Search

Satellite cells (SCs), muscle stem cells, display functional heterogeneity, and dramatic changes linked to their regenerative capabilities are associated with muscle-wasting diseases. SC behavior is related to endogenous expression of the myogenic transcription factor MYF5 and the propensity to enter into the cell cycle. Here, we report a role for miR-106b reinforcing MYF5 inhibition and blocking cell proliferation in a subset of highly quiescent SC population. miR-106b down-regulation occurs during SC activation and is required for proper muscle repair. In addition, miR-106b is increased in dystrophic mice, and intramuscular injection of antimiR in injured mdx mice enhances muscle regeneration promoting transcriptional changes involved in skeletal muscle differentiation. miR-106b inhibition promotes the engraftment of human muscle stem cells. Furthermore, miR-106b is also high in human dystrophic muscle stem cells and its inhibition improves intrinsic proliferative defects and increases their myogenic potential. This study demonstrates that miR-106b is an important modulator of SC quiescence, and that miR-106b may be a new target to develop therapeutic strategies to promote muscle regeneration improving the regenerative capabilities of injured dystrophic muscle.

Skeletal muscle trauma is a common injury with a range of severity. Adenosine, lidocaine and Mg2+ (ALM) is a protective solution and improves tissue perfusion and coagulopathy. Male Wistar rats were anesthetized and subjected to standardized skeletal muscle trauma of the left soleus muscle with the protection of the neurovascular structures. Seventy animals were randomly assigned to saline control or ALM. Immediately after trauma, a bolus of ALM solution was applied intravenously, followed by a one-hour infusion. After 1, 4, 7, 14 and 42 days, the biomechanical regenerative capacity was examined using incomplete tetanic force and tetany, and immunohistochemistry was used to examine for proliferation and apoptosis characteristics. Biomechanical force development showed a significant increase following ALM therapy for incomplete tetanic force and tetany on days 4 and 7. In addition, the histological evaluation showed a significant increase in proliferative BrdU-positive cells with ALM therapy on days 1 and 14. Ki67 histology also detected significantly more proliferative cells on days 1, 4, 7, 14 and 42 in ALM-treated animals. Furthermore, a simultaneous decrease in the number of apoptotic cells was observed using the TUNEL method. ALM solution showed significant superiority in biomechanical force development and also a significant positive effect on cell proliferation in traumatized skeletal muscle tissue and reduced apoptosis.

Approximately half of an adult human’s body weight is made up of muscles. Thus, restoring the functionality and aesthetics of lost muscle tissue is critical. The body is usually able to repair minor muscle injuries. However, when volumetric muscle loss occurs due to tumour extraction, for instance, the body will form fibrous tissue instead. Gelatin methacryloyl (GelMA) hydrogels have been applied for drug delivery, tissue adhesive, and various tissue engineering applications due to their tuneable mechanical properties. Here, we have synthesised GelMA from different gelatin sources (i.e., porcine, bovine, and fish) with varying bloom numbers, which refers to the gel strength, and investigated for the influence of the source of gelatin and the bloom number on biological activities and mechanical properties. The results indicated that the source of the gelatin and variable bloom numbers have an impact on GelMA hydrogel properties. Furthermore, our findings established that the bovine-derived gelatin methacryloyl (B-GelMA) has better mechanical properties than the other varieties composed of porcine and fish with 60 kPa, 40 kPa, and 10 kPa in bovine, porcine, and fish, respectively. Additionally, it showed a noticeably greater swelling ratio (SR) ~1100% and a reduced rate of degradation, improving the stability of hydrogels and giving cells adequate time to divide and proliferate to compensate for muscle loss. Furthermore, the bloom number of gelatin was also proven to influence the mechanical properties of GelMA. Interestingly, although GelMA made of fish had the lowest mechanical strength and gel stability, it demonstrated excellent biological properties. Overall, the results emphasise the importance of gelatin source and bloom number, allowing GelMA hydrogels to have a wide range of mechanical and excellent biological properties and making them suitable for various muscle tissue regeneration applications.

A well-functioning skeletal muscle is vital for independent and healthy living. However, aging is associated with a decline in muscle mass and regenerative capacity, known as sarcopenia. Sarcopenia is characterized by reduced force-generating capacity leading to lower mobility. Skeletal muscle has the ability to regenerate after injury, as well as to adapt its size to physical loads. This process of regeneration and adaptation declines with aging as a result of reduced muscle plasticity, leading to skeletal muscle loss and function. The etiology of sarcopenia is multifactorial, but decreased muscle stem cell (MuSC) function is thought to play a role in the development of sarcopenia. Little is known about the alterations leading to declined MuSC function with age. The aims of this thesis were: 1) To review the role of biophysical cues in determining MuSC function, and how aging affects the mechanosensitivity of MuSCs. 2) To investigate whether MuSCs are subjected to mechanical cues within their niche during muscle stretch-shortening, and whether mechanical cues produce a physiological response in MuSCs. 3) To explore the role of the glycocalyx in the MuSC mechanoresponse. 4) To assess the role of mechanical cues in MuSC function, and determine age-related alterations within MuSC that are involved in their declined regenerative function. 5) To determine the effects of tensile strain and shear stress on nitric oxide (NO) production and gene expression by myotubes, and the role of Ca2+ and ROCK signaling herein. The MuSC in the MuSC niche in connection to extra‐muscular connective tissue are likely be subjected to mechanical loads during physical exercise. The presence of several mechanosensory complexes within the MuSC membrane combined with evidence of mechanoresponsiveness of MuSCs indicates that mechanical loads and subsequent cellular deformations are important co‐determinants of MuSC function. We postulated that mechanosensing of the aging MuSC may become dysfunctional due to intrinsic changes of the MuSC by alterations in expression levels of transmembrane and/or cytoskeletal proteins (chapter 2). Our results showed that upon myofiber stretch, MuSCs within their native niche, attached to the basal lamina and sarcolemma of the host myofiber, are subjected to compression as well as tensile and shear deformations (chapter 3). Moreover, mechanical loading by pulsating fluid shear stress (PFSS) induces NO production, gene expression (i.e., c-Fos, Cdk4, and IL-6), and ERK 1/2 and p38 MAPK signaling. We also showed that the glycocalyx is likely involved in fluid shear stress-induced myoblast deformation and modulation of gene expression (chapter 4). The results of this thesis showed that aged MuSCs exhibited a reduced growth rate and integrin-α7 expression, as well as a lower number of phospho-paxillin clusters than young MuSCs. Furthermore, aged MuSCs exhibited reduced adhesion strength. Moreover, YAP signaling and mechanoresponsiveness were altered in aged MuSCs (chapter 5). We also showed that mechanical loading by cyclic strain (CS) and PFSS induced a differential mechanoresponse and production of factors involved in MuSC recruitment in primary myotubes (chapter 6). Moreover, calcium signaling was involved in PFSS-induced NO production and IL-6 expression, and ROCK activation in PFSS-induced COX2 and IL-6 expression. This thesis shows that besides biochemical cues, mechanical cues, which are inherent to skeletal muscles, are transduced to MuSCs and elicit a mechanoresponse in these cells. The process of mechanosensing is altered with aging in MuSCs. Our results highlight the importance of mechanical cues in regulating MuSC function and age-related cell-intrinsic alterations in MuSC function leading to declined muscle regeneration and muscle wasting. Taken together, we identified an impairment in mechanosensitivity of aged MuSCs as a previously unknown mechanism in the declined MuSC function and muscle regeneration, that most likely contributes to sarcopenia.

