injury, autocrine IL-33, an alarmin, has been observed to activate quiescent FAPs toward this inflammatory phenotype through the IL1RL1-MAPK/NF-κB signaling pathway. Il1rl1 deficiency results in decreased chemokine expression and recruitment of macrophages, accompanied by impaired muscle regeneration. These findings elucidate a novel mechanism involving the IL-33/IL1RL1 signaling pathway in promoting the activation of FAPs and facilitating muscle regeneration, which can aid the development of therapeutic strategies for muscle-related disorders and injuries.

Alcoholic cardiomyopathy: aspects of pathogenesis and clinic

Alcoholic cardiomyopathy is the most common form of myocardial damage caused by ethanol. Ethanol and its main active metabolite acetaldehyde have a direct toxic effect on the myocardium. The mechanisms of the cardiotoxic effect of ethanol are diverse and include membranotropic action, damage to cellular organelles, activation of lipid peroxidation, and a number of others. Dissolving in the lipids of biological membranes, ethanol changes their physico-chemical properties, disrupts the activity of membrane receptors, eventually disconnecting the connection of excitation with the contraction of cardiomyocytes. Particular importance in the pathogenesis of alcoholic cardiomyopathy is attached to the ability of ethanol to cause structural and functional changes in mitochondria, which is directly related to the violation of oxidative processes and energy supply of the myocardium as a whole. Under the influence of alcohol, free-radical lipid oxidation products content increases and antioxidants activity decreases, which indicates oxidative stress development. The deterioration of myocardial contractility due to chronic alcohol intoxication triggers a cascade of compensatory mechanisms in the form of cardiomyocyte hypertrophy and progressive fibrosis, while adaptive reactions become a factor in further pathological changes. Ethanol causes myocardial damage in a dose-dependent manner.The probability of alcoholic cardiomyopathy occurrence is a derivative of the total accumulated dose of alcohol and individual predisposition. Alcoholic cardiomyopathy is considered as a variant of secondary dilated cardiomyopathy with all its clinical manifestations and consequences: dilation of the heart chambers, low left ventricular ejection fraction, progressive circulatory insufficiency, and cardiac arrhythmias. A relatively favorable prognosis in alcoholic cardiomyopathy is provided due to partial reversibility of myocardial damage under the condition of abstinence from alcohol consumption. Specific drugs for the treatment of alcoholic cardiomyopathy have not been developed. New treatment strategies include the use of myocardial growth and regulation factors synthesized by cardiomyocytes, as well as the stimulation of heart muscle regeneration and repair processes.

Changes in protein fluxes in skeletal muscle during sequential stages of muscle regeneration after acute injury in male mice

Changes in protein turnover play an important role in dynamic physiological processes, including skeletal muscle regeneration, which occurs as an essential part of tissue repair after injury. The inability of muscle tissue to recapitulate this regenerative process can lead to the manifestation of clinical symptoms in various musculoskeletal diseases, including muscular dystrophies and pathological atrophy. Here, we employed a workflow that couples deuterated water (2H2O) administration with mass spectrometry (MS) to systematically measure in-vivo protein turnover rates across the muscle proteome in 8-week-old male C57BL6/J mice. We compared the turnover kinetics of over 100 proteins in response to cardiotoxin (CTX) induced muscle damage and regeneration at unique sequential stages along the regeneration timeline. This analysis is compared to gene expression data from mRNA-sequencing (mRNA-seq) from the same tissue. The data reveals quantitative protein flux signatures in response to necrotic damage, in addition to sequential differences in cell proliferation, energy metabolism, and contractile gene expression. Interestingly, the mRNA changes correlated poorly with changes in protein synthesis rates, consistent with post-transcriptional control mechanisms. In summary, the experiments described here reveal the signatures and timing of protein flux changes during skeletal muscle regeneration, as well as the inability of mRNA expression measurements to reveal changes in directly measured protein turnover rates. The results of this work described here provide a better understanding of the muscle regeneration process and could help to identify potential biomarkers or therapeutic targets.

Biomaterials-Based Technologies in Skeletal Muscle Tissue Engineering.