A well-functioning skeletal muscle is vital for independent and healthy living. However, aging is associated with a decline in muscle mass and regenerative capacity, known as sarcopenia. Sarcopenia is characterized by reduced force-generating capacity leading to lower mobility. Skeletal muscle has the ability to regenerate after injury, as well as to adapt its size to physical loads. This process of regeneration and adaptation declines with aging as a result of reduced muscle plasticity, leading to skeletal muscle loss and function. The etiology of sarcopenia is multifactorial, but decreased muscle stem cell (MuSC) function is thought to play a role in the development of sarcopenia. Little is known about the alterations leading to declined MuSC function with age. The aims of this thesis were: 1) To review the role of biophysical cues in determining MuSC function, and how aging affects the mechanosensitivity of MuSCs. 2) To investigate whether MuSCs are subjected to mechanical cues within their niche during muscle stretch-shortening, and whether mechanical cues produce a physiological response in MuSCs. 3) To explore the role of the glycocalyx in the MuSC mechanoresponse. 4) To assess the role of mechanical cues in MuSC function, and determine age-related alterations within MuSC that are involved in their declined regenerative function. 5) To determine the effects of tensile strain and shear stress on nitric oxide (NO) production and gene expression by myotubes, and the role of Ca2+ and ROCK signaling herein. The MuSC in the MuSC niche in connection to extra‐muscular connective tissue are likely be subjected to mechanical loads during physical exercise. The presence of several mechanosensory complexes within the MuSC membrane combined with evidence of mechanoresponsiveness of MuSCs indicates that mechanical loads and subsequent cellular deformations are important co‐determinants of MuSC function. We postulated that mechanosensing of the aging MuSC may become dysfunctional due to intrinsic changes of the MuSC by alterations in expression levels of transmembrane and/or cytoskeletal proteins (chapter 2). Our results showed that upon myofiber stretch, MuSCs within their native niche, attached to the basal lamina and sarcolemma of the host myofiber, are subjected to compression as well as tensile and shear deformations (chapter 3). Moreover, mechanical loading by pulsating fluid shear stress (PFSS) induces NO production, gene expression (i.e., c-Fos, Cdk4, and IL-6), and ERK 1/2 and p38 MAPK signaling. We also showed that the glycocalyx is likely involved in fluid shear stress-induced myoblast deformation and modulation of gene expression (chapter 4). The results of this thesis showed that aged MuSCs exhibited a reduced growth rate and integrin-α7 expression, as well as a lower number of phospho-paxillin clusters than young MuSCs. Furthermore, aged MuSCs exhibited reduced adhesion strength. Moreover, YAP signaling and mechanoresponsiveness were altered in aged MuSCs (chapter 5). We also showed that mechanical loading by cyclic strain (CS) and PFSS induced a differential mechanoresponse and production of factors involved in MuSC recruitment in primary myotubes (chapter 6). Moreover, calcium signaling was involved in PFSS-induced NO production and IL-6 expression, and ROCK activation in PFSS-induced COX2 and IL-6 expression. This thesis shows that besides biochemical cues, mechanical cues, which are inherent to skeletal muscles, are transduced to MuSCs and elicit a mechanoresponse in these cells. The process of mechanosensing is altered with aging in MuSCs. Our results highlight the importance of mechanical cues in regulating MuSC function and age-related cell-intrinsic alterations in MuSC function leading to declined muscle regeneration and muscle wasting. Taken together, we identified an impairment in mechanosensitivity of aged MuSCs as a previously unknown mechanism in the declined MuSC function and muscle regeneration, that most likely contributes to sarcopenia.

Skeletal muscles are highly resilient due to the capacity to regenerate damaged muscle tissue. Despite the regenerative capacity, pathological conditions can lead to reduced muscle mass and strength, also known as muscle wasting. Muscle wasting, seen in pathological conditions, is often progressive and concomitant with excessive or prolonged inflammation, fibrosis and fat infiltration. Aging is a risk factor for a large number of diseases that can result in muscle wasting. The prevalence of sarcopenia, an age-associated decline in skeletal muscle mass and strength in elderly, surges as life expectancy increases. In combination with muscle wasting caused by neuromuscular disorders, the burden of muscle wasting on society is significant. Gaining a better understanding of the cellular and molecular processes underlying muscle wasting conditions is critical for creating better treatments. Aging and thereby age-associated muscle wasting are multifactorial involving changes in different tissues and multiple cell systems. This thesis demonstrates how different omics approaches, like transcriptomics, proteomics and metabolomics, can elucidate the complexity of those molecular mechanisms and cellular processes involved in muscle wasting conditions. Employing a combination of different omics-techniques (multi-omics) specifically proved powerful and will continue to illuminate impaired mechanisms and consequently help to battle muscle wasting.

Pluripotent stem (PS) cells enable the scalable production of tissue-specific derivatives with therapeutic potential for various clinical applications, including muscular dystrophies. Given the similarity to human counterparts, the non-human primate (NHP) is an ideal preclinical model to evaluate several questions, including delivery, biodistribution, and immune response. While the generation of human-induced PS (iPS)-cell-derived myogenic progenitors is well established, there have been no data for NHP counterparts, probably due to the lack of an efficient system to differentiate NHP iPS cells towards the skeletal muscle lineage. Here, we report the generation of three independent Macaca fascicularis iPS cell lines and their myogenic differentiation using PAX7 conditional expression. The whole-transcriptome analysis confirmed the successful sequential induction of mesoderm, paraxial mesoderm, and myogenic lineages. NHP myogenic progenitors efficiently gave rise to myotubes under appropriate in vitro differentiation conditions and engrafted in vivo into the TA muscles of NSG and FKRP-NSG mice. Lastly, we explored the preclinical potential of these NHP myogenic progenitors in a single wild-type NHP recipient, demonstrating engraftment and characterizing the interaction with the host immune response. These studies establish an NHP model system through which iPS-cell-derived myogenic progenitors can be studied.

Skeletal muscle formation is an extremely important step in animal growth and development. Recent studies have found that TMEM8c (also known as Myomaker, MYMK), a muscle-specific transmembrane protein, can promote myoblast fusion and plays a key role in the normal development of skeletal muscle. However, the effect of Myomaker on porcine (Sus scrofa) myoblast fusion and the underlying regulatory mechanisms remain largely unknown. Therefore, in this study, we focused on the role and corresponding regulatory mechanism of the Myomaker gene during skeletal muscle development, cell differentiation, and muscle injury repair in pigs. We obtained the entire 3′ UTR sequence of porcine Myomaker using the 3′ RACE approach and found that miR-205 inhibited porcine myoblast fusion by targeting the 3′ UTR of Myomaker. In addition, based on a constructed porcine acute muscle injury model, we discovered that both the mRNA and protein expression of Myomaker were activated in the injured muscle, while miR-205 expression was significantly inhibited during skeletal muscle regeneration. The negative regulatory relationship between miR-205 and Myomaker was further confirmed in vivo. Taken together, the present study reveals that Myomaker plays a role during porcine myoblast fusion and skeletal muscle regeneration and demonstrates that miR-205 inhibits myoblast fusion through targeted regulation of the expression of Myomaker.

International audience; Background: Intensive care unit (ICU)-acquired weakness is characterized by muscle atrophy and impaired contractility that may persist after ICU discharge. Dysregulated muscle repair and regeneration gene co-expression networks are present in critical illness survivors with persistent muscle wasting and weakness. We aimed to identify microRNAs (miRs) regulating the gene networks and determine their role in the self-renewal of muscle in ICU survivors.Methods: Muscle whole-transcriptome expression was assessed with microarrays in banked quadriceps biopsies obtained at 7 days and 6 months post-ICU discharge from critically ill patients (n = 15) in the RECOVER programme and healthy individuals (n = 8). We conducted an integrated miR-messenger RNA analysis to identify miR/gene pairs associated with muscle recovery post-critical illness and evaluated their impact on myoblast proliferation and differentiation in human AB1167 and murine C2C12 cell lines in vitro. Select target genes were validated with quantitative PCR.Results: Twenty-two miRs were predicted to regulate the Day 7 post-ICU muscle transcriptome vs. controls. Thirty per cent of all differentially expressed genes shared a 3'UTR regulatory sequence for miR-424-3p/5p, which was 10-fold down-regulated in patients (P < 0.001) and correlated with quadriceps size (R = 0.86, P < 0.001), strength (R = 0.75, P = 0.007), and physical function (Functional Independence Measures motor subscore, R = 0.92, P < 0.001) suggesting its potential role as a master regulator of early recovery of muscle mass and strength following ICU discharge. Network analysis demonstrated enrichment for cellular respiration and muscle fate commitment/development related genes. At 6 months post-ICU discharge, a 14-miR expression signature, including miRs-490-3p and -744-5p, identified patients with muscle mass recovery vs. those with sustained atrophy. Constitutive overexpression of the novel miR-490-3p significantly inhibited AB1167 and C2C12 myoblast proliferation (cell count AB1167 miR-490-3p mimic or scrambled-miR transfected myoblasts 7926 ± 4060 vs. 14 159 ± 3515 respectively, P = 0.006; proportion Ki67-positive nuclei AB1167 miR-490-3p mimic or scrambled-miR transfected myoblasts 0.38 ± 0.07 vs. 0.54 ± 0.06 respectively, P < 0.001; proliferating cell nuclear antigen expression AB1167 miR-490-3p mimic or scrambled-miR transfected myoblasts 11.48 ± 1.97 vs. 16.75 ± 1.19 respectively, P = 0.040). Constitutive overexpression of miR-744-5p, a known regulator of myogenesis, significantly inhibited AB1167 and C2C12 myoblast differentiation (fusion index AB1167 miR-744-5p mimic or scrambled-miR transfected myoblasts 8.31 ± 7.00% vs. 40.29 ± 9.37% respectively, P < 0.001; myosin heavy chain expression miR-744-5p mimic or scrambled-miR transfected myoblasts 0.92 ± 0.39 vs. 13.53 ± 5.5 respectively, P = 0.01).Conclusions: Combined functional transcriptomics identified 36 miRs including miRs-424-3p/5p, -490-3p, and -744-5p as potential regulators of gene networks associated with recovery of muscle mass and strength following critical illness. MiR-490-3p is identified as a novel regulator of myogenesis.