For many clinically prevalent severe injuries, the inherent regenerative capacity of skeletal muscle remains inadequate. Skeletal muscle tissue engineering (SMTE) seeks to meet this clinical demand. With continuous progress in biomedicine and related technologies including micro/nanotechnology and 3D printing, numerous studies have uncovered various intrinsic mechanisms regulating skeletal muscle regeneration and developed tailored biomaterial systems based on these understandings. Here, the skeletal muscle structure and regeneration process are discussed and the diverse biomaterial systems derived from various technologies are explored in detail. Biomaterials serve not merely as local niches for cell growth, but also as scaffolds endowed with structural or physicochemical properties that provide tissue regenerative cues such as topographical, electrical, and mechanical signals. They can also act as delivery systems for stem cells and bioactive molecules that have been shown as key participants in endogenous repair cascades. To achieve bench-to-bedside translation, the typical effect enabled by biomaterial systems and the potential underlying molecular mechanisms are also summarized. Insights into the roles of biomaterials in SMTE from cellular and molecular perspectives are provided. Finally, perspectives on the advancement of SMTE are provided, for which gene therapy, exosomes, and hybrid biomaterials may hold promise to make important contributions.

Unlocking Strength: The Powerful Impact of Ashwagandha (Withania somnifera) Supplementation on Resistance Training

Withania somnifera, commonly known as ashwagandha, has gained popularity in recent years among individuals interested in healthy lifestyles, natural medicine, and sports. This review aims to summarize the current knowledge regarding the effects of ashwagandha supplementation on various aspects of physical performance, particularly in the context of resistance training. Studies have shown that ashwagandha can positively impact muscle strength, with significant increase of muscle force and power output. Additionally, it has demonstrated potential in modulating body composition, with reduction of body fat percentage noted in certain populations. However, results of studies on ashwagandha’s effect on muscle size remain inconclusive and may depend on various factors such as training status and individual responses. In terms of endurance, ashwagandha supplementation has been associated with improvement in maximal oxygen uptake, indicating enhanced aerobic capacity and delayed exhaustion during endurance activities. Furthermore, ashwagandha was shown to aid muscle recovery by reducing exercise-induced muscle damage and inflammatory response, potentially accelerating the muscle regeneration process. Ashwagandha may also influence hormone levels, such as testosterone and cortisol, with potential implications for muscle growth and recovery. However, further research is needed to fully understand the mechanisms underlying these effects. Overall, ashwagandha supplementation appears to hold promise for enhancing physical performance and overall well-being. Further research is needed to elucidate optimal dosages, long-term safety, and potential interactions with other supplements or medications.

Morphometric evaluation of mitochondrial networks in regenerating myofibers using photothermal microscopy

Background: Mitochondria constitute their interconnected network through dynamic mitochondrial processes such as fission and fusion in response to changes in their intracellular environment, such as energy demand. Transmission electron microscopy is the standard method for evaluating mitochondrial morphology. We have developed a simple method to evaluate mitochondrial morphology using tissue sections for bright-field observation. In this study, we attempted to image light-absorbing molecules (cytochrome c) in mitochondria using photothermal (PT) microscopy. Purpose: This method can observe an entire myocyte, so this study attempted to evaluate the entire mitochondrial network of immature (regenerating) myocytes during the skeletal muscle regeneration process. Methods: The gastrocnemius muscle of anesthetized male Wistar rats (13 weeks old) was subjected to 300 controlled eccentric contractions (ECC) using electrical stimulation. 7 days after ECC loading, in situ perfusion fixation was performed, and transverse sections (1 μm) from red (deep regions) and white (superficial regions) were prepared after Epon embedding. The sections were observed by PT microscopy, and quantitative analysis of mitochondrial morphology was performed. Mitochondrial content was expressed as a percentage of the fiber total area in transverse sections. The PT imaging system uses lasers with wavelengths of 515 nm and 638 nm constituting the PT pump and the probe beams, respectively. Results: After 7 days of ECC, many myofibers with small fiber diameters and central nuclei, which are characteristic of the recovery process after muscle damage, were observed. The cross-sectional area of the regenerating muscle (superficial region 389.9 ± 15.2 μm2, deep region 366.2 ± 17.5 μm2) was significantly smaller than that of the normal muscle (superficial region 2211.1 ± 127.3 μm2, deep region 1396.1 ± 72.9 μm2). Mitochondrial content of the regenerating muscle (superficial region 26.1 ± 1.1%, deep region 22.7 ± 0.8%) was significantly lower than that of the normal muscle (superficial region 31.8 ± 1.9%, deep region 34.9 ± 1.8%). In normal muscle, a linear network of mitochondria along myofibrils was observed. On the other hand, no spatially distributed mitochondria were observed in the cytoplasm of the regenerating muscle. Furthermore, a mitochondrial network was formed around the periphery of the central nucleus. Conclusion: In muscle tissue imaging using PT microscopy, myofibers in the regenerative process have less mitochondrial content than mature muscle fibers and lack the mitochondrial network along myofibrils that characterizes mature muscle. On the other hand, the central nucleus of immature muscle was surrounded by highly developed mitochondria. The mitochondrial morphological characteristics of regenerating muscle fibers may offer a unique window through which to better understand mitochondrial design and function and their plasticity during maturation. This study was supported in part by a Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) KAKENHI Grant (No. JP20H04074, 21K19703). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Loss of FAM210A in Satellite Cell Drives Mitochondrial Dysfunction and Muscle Weakness in Experimental Peripheral Artery Disease

Introduction: Peripheral artery disease (PAD) affects approximately 200 million people globally. Chronic limb-threatening ischemia (CLTI) represents the most advanced clinical phenotype of PAD. Mitochondrial metabolism defects are apparent in the satellite cells of CLTI patients, and the muscle regeneration process demands increased energy, with satellite cell mitochondrial metabolism and dynamics playing a crucial role. However, our current understanding of the role of satellite cells and muscle regeneration in PAD pathology is notably inadequate. Recent studies have shown that the absence of FAM210A, a newly identified mitochondrial protein, can trigger morphological and energetic abnormalities in mitochondria, resulting in muscle function deterioration and promoting muscle wasting. Given the impaired mitochondrial function in satellite cells in CLTI patients and the heightened energy demand regulated by mitochondrial dynamics during muscle regeneration in satellite cell, it is therefore essential to exploring the role of FAM210A in satellite cells in CLTI limb pathology. The objective of this study was to investigate the impact of the loss of FAM210A in satellite cells in mice with experimental PAD. Methods: Male and female conditional FAM210A satellite cell knock out (FAM210ASCKO) and FAM210A floxed (FAM210Afl/fl) mice (N=3-5) were used for this project. Femoral artery ligation surgery was performed to induce PAD. Tamoxifen was administered for 5 days intraperitoneally one week prior to the surgery. Mice were euthanized twenty-eight days after surgery. Outcome measures included limb perfusion assessment, muscle weight, muscle force production, and mitochondrial respiration. Results: FAM210ASCKO mice had significantly decreased muscle mass of the tibialis anterior and extensor digitorum longus ( p<0.0001 and p=0.0004 respectively). More importantly, maximal muscle force with in-situ contraction in tibialis anterior was significantly lower in FAM210ASCKO mice compared to FAM210Afl/fl mice (FAM210Afl/fl mice: 1085.16 ± 331.08 mN; FAM210ASCKO mice: 430.77 ± 467.91 mN; p=0.0139). Furthermore, analysis of mitochondrial oxidative phosphorylation (OXPHOS) revealed that FAM210ASCKO mice had a significant reduction compared to FAM210Afl/fl mice (48%) in skeletal muscle mitochondrial bioenergetic function at various levels of energy demand mimicking from resting to maximal exercise when carbohydrate was used to energize mitochondria ( p=0.034). Importantly, the analysis of mitochondrial membrane potential consistently reveals a substantial reduction in FAM210ASCKO mice compared to FAM210Afl/fl mice, which can partially explain the diminished mitochondrial bioenergetic function observed in ischemic FAM210ASCKO mice. Conclusion: The results of this study indicate that the loss of FAM210A in satellite cells exacerbates ischemic muscle pathology evidenced by muscle weakness and mitochondrial failure in FAM210ASCKO mice with PAD. Future work is needed to elucidate the mechanisms by which FAM210A regulate ischemic satellite cell function and the therapeutic potential of preventing ischemia-induced loss of FAM210A in PAD. This study was supported by National Institutes of Health (NIH) grant R01-HL149704 (to Dr Ryan). Mr Thome was supported by NIH grant F31-DK128920. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Mediator Med23 controls a transcriptional switch for muscle stem cell proliferation and differentiation in muscle regeneration