Duchenne muscular dystrophy (DMD) is a devastating disorder and is considered to be one of the worst forms of inherited muscular dystrophies. DMD occurs as a result of mutations in the dystrophin gene, leading to progressive muscle fiber degradation and weakness. Although DMD pathology has been studied for many years, there are aspects of disease pathogenesis and progression that have not been thoroughly explored yet. The underlying issue with this is that the development of further effective therapies becomes stalled. It is becoming more evident that extracellular vesicles (EVs) may contribute to DMD pathology. EVs are vesicles secreted by cells that exert a multitude of effects via their lipid, protein, and RNA cargo. EV cargo (especially microRNAs) is also said to be a good biomarker for identifying the status of specific pathological processes that occur in dystrophic muscle, such as fibrosis, degeneration, inflammation, adipogenic degeneration, and dilated cardiomyopathy. On the other hand, EVs are becoming more prominent vehicles for custom-engineered cargos. In this review, we will discuss the possible contribution of EVs to DMD pathology, their potential use as biomarkers, and the therapeutic efficacy of both, EV secretion inhibition and custom-engineered cargo delivery. ispartof: FRONTIERS IN PHYSIOLOGY vol:14 ispartof: location:Switzerland status: published

Background Skeletal muscle regeneration is an adaptive response to injury that is crucial to the maintenance of muscle mass and function. A p21‐activated kinase 4 (PAK4) serine/threonine kinase is critical to the regulation of cytoskeletal changes, cell proliferation, and growth. However, PAK4's role in myoblast differentiation and regenerative myogenesis remains to be determined. Methods We used a mouse model of myotoxin (notexin)‐induced muscle regeneration. In vitro myogenesis was performed in the C2C12 myoblast cell line, primary myoblasts, and primary satellite cells. In vivo overexpression of PAK4 or kinase‐inactive mutant PAK4S474A was conducted in skeletal muscle to examine PAK4's kinase‐dependent effect on muscle regeneration. The regeneration process was evaluated by determining the number and size of multinucleated myofibres and expression patterns of myogenin and eMyHC. To explore whether PAK4 inhibition improves muscle regeneration, mice were injected intramuscularly with siRNA that targeted PAK4 or orally administered with a chemical inhibitor of PAK4. Results p21‐activated kinase 4 was highly expressed during the myoblast stage, but expression gradually and substantially decreased as myoblasts differentiated into myotubes. PAK4 overexpression, but not kinase‐inactive mutant PAK4S474A overexpression, significantly impeded myoblast fusion and MyHC‐positive myotube formation in C2C12 cells, primary myoblasts, and satellite cells (P

The resident stem cell for skeletal muscle is the satellite cell. On the 50th anniversary of its discovery in 1961, we described the history of skeletal muscle research and the seminal findings made during the first 20 years in the life of the satellite cell (Scharner and Zammit 2011, doi: 10.1186/2044-5040-1-28). These studies established the satellite cell as the source of myoblasts for growth and regeneration of skeletal muscle. Now on the 60th anniversary, we highlight breakthroughs in the second phase of satellite cell research from 1980 to 2000. These include technical innovations such as isolation of primary satellite cells and viable muscle fibres complete with satellite cells in their niche, together with generation of many useful reagents including genetically modified organisms and antibodies still in use today. New methodologies were combined with description of endogenous satellite cells markers, notably Pax7. Discovery of the muscle regulatory factors Myf5, MyoD, myogenin, and MRF4 in the late 1980s revolutionized understanding of the control of both developmental and regerenative myogenesis. Emergence of genetic lineage markers facilitated identification of satellite cells in situ, and also empowered transplantation studies to examine satellite cell function. Finally, satellite cell heterogeneity and the supportive role of non-satellite cell types in muscle regeneration were described. These major advances in methodology and in understanding satellite cell biology provided further foundations for the dramatic escalation of work on muscle stem cells in the 21st century.

Volumetric muscle loss (VML) VML is defined as the loss of a critical mass of skeletal muscle that overwhelms the muscle's natural healing mechanisms, leaving patients with permanent functional deficits and deformity. The treatment of these defects is complex, as skeletal muscle is a composite structure that relies closely on the action of supporting tissues such as tendons, vasculature, nerves, and bone. The gold standard of treatment for VML injuries, an autologous muscle flap transfer, suffers from many shortcomings but nevertheless remains the best clinically available avenue to restore function. This review will consider the use of composite tissue engineered constructs, with multiple components that act together to replicate the function of an intact muscle, as an alternative to autologous muscle flaps. We will discuss recent advances in the field of tissue engineering that enable skeletal muscle constructs to more closely reproduce the functionality of an autologous muscle flap by incorporating vasculature, promoting innervation, and reconstructing the muscle-tendon boundary. Additionally, our understanding of the cellular composition of skeletal muscle has evolved to recognize the importance of a diverse variety of cell types in muscle regeneration, including fibro/adipogenic progenitors and immune cells like macrophages and regulatory T cells. We will address recent advances in our understanding of how these cell types interact with, and can be incorporated into, implanted tissue engineered constructs.

Understanding the mechanisms of tissue and organ regeneration in adult animals and humans is of great interest from a basic biology as well as a medical, therapeutical point of view. It is increasingly clear that the relatively limited ability to regenerate tissues and organs in mammals as oppose to lower vertebrates is the consequence of evolutionary trade-offs and changes during development and aging. Thus, the coordinated interaction of the immune system, particularly the innate part of it, and the injured, degenerated parenchymal tissues such as skeletal muscle, liver, lung, or kidney shape physiological and also pathological processes. In this review, we provide an overview of how morphologically and functionally complete (ad integrum) regeneration is achieved using skeletal muscle as a model. We will review recent advances about the differentiation, activation, and subtype specification of circulating monocyte to resolution or repair-type macrophages during the process we term regenerative inflammation, resulting in complete restoration of skeletal muscle in murine models of toxin-induced injury.

Developing a universal culture platform that manipulates cell fate is one of the most important tasks in the investigation of the role of the cellular microenvironment. This study focuses on the application of topographical and electrical field stimuli to human myogenic precursor cell (hMPC) cultures to assess the influences of the adherent direction, proliferation, and differentiation, and induce preconditioning-induced therapeutic benefits. First, a topographical surface of commercially available culture dishes was achieved by femtosecond laser texturing. The detachable biphasic electrical current system was then applied to the hMPCs cultured on laser-textured culture dishes. Laser-textured topographies were remarkably effective in inducing the assembly of hMPC myotubes by enhancing the orientation of adherent hMPCs compared with flat surfaces. Furthermore, electrical field stimulation through laser-textured topographies was found to promote the expression of myogenic regulatory factors compared with nonstimulated cells. As such, we successfully demonstrated that the combined stimulation of topographical and electrical cues could effectively enhance the myogenic maturation of hMPCs in a surface spatial and electrical field-dependent manner, thus providing the basis for therapeutic strategies., Graphical abstract Image 1, Highlights • We applied simultaneous topographical and electrical stimuli on human myogenic precursor cells. • Combined topographical/electrical stimulation enhanced the maturation of hMPCs. • hMPCs that simultaneously experienced stimulations contributed to muscle regeneration and angiogenesis in vivo. • Laser modification for commercially available plastic culture products provides the basis for future therapeutic strategies.