Muscle stem cells (MuSCs) contribute to a robust muscle regeneration process after injury, which is highly orchestrated by the sequential expression of multiple key transcription factors. However, it remains unclear how key transcription factors and cofactors such as the Mediator complex cooperate to regulate myogenesis. Here, we show that the Mediator Med23 is critically important for MuSC-mediated muscle regeneration. Med23 is increasingly expressed in activated/proliferating MuSCs on isolated myofibers or in response to muscle injury. Med23 deficiency reduced MuSC proliferation and enhanced its precocious differentiation, ultimately compromising muscle regeneration. Integrative analysis revealed that Med23 oppositely impacts Ternary complex factor (TCF)-targeted MuSC proliferation genes and myocardin-related transcription factor (MRTF)-targeted myogenic differentiation genes. Consistently, Med23 deficiency decreases the ETS-like transcription factor 1 (Elk1)/serum response factor (SRF) binding at proliferation gene promoters but promotes MRTF-A/SRF binding at myogenic gene promoters. Overall, our study reveals the important transcriptional control mechanism of Med23 in balancing MuSC proliferation and differentiation in muscle regeneration.

Uremia Impedes Skeletal Myocyte Myomixer Expression and Fusogenic Activity: Implication for Uremic Sarcopenia

In patients with chronic kidney disease (CKD), skeletal muscle mass and function are known to occasionally decline. However, the muscle regeneration and differentiation process in uremia has not been extensively studied. In mice with CKD induced by adenine-containing diet, the tibialis anterior muscle injured using a barium chloride injection method recovered poorly as compared to control mice. In the cultured murine skeletal myocytes, stimulation with indoxyl sulfate (IS), a representative uremic toxin, morphologically jeopardized the differentiation, which was counteracted by L-ascorbic acid (L-AsA) treatment. Transcriptome analysis of cultured myocytes identified a set of genes whose expression was down-regulated by IS stimulation but up-regulated by L-AsA treatment. Gene silencing of myomixer, one of the genes in the set, impaired myocyte fusion during differentiation. By contrast, lentiviral overexpression of myomixer compensated for a hypomorphic phenotype caused by IS treatment. The split-luciferase technique demonstrated that IS stimulation negatively affected early myofusion activity that was rescued by L-AsA treatment. Lastly, in mice with CKD compared with control mice, myomixer expression in the muscle tissue in addition to the muscle weight after the injury was reduced, both of which were restored with L-AsA treatment. Collectively, data showed that the uremic milieu impairs the expression of myomixer and impedes the myofusion process. Considering frequent musculoskeletal injuries in uremic patients, defective myocyte fusion followed by delayed muscle damage recovery could underlie their muscle loss and weakness.

Determining the Algorithm of Rehabilitation Procedures in Patients with Brachial Plexus Injuries Based on the Prospective Single-Centre Clinical Neurophysiology Studies: Preliminary Results

The clinical neurophysiological tests allow us to determine the type, extent, and nature of brachial plexus damage. They are crucial in decision making regarding surgical procedures or conservative treatment. This report aimed to present an algorithm for rehabilitation procedures in patients with brachial plexus injury of various origins based on the results of neurophysiology findings for the selection of procedures supporting the process of nerve and muscle regeneration. The research group consisted of patients whose medical documentation was analysed concerning the reason, level, and localization of damage to the brachial plexus structures, surgical and or rehabilitative treatment, as well as the MRI results. Among the group of fourteen patients, the clinical studies showed the greatest incidence of brachial plexus injuries of the mixed (both pre- and postganglionic), all trunks, and cervical root injuries, respectively. Results of the motor evoked potentials (MEP) and electroneurography (ENG) recordings induced at levels of spinal roots and Erb’s point showed a decrease of more than 40% in amplitudes on the symptomatic side in comparison to the asymptomatic side. This diffeence was recorded for the axillary and radial innervation and the C5, C6, and C7 root domains, ranging from 57% to 66%; the lowest decrease was recorded following electrical stimulation at Erb’s point for the ulnar nerve (34%). The latency prolongation on the symptomatic side in CMAP and MEP tests ranged from 0.2 to 1.7 ms, with the most following magnetic stimulation of the C5 cervical root for the axillary innervation. Most of the results indicated the axonotmesis and neuropraxia type of injury in motor fibers (40%) confirmed by EMG results. The sensory conduction studies (SNCS) in distal nerve branches did not confirm the severe advancement of the brachial plexus injury (63%). The proposed algorithm of the physiotherapeutic procedures should be mainly targeted for recovery of motor dysfunction as the consequence of brachial plexus injury. Rehabilitation should incorporate the treatment supporting nerve regeneration, muscle strengthening, and maintaining functional ranges of motion of the injured extremities. The rehabilitation treatment for patients with brachial plexus injuries is an individualised process, and the selection of procedures and the effectiveness of the treatment undertaken should be confronted with results of neurophysiological tests verifying the motor neural transmission from the level of the cervical motor centre to the effector, peripheral nerve function, and muscle’s motor unit activity.

Differential effects of the venoms of Russell’s viper and Indian cobra on human myoblasts

Local tissue damage following snakebite envenoming remains a poorly researched area. To develop better strategies to treat snakebites, it is critical to understand the mechanisms through which venom toxins induce envenomation effects including local tissue damage. Here, we demonstrate how the venoms of two medically important Indian snakes (Russell's viper and cobra) affect human skeletal muscle using a cultured human myoblast cell line. The data suggest that both venoms affect the viability of myoblasts. Russell’s viper venom reduced the total number of cells, their migration, and the area of focal adhesions. It also suppressed myogenic differentiation and induced muscle atrophy. While cobra venom decreased the viability, it did not largely affect cell migration and focal adhesions. Cobra venom affected the formation of myotubes and induced atrophy. Cobra venom-induced atrophy could not be reversed by small molecule inhibitors such as varespladib (a phospholipase A2 inhibitor) and prinomastat (a metalloprotease inhibitor), and soluble activin type IIb receptor (a molecule used to promote regeneration of skeletal muscle), although the antivenom (raised against the Indian ‘Big Four’ snakes) has attenuated the effects. However, all these molecules rescued the myotubes from Russell’s viper venom-induced atrophy. This study demonstrates key steps in the muscle regeneration process that are affected by both Indian Russell’s viper and cobra venoms and offers insights into the potential causes of clinical features displayed in envenomed victims. Further research is required to investigate the molecular mechanisms of venom-induced myotoxicity under in vivo settings and develop better therapies for snakebite-induced muscle damage.

CREG1 deficiency impaired myoblast differentiation and skeletal muscle regeneration.

CREG1 (cellular repressor of E1A-stimulated genes 1) is a protein involved in cellular differentiation and homeostasis regulation. However, its role in skeletal muscle satellite cells differentiation and muscle regeneration is poorly understood. This study aimed to investigate the role of CREG1 in myogenesis and muscle regeneration. RNA sequencing data (GSE8479) was analysed from the Gene Expression Omnibus database (GEO, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi). We generated Creg1 knockdown and skeletal muscle satellite cells specific Creg1 overexpression mice mediated by adeno-associated virus serotype 9 (AAV9), skeletal muscle mature myofibre Creg1 knockout mice (myoblast/Creg1MKO), and control mice Creg1flox/flox (Creg1fl/fl) as in vivo models. The mice were injected into tibialis anterior (TA) muscle with 100μL of 10μM cardiotoxin to establish a muscle regeneration model. Creg1fl/fl and Creg1MKO mice were treated with AAV-sh-C-Cbl (2×1010 genomic copies/mouse) to silence C-Cbl in the TA muscle. 293T and C2C12 cells were transfected with plasmids using lipofectamine RNAi MAX in vitro. Mass spectrometry analyses and RNA sequencing transcriptomic assay were performed. We analysed the transcriptional profiles of the skeletal muscle biopsies from healthy older (N=25) and younger (N=26) adult men and women in GSE8479 database, and the results showed that Creg1 was associated with human sarcopenia. We found that Creg1 knockdown mice regenerated less newly formed fibres in response to cardiotoxin injection (~30% reduction, P<0.01); however, muscle satellite cells specific Creg1 overexpression mice regenerated more newly formed fibres (~20% increase, P<0.05). AMPKa1 is known as a key mediator in the muscle regeneration process. Our results revealed that CREG1 deficiency inhibited AMPKa1 signalling through C-CBL E3-ubiquitin ligase-mediated AMPKa1 degradation (P<0.01). C-CBL-mediated AMPKa1 ubiquitination was attributed to the K48-linked polyubiquitination of AMPKa1 at K396 and that the modification played an important role in the regulation of AMPKa1 protein stability. We also found that Creg1MKO mice regenerated less newly formed fibres compared with Creg1fl/fl mice (~30% reduction, P<0.01). RNA-seq analysis showed that CREG1 deletion in impaired muscles led to the upregulation of inflammation and DKK3 expression. The TA muscles of Creg1MKO mice were injected with AAV-vector or AAV-shC-Cbl, silencing C-CBL (P<0.01) in the skeletal muscles of Creg1MKO mice significantly improved muscle regeneration induced by CTX injury (P<0.01). Our findings suggest that CREG1 may be a potential therapeutic target for skeletal muscle regeneration.