BCAAs supplementation has been widely used for post-exercise recovery. However, no evidence is currently available to answer the question of whether BCAAs supplementation can attenuate muscle damage and ameliorate recovery after a bout of change of direction (COD) sprinting, which is an exercise motion frequently used during team sport actions. This study aimed to assess the effect of BCAAs supplementation on muscle damage markers, subjective muscle soreness, neuromuscular performance, and the vascular health of collegiate basketball players during a 72 h recovery period after a standardized COD protocol. Participants orally received either BCAAs (0.17 g/kg BCAAs + 0.17 g/kg glucose) or placebo (0.34 g/kg glucose) supplementation before and immediately after a COD exercise protocol in a randomized, crossover, double-blind, and placebo-controlled manner. Creatine kinase increased immediately after exercise and peaked at 24 h, muscle soreness remained elevated until 72 h, whilst arterial stiffness decreased after exercise for both supplemented conditions. A negligibly lower level of interleukin-6 was found in the BCAAs supplemented condition. In conclusion, the results of this study do not support the benefits of BCAAs supplementation on mitigating muscle damage and soreness, neuromuscular performance, and arterial stiffness after COD for basketball players.

Increased oxidative stress can slow down the regeneration of skeletal muscle and affect the activity of muscle satellite cells (mSCs). Therefore, we evaluated the role of the NRF2 transcription factor (encoded by the Nfe2l2 gene), the main regulator of the antioxidant response, in muscle cell biology. We used (i) an immortalized murine myoblast cell line (C2C12) with stable overexpression of NRF2 and (ii) primary mSCs isolated from wild-type and Nfe2l2 (transcriptionally)-deficient mice (Nfe2l2tKO). NRF2 promoted myoblast proliferation and viability under oxidative stress conditions and decreased the production of reactive oxygen species. Furthermore, NRF2 overexpression inhibited C2C12 cell differentiation by down-regulating the expression of myogenic regulatory factors (MRFs) and muscle-specific microRNAs. We also showed that NRF2 is indispensable for the viability of mSCs since the lack of its transcriptional activity caused high mortality of cells cultured in vitro under normoxic conditions. Concomitantly, Nfe2l2tKO mSCs grown and differentiated under hypoxic conditions were viable and much more differentiated compared to cells isolated from wild-type mice. Taken together, NRF2 significantly influences the properties of myoblasts and muscle satellite cells. This effect might be modulated by the muscle microenvironment.

Eden najbolj raziskanih genov, povezanih s športno aktivnostjo, je gen ACTN3, ki kodira specifično v hitrih glikolitičnih mišičnih vlaknih izražen protein α-aktinin-3. Pogost polimorfizem tega gena je R577X, ki povzroči spremembo kodona za arginin (R) v terminacijski kodon (X). Namen diplomskega dela je pregled obstoječe literature, ki opisuje vpliv ničtega polimorfizma ACTN3 R577X na različne vidike telesne dejavnosti. Fenotipske posledice odsotnosti α-aktinina-3 so spremenjene strukturne lastnosti, metabolizem in signalne poti hitrih mišičnih vlaken, spremenjena vsebnost glikogena v mišicah, spremenjena kinetika Ca2+ v povezavi s sarkoplazemskim retikulumom ter zmanjšana mineralna kostna gostota. Homozigotni nosilci mutacije (genotip XX) imajo manjši premer hitrih mišičnih vlaken, večje zaloge mišičnega glikogena, zmanjšano kostno maso, v njihovih hitrih mišičnih vlaknih pa prevladuje aerobni metabolizem. Tako naj bi imeli športniki z genotipom XX večjo mišično vzdržljivost, medtem ko naj bi imeli športniki z genotipom RR večjo mišično moč in eksplozivnost. Nekatere raziskave obenem kažejo, da alel X vpliva tudi na povečano dovzetnost posameznika na vzdržljivostni trening, povečano hitrost regeneracije po športni aktivnosti ter zagotavlja boljšo telesno pripravljenost starostnikov. Po drugi strani pa alel X vpliva na povečano resnost in pogostost poškodb mišic, sklepov in kosti, nastalih med vadbo, ter na slabše zdravstveno stanje starostnikov. One of the most studied genes related to sport is the ACTN3 gene, which encodes the α-actinin-3 protein expressed specifically in fast glycolytic muscle fibers. A common polymorphism of this gene is R577X, which results in a change of codon for arginine (R) to termination codon (X). The aim of the thesis is to review the existing literature describing the influence of the ACTN3 R577X null polymorphism on various aspects of physical activity. The absence of α-actinin-3 has phenotypic consequences such as altered structural properties, metabolism and signaling pathways of fast-twitch muscle fibers, altered glycogen content in muscles, different Ca2+ kinetics in connection with the sarcoplasmic reticulum and reduced bone mineral density. Homozygous carriers of the mutation (genotype XX) have a smaller diameter of fast-twitch muscle fibers, larger stores of muscle glycogen and reduced bone mass. Moreover aerobic metabolism dominates in their fast-twitch muscle fibers. Because of that athletes with the XX genotype should have greater muscular endurance, while athletes with the RR genotype should have greater muscular strength and explosiveness. Research has also shown that X allele affects individual's increased susceptibility to endurance training, faster regeneration after sports activity and ensures better physical fitness of the elderly. However, X allele is also associated with increased severity and frequency of muscle, joint and bone injuries caused by exercise and the poor health of the elderly.

The fundamental basis for the human function is provided by skeletal muscle. Advancing age causes selective fiber atrophy, motor unit loss, and hybrid fiber formation resulting in hampered mass and strength, thus referred to as sarcopenia. Influence on the loss of independence of aged adults, contribute toward inclined healthcare costs conveys the injurious impact. The current understating of age-related skeletal muscle changes are addressed in this review, and further discusses mechanisms regulating protein turnover, although they do not completely define the process yet. Moreover, the reduced capacity of muscle regeneration due to impairment of satellite cell activation and proliferation with neuronal, immunological, hormonal factors were brought into the light of attention. Nevertheless, complete understating of sarcopenia requires disentangling it from disuse and disease. Nutritional intervention is considered a potentially preventable factor contributing to sarcopenia. Seafood is a crucial player in the fight against hunger and malnutrition, where it consists of macro and micronutrients. Hence, the review shed light on seafood as a nutritional intrusion in the treatment and prevention of sarcopenia. Understanding multiple factors will provide therapeutic targets in the prevention, treatment, and overcoming adverse effects of sarcopenia.