CD155 is essential for skeletal muscle regeneration by regulating satellite cell proliferation and differentiation.

CD155, a member of the immunoglobulin superfamily, is closely related to cell proliferation, adhesion, and migration. CD155 is overexpressed on the surface of cancer cells to promote cell proliferation and is upregulated in damaged tissues as a stress-induced molecule. The process of skeletal muscle regeneration after injury is complex and involves injurious stimulation and subsequent satellite cell proliferation. However, the role of CD155 in this process remains unelucidated. This study aimed to explore the role of CD155 in injured skeletal muscle regeneration and to clarify its effect on satellite cell proliferation and differentiation. Here, quantitative real-time polymerase chain reaction (RT-qPCR) and immunofluorescence results indicated that CD155 expression in satellite cells increased after skeletal muscle injury. CD155 knockout in mice impaired the regeneration of skeletal muscle. A bone marrow transplantation mouse model was constructed and revealed that CD155 on skeletal muscle tissues, not immune cells, affected muscle regeneration. Invitro, CD155 knockdown in myoblasts inhibited their proliferation and differentiation. The transcriptomic analysis also indicated that CD155 absence can impair the biological proliferation and differentiation process of myoblasts. Our research demonstrates that CD155 directly promotes injured muscle regeneration by regulating satellite cell proliferation and differentiation, which may be a potential therapeutic molecule for skeletal muscle injury.

CircUBE3C modulates myoblast development by binding to miR-191 and upregulating the expression of p27.

Noncoding RNAs, including miRNAs (microRNAs) and circRNAs (circular RNA), are crucial regulators of myoblast proliferation and differentiation during muscle development. However, the specific roles and molecular mechanisms of circRNAs in muscle development remain poorly understood. Based on the existing circRNA-miRNA-mRNA network, our study focuses on circUBE3C, exploring its differential expression in fetal and adult muscle tissue of the cattle and investigating its impact on myoblast proliferation, apoptosis, and differentiation. The functional analysis of overexpression plasmids and siRNAs (small interfering RNAs) targeting circUBE3C was comprehensively evaluated by employing an array of advanced assays, encompassing CCK-8 (cell counting kit-8), EdU (5-ethynyl-20-deoxyuridine), flow cytometry, western blot analysis, and RT-qPCR. In vivo investigations indicated that overexpression of circUBE3C impedes the process of skeletal muscle regeneration. Mechanistically, we demonstrated that circUBE3C interacts with miR-191 and alleviates the suppression of p27 through cytoplasmic separation, bioinformatics prediction, dual-luciferase reporter assay, and RIP (RNA immunoprecipitation). Our findings indicate that the novel circRNA circUBE3C competitively binds to miR-191, thereby inhibiting proliferation and promoting apoptosis in bovine primary myoblasts and unveiling a regulatory pathway in bovine skeletal muscle development. These findings expand our understanding of circRNA functions in mammals and provide a basis for further exploration of their role in myogenesis and muscle diseases.

Effects of the immune system on muscle regeneration.