Background Transmembrane proteins are vital for intercellular signalling and play important roles in the control of cell fate. However, their physiological functions and mechanisms of action in myogenesis and muscle disorders remain largely unexplored. It has been found that transmembrane protein 182 (TMEM182) is dramatically up‐regulated during myogenesis, but its detailed functions remain unclear. This study aimed to analyse the function of TMEM182 during myogenesis and muscle regeneration. Methods RNA sequencing, quantitative real‐time polymerase chain reaction, and immunofluorescence approaches were used to analyse TMEM182 expression during myoblast differentiation. A dual‐luciferase reporter assay was used to identify the promoter region of the TMEM182 gene, and a chromatin immunoprecipitation assay was used to investigate the regulation TMEM182 transcription by MyoD. We used chickens and TMEM182‐knockout mice as in vivo models to examine the function of TMEM182 in muscle growth and muscle regeneration. Chickens and mouse primary myoblasts were used to extend the findings to in vitro effects on myoblast differentiation and fusion. Co‐immunoprecipitation and mass spectrometry were used to identify the interaction between TMEM182 and integrin beta 1 (ITGB1). The molecular mechanism by which TMEM182 regulates myogenesis and muscle regeneration was examined by Transwell migration, cell wound healing, adhesion, glutathione‐S‐transferse pull down, protein purification, and RNA immunoprecipitation assays. Results TMEM182 was specifically expressed in skeletal muscle and adipose tissue and was regulated at the transcriptional level by the myogenic regulatory factor MyoD1. Functionally, TMEM182 inhibited myoblast differentiation and fusion. The in vivo studies indicated that TMEM182 induced muscle fibre atrophy and delayed muscle regeneration. TMEM182 knockout in mice led to significant increases in body weight, muscle mass, muscle fibre number, and muscle fibre diameter. Skeletal muscle regeneration was accelerated in TMEM182‐knockout mice. Furthermore, we revealed that the inhibitory roles of TMEM182 in skeletal muscle depend on ITGB1, an essential membrane receptor involved in cell adhesion and muscle formation. TMEM182 directly interacted with ITGB1, and this interaction required an extracellular hybrid domain of ITGB1 (aa 387–470) and a conserved region (aa 52–62) within the large extracellular loop of TMEM182. Mechanistically, TMEM182 modulated ITGB1 activation by coordinating the association between ITGB1 and laminin and regulating the intracellular signalling of ITGB1. Myogenic deletion of TMEM182 increased the binding activity of ITGB1 to laminin and induced the activation of the FAK‐ERK and FAK‐Akt signalling axes during myogenesis. Conclusions Our data reveal that TMEM182 is a novel negative regulator of myogenic differentiation and muscle regeneration.

Myocardin‐related transcription factor‐A/serum response factor (MRTF‐A/SRF), a well‐known transcriptional programme, has been proposed to play crucial roles in skeletal muscle development and function. However, whether MRTF‐A participates in muscle regeneration and the molecular mechanisms are not completely understood. Here, we show that MRTF‐A levels are highly correlated with myogenic genes using a RNA‐seq assay, which reveal that MRTF‐A knockdown in C2C12 cells significantly reduces PAX7 expression. Subsequent in vitro and in vivo data show that MRTF‐A and PAX7 present identical expression patterns during myoblast differentiation and CTX‐induced muscle injury and repair. Remarkably, MRTF‐A overexpression promotes myoblast proliferation, while inhibiting cell differentiation and the expression of MyoD and MyoG. MRTF‐A loss of function produces the opposite effect. Moreover, mice with lentivirus (MRTF‐A) injection possesses more PAX7+ satellite cells, but less differentiating MyoD+ and MyoG+ cells, leading subsequently to diminished muscle regeneration. Our mechanistic results reveal that MRTF‐A contributes to PAX7‐mediated myoblast self‐renewal, proliferation, and differentiation by binding to its distal CArG box. Overall, we propose that MRTF‐A functions as a novel PAX7 regulator upon myoblast commitment to differentiation, which could provide pathways for dictating muscle stem cell fate and open new avenues to explore stem cell‐based therapy for muscle degenerative diseases.

Duchenne muscular dystrophy (DMD) is a progressive and lethal disease, caused by X‐linked mutations of the dystrophin encoding gene. The lack of dystrophin leads to muscle weakness, degeneration, fibrosis, and progressive loss of skeletal, cardiac, and respiratory muscle function resulting in premature death due to the cardiac and respiratory failure. There is no cure for DMD and current therapies neither cure nor arrest disease progression. Thus, there is an urgent need to develop new approaches and safer therapies for DMD patients. We have previously reported functional improvements which correlated with increased dystrophin expression following transplantation of dystrophin expressing chimeric (DEC) cells of myoblast origin to the mdx mouse models of DMD. In this study, we demonstrated that systemic‐intraosseous transplantation of DEC human cells derived from myoblasts of normal and DMD‐affected donors, increased dystrophin expression in cardiac, respiratory, and skeletal muscles of the mdx/scid mouse model of DMD. DEC transplant correlated with preservation of ejection fraction and fractional shortening on echocardiography, improved respiratory function on plethysmography, and improved strength and function of the limb skeletal muscles. Enhanced function was associated with improved muscle histopathology, revealing reduced mdx pathology, fibrosis, decreased inflammation, and preserved muscle morphology and architecture. Our findings confirm that DECs generate a systemic protective effect in DMD‐affected target organs. Therefore, DECs represents a novel therapeutic approach with the potential to preserve or enhance multiorgan function of the skeletal, cardiac, and respiratory muscles critical for the well‐being of DMD patients., Systemic‐intraosseous transplantation of human dystrophin expressing chimeric (DEC) cells (0.5 × 106 and 1 × 106) results in significant increase in dystrophin expression in heart, diaphragm, and gastrocnemius muscle (GM) which correlates with the reduction of centrally nucleated fibers in diaphragm and GM indicating reduced muscle pathology and further confirming beneficial effect of DEC therapy at 90 days after systemic transplant to mdx/scid mouse model of DMD.

Muscle stem cells (MuSCs) are tissue-resident stem cells required for growth and repair of skeletal muscle, that are otherwise maintained in a cell-cycle-arrested state called quiescence. While quiescence was originally believed to be a state of cellular inactivity, increasing evidence suggests that quiescence is dynamically regulated and contributes to stemness, the long-term capacity to maintain regenerative functions. Here, we review the current understanding of MuSC quiescence and highlight recently discovered molecular markers, which differentiate depth of quiescence and influence self-renewal capacity. We also discuss how quiescent MuSCs integrate paracrine factors from their niche and dynamically regulate cell signaling, metabolism and proteostasis as they anticipate physiological needs, and how perturbing these cues during aging impairs muscle regeneration.

CRISPR gene therapy is one promising approach for treatment of Duchenne muscular dystrophy (DMD), which is caused by a large spectrum of mutations in the dystrophin gene. To broaden CRISPR gene editing strategies for DMD treatment, we report the efficient restoration of dystrophin expression in induced myotubes by SpCas9 and dual guide RNAs (gRNAs). We first sequenced 32 deletion junctions generated by this editing method and revealed that non-homologous blunt-end joining represents the major indel type. Based on this predictive repair outcome, efficient in-frame deletion of a part of DMD exon 51 was achieved in HEK293T cells with plasmids expressing SpCas9 and dual gRNAs. More importantly, we further corrected a frameshift mutation in human DMD (exon45del) fibroblasts with SpCas9-dual gRNA ribonucleoproteins. The edited DMD fibroblasts were transdifferentiated into myotubes by lentiviral-mediated overexpression of a human MYOD transcription factor. Restoration of DMD expression at both the mRNA and protein levels was confirmed in the induced myotubes. With further development, the combination of SpCas9-dual gRNA-corrected DMD patient fibroblasts and transdifferentiation may provide a valuable therapeutic strategy for DMD., Graphical abstract, Duchenne muscular dystrophy (DMD) is caused by dystrophin deficiency and affects ∼1 in 5,000 boys. In this study, Yonglun Luo and colleagues report a CRISPR approach to efficiently correct DMD mutations and restore dystrophin expression in human muscle cells, providing a potential therapeutic approach for DMD.

The aim of the present study was directed to evaluate the therapeutic efficacy of chrysin (CH) on acrylamide (ACR) induced striated muscles dystrophy of rat’s tongue. subjects and methods: Forty adult male albino rats (200–250 g) were divided into four equal group(s) (G(s)), used for two successive periods (20 days: A , 40 days: B). G1 were fed with standard diet, without receiving any kind of treatment and considered as negative control group. G2 were received 15 mg/kg body weight of ACR orally once daily for 20 days (G2A) and for 40 days (G2B) and considered as positive control group. G3 were treated as G2, in addition; they were received 50 mg/kg intraperitoneal of CH two weeks before, as well as during application of ACR on alternative days and considered as a chemopreventive group. G4 were treated as G2, in addition; they were received 50 mg/kg intraperitoneal of CH, at first, third and seventh days after application of ACR and considered as a treatment group. Results: G1 has no observable abnormalities. G2A and G2B revealed an increase in collagen fibers with degenerative changes. G3A and G3B revealed decreased collagen fibers with muscle regeneration. G4A revealed localized red swelling of tongue mucosa, diffuse in G4B. Both had limited areas of collagen fibers. Conclusion: Striated muscle damage in rat's tongue by ACR becomes worse with prolonged duration is realized. CH has a significant protective efficacy of when compared with that of treatment on ACR induced striated muscle injury of rat’s tongue.

Muscle regeneration after damage or during myopathies requires a fine cooperation between myoblast proliferation and myogenic differentiation. A growing body of evidence suggests that microRNAs play critical roles in myocyte proliferation and differentiation transcriptionally. However, the molecular mechanisms underlying the orchestration are not fully understood. Here, we showed that miR-130b is able to repress myoblast proliferation and promote myogenic differentiation via targeting Sp1 transcription factor. Importantly, overexpression of miR-130b is capable of improving the recovery of damaged muscle in a freeze injury model. Moreover, miR-130b expression is declined in the muscle of muscular dystrophy patients. Thus, these results indicated that miR-130b may play a role in skeletal muscle regeneration and myopathy progression. Together, our findings suggest that the miR-130b/Sp1 axis may serve as a potential therapeutic target for the treatment of patients with muscle damage or severe myopathies.

Background Skeletal muscle wasting is a devastating consequence of cancer that affects up to 80% of cancer patients and associates with reduced survival. Herein, we investigated the biological significance of Forkhead box P1 (FoxP1), a transcriptional repressor that we demonstrate is up‐regulated in skeletal muscle in multiple models of cancer cachexia and in cachectic cancer patients. Methods Inducible, skeletal muscle‐specific FoxP1 over‐expressing (FoxP1iSkmTg/Tg) mice were generated through crossing conditional Foxp1a transgenic mice with HSA‐MCM mice that express tamoxifen‐inducible Cre recombinase under control of the skeletal muscle actin promoter. To determine the requirement of FoxP1 for cancer‐induced skeletal muscle wasting, FoxP1‐shRNA was packaged and targeted to muscles using AAV9 delivery prior to inoculation of mice with Colon‐26 Adenocarcinoma (C26) cells. Results Up‐regulation of FoxP1 in adult skeletal muscle was sufficient to induce features of cachexia, including 15% reduction in body mass (P

Summary The main and more frequent problem in face lift is recurrence of platysma bands, and of skin excess at the bitterness and naso-jugal folds. To improve face lift stability, a new aging analysis is proposed. The visible sign of face aging is skin excess. SMAS retraction is a much less visible sign. Nevertheless, botulinum toxin injections elongate muscles of the SMAS and rejuvenate the face. In the Face Recurve Concept, MRI studies prove the mimic muscles retraction that comes with aging. Face lift techniques that are used today include SMAS plication, traction or excision, which increases discrepancy between the envelop, the skin and the core, the SMAS. As skin excision is poorly efficient because performed posteriorly, far from the anterior skin excess located at the medial neck or at the bitterness fold, the association to SMAS retraction exacerbates the gradient difference between envelop and core. This analysis shows first of all that it is paramount to preserve the SMAS. Secondly, that the sub-skin dissection has to be executed moving beyond the paramedian folds. Then permanent tensor threads anchored in the malar, parotid and mastoid aponeurosis with a U pass flatten the folds and reposition sub cutaneous tissue excess backward to the SMAS. No action is performed on the SMAS, only an horizontal section of the platysma at the hyoid level will avoid platysma band recurrence. Botulinum toxin injections performed just after block muscle regeneration. 35 patients have been operated on a one year period. Results are a lot more stable and swelling is lowered down to the minimum.

Laser light can stimulate cell proliferation and restoration of various tissues after injury. The age aspect of laser irradiation effects at muscle regeneration, in particular the response of myosatellitocytes and myocytes, remains unexplored. The purpose of the research was to study the effect of laser light at the area of nuclei and the number of myosatellitocytes, as well as the area of myocyte nuclei in a model of posttraumatic regeneration of skeletal muscle in animals of different age groups. Material and methods . 32 laboratory mongrel rats, divided into 2 groups – young (3 months) and old (30 months) – were taken into the experiment. The formed surgical injuries at the caviar muscle on the left limb were experimental; they were irradiated with laser light (1060 nm, power 2.0 W, continuous mode, exposure 60 s, one time); injuries on the right limb were dynamic control. The animals were removed from the experiment on days 7 and 14. Tissue samples were fixed in formalin, histological sections were prepared, hematoxylin-eosin and Van Gizon picrofuxin were stained, and a digital image of objects was morphometrically analyzed. Results . It was found that after laser irradiation of the muscle, the area of nuclei and the number of myosatellites increase significantly compared to the control group, regardless of animal’s age. The response to myocyte nuclei to laser light was seen in both age groups, but it was significantly higher in younger animals. The obtained results demonstrate that laser radiation has the potential to activate muscle regeneration regardless of animal’s age.

A harántcsíkolt izomszövetből felépülő vázizmok akaratlagos összehúzódásai a test mozgásait okozzák és jól irányíthatók. A vázizom egyik legjellegzetesebb tulajdonsága, hogy nagyfokú in vivo és in vitro regenerációs képességgel rendelkezik, viszont nagyobb mennyiségű izomvesztés esetén a regenerációs folyamat kimerül, intervenciós támogatást igényel. Ebből következik, hogy az izomsérülés folyamatos kihívást jelent a klinikum számára. Az elmúlt században, különösen az utóbbi néhány évtizedben számos technikát dolgoztak ki az izomregeneráció elősegítése érdekében, ideértve a különböző műtéti technikákat, a fizikoterápiát, a biológiai anyagokat, az izomszövettenyésztést, krioterápiát, mágneses mező alkalmazását, valamint a sejtterápiát. Ugyanakkor továbbra is nagy igény mutatkozik új módszerek és anyagok kifejlesztésére, melyek elősegítik a vázizom megfelelő érését és a funkcionális regenerációt. Ebben a tanulmányban átfogó képet adunk az izomszövetveszteség epidemiológiájáról, rávilágítunk a klinikai kezelés aktuális stratégiáira, és megvitatjuk az izomregeneráció új módszereit, a jövőbeli klinikai átalakulásuk kihívásait.

Graphical abstract The intrinsic regenerative capacity of skeletal muscle makes it an excellent target for cell therapy. However, the potential of muscle tissue to renew is typically exhausted and insufficient in muscular dystrophies (MDs), a large group of heterogeneous genetic disorders showing progressive loss of skeletal muscle fibers. Cell therapy for MDs has to rely on suppletion with donor cells with high myogenic regenerative capacity. Here, we provide an overview on stem cell lineages employed for strategies in MDs, with a focus on adult stem cells and progenitor cells resident in skeletal muscle. In the early days, the potential of myoblasts and satellite cells was explored, but after disappointing clinical results the field moved to other muscle progenitor cells, each with its own advantages and disadvantages. Most recently, mesoangioblasts and pericytes have been pursued for muscle cell therapy, leading to a handful of preclinical studies and a clinical trial. The current status of (pre)clinical work for the most common forms of MD illustrates the existing challenges and bottlenecks. Besides the intrinsic properties of transplantable cells, we discuss issues relating to cell expansion and cell viability after transplantation, optimal dosage, and route and timing of administration. Since MDs are genetic conditions, autologous cell therapy and gene therapy will need to go hand-in-hand, bringing in additional complications. Finally, we discuss determinants for optimization of future clinical trials for muscle cell therapy. Joined research efforts bring hope that effective therapies for MDs are on the horizon to fulfil the unmet clinical need in patients.