Muscle regeneration is a complex process orchestrated by multiple steps. Recent findings indicate that inflammatory responses could play central roles in bridging initial muscle injury responses and timely muscle injury reparation. The various types of immune cells and cytokines have crucial roles in muscle regeneration process. In this review, we provide an overview of the functions of acute inflammation in muscle regeneration.

Muscle stem cell niche dynamics during muscle homeostasis and regeneration.

The process of skeletal muscle regeneration involves a coordinated interplay of specific cellular and molecular interactions within the injury site. This review provides an overview of the cellular and molecular components in regenerating skeletal muscle, focusing on how these cells or molecules in the niche regulate muscle stem cell functions. Dysfunctions of muscle stem cell-to-niche cell communications during aging and disease will also be discussed. A better understanding of how niche cells coordinate with muscle stem cells for muscle repair will greatly aid the development of therapeutic strategies for treating muscle-related disorders.

Folic acid promotes myoblast migration through Folr1 interacting with RhoA and improves skeletal muscle regeneration

Folic acid (FA) plays multiple roles in many biological processes. Myoblast intakes FA through endocytosis by folate receptor 1 (Folr1), which has vital role in regulating muscle development. However, the mechanism remains unclear. In this study, FA supplement experiment showed that FA promoted C2C12 cell differentiation through Folr1. Further exploration found that FA could promote C2C12 cell migration through Folr1 interacting with RhoA. Then stimulated RhoA/ROCK/LIMK/cofilin signaling pathway, and affected the distribution of microfilaments to improve C2C12 cell migration. Meanwhile, mice experiments also proved that FA could activate the expression of RhoA/ROCK/LIMK/cofilin signaling pathway in vivo and accelerate the process of skeletal muscle regeneration in mice. This discovery is helpful to further understand the molecular mechanism of FA regulating muscle development and provide new ideas for the treatment of muscle diseases.

Simulated altitude is medicine: intermittent exposure to hypobaric hypoxia and cold accelerates injured skeletal muscle recovery.

Muscle injuries are the leading cause of sports casualties. Because of its high plasticity, skeletal muscle can respond to different stimuli to maintain and improve functionality. Intermittent hypobaric hypoxia (IHH) improves muscle oxygen delivery and utilization. Hypobaria coexists with cold in the biosphere, opening the possibility to consider the combined use of both environmental factors to achieve beneficial physiological adjustments. We studied the effects of IHH and cold exposure, separately and simultaneously, on muscle regeneration. Adult male rats were surgically injured in one gastrocnemius and randomly assigned to the following groups: (1) CTRL: passive recovery; (2) COLD: intermittently exposed to cold (4°C); (3) HYPO: submitted to IHH (4500m); (4) COHY: exposed to intermittent simultaneous cold and hypoxia. Animals were subjected to these interventions for 4h/day for 9 or 21days. COLD and COHY rats showed faster muscle regeneration than CTRL, evidenced after 9days at histological (dMHC-positive and centrally nucleated fibre reduction) and functional levels after 21days. HYPO rats showed a full recovery from injury (at histological and functional levels) after 9days, while COLD and COHY needed more time to induce a total functional recovery. IHH can be postulated as an anti-fibrotic treatment since it reduces collagen I deposition. The increase in the pSer473Akt/total Akt ratio observed after 9days in COLD, HYPO and COHY, together with the increase in the pThr172AMPKα/total AMPKα ratio observed in the gastrocnemius of HYPO, provides clues to the molecular mechanisms involved in the improved muscle regeneration. KEY POINTS: Only intermittent hypobaric exposure accelerated muscle recovery as early as 9days following injury at histological and functional levels. Injured muscles from animals treated with intermittent (4h/day) cold, hypobaric hypoxia or a simultaneous combination of both stimuli regenerated histological structure and recovered muscle function 21days after injury. The combination of cold and hypoxia showed a blunting effect as compared to hypoxia alone in the time course of the muscle recovery. The increased expression of the phosphorylated forms of Akt observed in all experimental groups could participate in the molecular cascade of events leading to a faster regeneration. The elevated levels of phosphorylated AMPKα in the HYPO group could play a key role in the modulation of the inflammatory response during the first steps of the muscle regeneration process.