Balenine is one of the endogenous imidazole dipeptides derived from marine products. It is composed of beta-alanine and 3-methyl-L-histidine, which exist mainly in the muscles of marine organisms. The physiological functions of dietary balenine are not well-known. In this study, we investigated whether the supplementation of dietary balenine was associated with muscle function in a cardiotoxin-indued muscle degeneration/regeneration model. Through morphological observation, we found that the supplementation of balenine-enriched extract promoted the regeneration stage. In addition, the expression of regeneration-related myogenic marker genes, such as paired box protein 7, MyoD1, myogenin, and Myh3, in a group of mice fed a balenine-enriched extract diet was higher than that in a group fed a normal diet. Moreover, the supplementation of balenine-enriched extract promoted the expression of anti-inflammatory cytokines as well as pro-inflammatory cytokines at the degeneration stage. Interestingly, phagocytic activity in the balenine group was significantly higher than that in the control group in vitro. These results suggest that balenine may promote the progress of muscle regeneration by increasing the phagocytic activity of macrophages., Marine Drugs, 20 (5), art. no. 313; 2022

The use of peptides as drugs has progressed over time and continues to evolve as treatment paradigms change and new drugs are developed. Myostatin (MSTN) inhibition therapy has shown great promise for the treatment of muscle wasting diseases. Here, we report the MSTN-derived novel peptides MIF1 (10-mer) and MIF2 (10-mer) not only enhance myogenesis by inhibiting MSTN and inducing myogenic-related markers but also reduce adipogenic proliferation and differentiation by suppressing the expression of adipogenic markers. MIF1 and MIF2 were designed based on in silico interaction studies between MSTN and its receptor, activin type IIB receptor (ACVRIIB), and fibromodulin (FMOD). Of the different modifications of MIF1 and MIF2 examined, Ac-MIF1 and Ac-MIF2-NH2 significantly enhanced cell proliferation and differentiation as compared with non-modified peptides. Mice pretreated with Ac-MIF1 or Ac-MIF2-NH2 prior to cardiotoxin-induced muscle injury showed more muscle regeneration than non-pretreated controls, which was attributed to the induction of myogenic genes and reduced MSTN expression. These findings imply that Ac-MIF1 and Ac-MIF2-NH2 might be valuable therapeutic agents for the treatment of muscle-related diseases.

Ischemia-reperfusion injury (IRI) is a critical condition associated with serious clinical manifestations. Extensive research has focused on the strategies increasing organ tolerance to IRI. Preconditioning (PC) has been shown to provide protection to various organs toward IRI. However, the underlying mechanisms remain unknown. This study aimed to evaluate the role of PC on muscle regeneration after IRI and the potential underlying mechanisms. Three-month-old male UCP-1 reporter mice underwent unilateral hindlimb IRI with or without PC, the tissue viability and injury index were measured at 24 h after IRI. Hindlimb gait, muscle contractility, muscle histology were analyzed at 2 weeks after IRI. In another group of animals, β3 adrenergic receptor (β3AR) agonist amibegron and β3AR antagonist SR-59230A were administrated before PC/IRI, the hindlimb function and muscle regeneration were evaluated at 2 weeks after IRI. Our results showed that PC has little effect on improving the tissue viability at the acute phase of IRI, but it showed a long-term beneficial role of improving hindlimb function and muscle regeneration as evidenced by increased central nuclei regenerating myofibers. The effects of PC are related to inducing muscle fibro-adipogenic progenitor (FAP) brown/beige-like adipocyte (BAT) differentiation. Amibegron treatment displayed a similar role of PC while SR-59230A abolished the effect of PC. This study suggests PC has a beneficial role in promoting muscle regeneration after IRI through β3AR signaling pathway-stimulated FAP-BAT differentiation.

Background With organismal aging, the hypothalamic–pituitary–gonadal (HPG) activity gradually decreases, resulting in the systemic functional declines of the target tissues including skeletal muscles. Although the HPG axis plays an important role in health span, how the HPG axis systemically prevents functional aging is largely unknown. Methods We generated muscle stem cell (MuSC)‐specific androgen receptor (Ar) and oestrogen receptor 2 (Esr2) double knockout (dKO) mice and pharmacologically inhibited (Antide) the HPG axis to mimic decreased serum levels of sex steroid hormones in aged mice. After short‐term and long‐term sex hormone signalling ablation, the MuSCs were functionally analysed, and their aging phenotypes were compared with those of geriatric mice (30‐month‐old). To investigate pathways associated with sex hormone signalling disruption, RNA sequencing and bioinformatic analyses were performed. Results Disrupting the HPG axis results in impaired muscle regeneration [wild‐type (WT) vs. dKO, P

The aim of the present study was to evaluate the effects of photobiomodulation (low-level laser therapy (LLLT)) and aquatic exercise on the expression of genes related to muscle regeneration in rats. Wistar rats were divided into five groups: control group (n = 15), non-treated injury group (n = 15), injury+LLLT group (n = 15), injury+aquatic exercise group (n = 15), and injury+LLLT+aquatic exercise group (n = 15). Cryoinjury was performed on the belly of the tibialis anterior (TA) muscle. LLLT was performed daily with an AlGaAs laser (830 nm; beam spot of 0.0324 cm2, output power of 100 mW, energy density of 180 J/cm2, and 58-s exposure time). Animals were euthanized at 7, 14, and 21 days. The TA muscles were removed for gene expression analysis of TGF-β, Myogenin, and MyoD. The results were statistically analyzed at a significance level of 5%. The cryoinjury increased the expression of genes related to muscle regeneration—MyoD, Myogenin, and TGF-β—compared to the control group (p < 0.05); the photobiomodulation increased the expression of these genes at day 7 (p < 0.05), decreasing until day 21; and the aquatic exercise increases the expression of the three genes over time. When the two treatments were combined, the expression of the analyzed genes also increased over time. In summary, the results of our study suggest that photobiomodulation (LLLT), when applied alone in cryoinjury, is able to increase the gene expression of MyoD, Myogenin, and TGF-β at the acute phase, while when combined with aquatic exercises, there is an increase in expression of these genes specially at the long-term treatment.

The ability of skeletal muscle to regenerate declines significantly with aging. The expression of aryl hydrocarbon receptor nuclear translocator (ARNT), a critical component of the hypoxia signaling pathway, was less abundant in skeletal muscle of old (23‐25 months old) mice. This loss of ARNT was associated with decreased levels of Notch1 intracellular domain (N1ICD) and impaired regenerative response to injury in comparison to young (2‐3 months old) mice. Knockdown of ARNT in a primary muscle cell line impaired differentiation in vitro. Skeletal muscle‐specific ARNT deletion in young mice resulted in decreased levels of whole muscle N1ICD and limited muscle regeneration. Administration of a systemic hypoxia pathway activator (ML228), which simulates the actions of ARNT, rescued skeletal muscle regeneration in both old and ARNT‐deleted mice. These results suggest that the loss of ARNT in skeletal muscle is partially responsible for diminished myogenic potential in aging and activation of hypoxia signaling holds promise for rescuing regenerative activity in old muscle.

Summary Muscle satellite cells are normally quiescent but are rapidly activated following muscle damage. Here, we investigated whether damaged myofibers influence the activation of satellite cells. Our findings revealed that satellite cells are directly activated by damaged-myofiber-derived factors (DMDFs). DMDFs induced satellite cells to enter the cell cycle; however, the cells stayed at the G1 phase and did not undergo S phase, and these cells were reversible to the quiescent-like state. Proteome analysis identified metabolic enzymes, including GAPDH, as DMDFs, whose recombinant proteins stimulated the activation of satellite cells. Satellite cells pre-exposed to the DMDFs demonstrated accelerated proliferation ex vivo. Treatment with recombinant GAPDH prior to muscle injury promoted expansion of the satellite cell population in vivo. Thus, our results indicate that DMDFs are not only a set of biomarkers for muscle damage, but also act as moonlighting proteins involved in satellite cell activation at the initial step of muscle regeneration., Graphical Abstract, Highlights • Damaged-myofiber-derived factors (DMDFs) activate satellite cells • DMDFs induce activation, but not proliferation, of satellite cells • Satellite cells activated by DMDFs are reversible to the quiescent-like state • DMDFs play an important role in the initial step of muscle regeneration, In this article, Tsuchiya et al. demonstrate that muscle satellite cells are directly activated by damaged-myofiber-derived factors (DMDFs). DMDFs promote population expansion of satellite cells during muscle regeneration. DMDFs are not only a set of biomarkers for muscle damage, but also act as functional proteins involved in satellite cell activation at the initial step of muscle regeneration.

A central challenge in tissue engineering is obtaining a suitable cell type with a capable delivery vehicle to replace or repair damaged or diseased tissues with tissue mimics. Notably, for skeletal muscle tissue engineering, given the inadequate availability and regenerative capability of endogenous myogenic progenitor cells as well as the tumorigenic risks presented by the currently available pluri- and multipotent stem cells, seeking a safe regenerative cell source is urgently demanded. To conquer this problem, we previously established a novel reprogramming technology that can generate multipotent cells from dermal fibroblasts using a single protein, fibromodulin (FMOD). The yield FMOD-reprogrammed (FReP) cells exhibit exceeding myogenic capability without tumorigenic risk, making them a promising and safe cell source for skeletal muscle establishment. In addition to using the optimal cell for implantation, it is equally essential to maintain cellular localization and retention in the recipient tissue environment for critical-sized muscle tissue establishment. In this study, we demonstrate that the photopolymerizable methacrylated glycol chitosan (MeGC)/type I collagen (ColI)-hydrogel provides a desirable microenvironment for encapsulated FReP cell survival, spreading, extension, and formation of myotubes in the hydrogel three-dimensionally in vitro, without undesired osteogenic, chondrogenic, or tenogenic differentiation. Furthermore, gene profiling revealed a paired box 7 (PAX7) → myogenic factor 5 (MYF5) → myogenic determination 1 (MYOD1) → myogenin (MYOG) → myosin cassette elevation in the encapsulated FReP cells during myogenic differentiation, which is similar to that of the predominant driver of endogenous skeletal muscle regeneration, satellite cells. These findings constitute the evidence that the FReP cell-MeGC/ColI-hydrogel construct is a promising tissue engineering mimic for skeletal muscle generation in vitro, and thus possesses the extraordinary potential for further in vivo validation. IMPACT STATEMENT: The present study revealed the promising potential of methacrylated glycol chitosan (MeGC)/ColI-hydrogel-encapsulated fibromodulin-reprogrammed (FReP) cells for skeletal muscle tissue engineering. Moreover, the excellent biocompatibility of MeGC/ColI-hydrogel with FReP cells—a novel induced multipotent cell type that is generated through innovative protein-based technology—makes it a suitable cell delivery vehicle for FReP cell-based tissue engineering. The development of various photopolymerizable MeGC/ColI-hydrogel/FReP cell constructs may also shift the paradigm of regenerative medicine into a feasible, safe, and efficient procedure with controllable in situ cell delivery.

Estrogens are female sex hormones that are important for comprehensively maintaining muscle function, and an insufficiency affects muscle strength and regeneration in females. However, it is still unclear whether estrogen signaling is mediated through receptors. To investigate the specific role of estrogen receptor β (ERβ) in skeletal muscle and satellite cells (muscle stem cells), we generated muscle-specific ERβ-knockout (mKO) and satellite cell-specific ERβ-knockout (scKO) mice, respectively. Young female mKO mice displayed a decrease in fast-type dominant muscle mass. Female, but not male, scKO mice exhibited impaired muscle regeneration following acute muscle injury, probably due to reduced proliferation and increased apoptosis of satellite cells. RNA-sequencing analysis revealed that loss of ERβ in satellite cells altered gene expression of extracellular matrix components, including laminin and collagen. The results indicate that the estrogen-ERβ pathway is a sex-specific regulatory mechanism that controls muscle growth and regeneration in female mice., 長崎大学学位論文 学位記番号:博(医歯薬)甲第1268号 学位授与年月日:令和2年9月2日, Author: Daiki Seko, Ryo Fujita, Yuriko Kitajima, Kodai Nakamura, Yuuki Imai, Yusuke Ono, Citation: Stem Cell Reports, 15(3), pp.577-586; 2020

Skeletal muscle injuries occur often in athletics and in daily life. In minor injuries, muscles are able to regenerate completely and recover their functional capabilities. However, in the case of severe injuries, the injured muscle cannot recover to a functional level because of the formation of fibrous scar tissue. The physical barrier of scars is significantly challenged in both research and clinical treatment. Fibrous scar tissue not only limits cells’ migration, but also contributes to normal tissue biomechanical properties. This scar formation creates an unsuitable environment for tissue structure resulting in frequent pain. Antifibrosis treatment is one of the major strategies used to augment muscle regeneration and accelerate its functional recovery. This review will discuss the currently available methods for improving muscle regeneration with a specific focus on antifibrosis applications. We also discussed several novel hypotheses and clinical applications in muscle fibrosis treatment currently in practice.

Summary Estrogens are female sex hormones that are important for comprehensively maintaining muscle function, and an insufficiency affects muscle strength and regeneration in females. However, it is still unclear whether estrogen signaling is mediated through receptors. To investigate the specific role of estrogen receptor β (ERβ) in skeletal muscle and satellite cells (muscle stem cells), we generated muscle-specific ERβ-knockout (mKO) and satellite cell-specific ERβ-knockout (scKO) mice, respectively. Young female mKO mice displayed a decrease in fast-type dominant muscle mass. Female, but not male, scKO mice exhibited impaired muscle regeneration following acute muscle injury, probably due to reduced proliferation and increased apoptosis of satellite cells. RNA-sequencing analysis revealed that loss of ERβ in satellite cells altered gene expression of extracellular matrix components, including laminin and collagen. The results indicate that the estrogen-ERβ pathway is a sex-specific regulatory mechanism that controls muscle growth and regeneration in female mice., Graphical Abstract, Highlights • ERβ controls muscle growth in young female mice • ERβ is essential for muscle regeneration in female mice • Inactivation of ERβ causes an increase in apoptosis • ERβ is required for satellite cell population expansion, In this article, Seko et al. demonstrate that ERβ is a female-specific regulator of muscle growth and regeneration. Muscle-specific ERβ-deleted young female mice exhibited a decrease in muscle mass, while satellite cell-specific ERβ inactivation resulted in impaired muscle regeneration following acute muscle injury in female mice due to reduced proliferation and increased apoptosis of satellite cells.

Rationale: The adult skeletal muscle can self-repair efficiently following mechanical or pathological damage due to its remarkable regenerative capacity. However, regulatory mechanisms underlying muscle regeneration are complicated and have not been fully elucidated. Alternative splicing (AS) is a major mechanism responsible for post-transcriptional regulation. Many aberrant AS events have been identified in patients with muscular dystrophy which is accompanied by abnormal muscle regeneration. However, little is known about the correlation between AS and muscle regeneration. It has been reported that RNA binding motif protein 24 (Rbm24), a tissue-specific splicing factor, is involved in embryo myogenesis while the role of Rbm24 in adult myogenesis (also called muscle regeneration) is poorly understood. Methods: To investigate the role of Rbm24 in adult skeletal muscle, we generated Rbm24 conditional knockout mice and satellite cell-specific knockout mice. Furthermore, a cardiotoxin (CTX)-induced injury model was utilized to assess the effects of Rbm24 on skeletal muscle regeneration. Genome-wide RNA-Seq was performed to identify the changes in AS following loss of Rbm24. Results: Rbm24 knockout mice displayed abnormal regeneration 4 months after tamoxifen treatment. Using RNA-Seq, we found that Rbm24 regulated a complex network of AS events involved in multiple biological processes, including myogenesis, muscle regeneration and muscle hypertrophy. Moreover, using a CTX-induced injury model, we showed that loss of Rbm24 in skeletal muscle resulted in myogenic fusion and differentiation defects and significantly delayed muscle regeneration. Furthermore, satellite cell-specific Rbm24 knockout mice recapitulated the defects in regeneration seen in the global Rbm24 knockout mice. Importantly, we demonstrated that Rbm24 regulated AS of Mef2d, Naca, Rock2 and Lrrfip1 which are essential for myogenic differentiation and muscle regeneration. Conclusions: The present study demonstrated that Rbm24 regulates dynamic changes in AS and is essential for adult skeletal muscle regeneration.

Background Perturbation in cell adhesion and growth factor signalling in satellite cells results in decreased muscle regenerative capacity. Cdon (also called Cdo) is a component of cell adhesion complexes implicated in myogenic differentiation, but its role in muscle regeneration remains to be determined. Methods We generated inducible satellite cell‐specific Cdon ablation in mice by utilizing a conditional Cdon allele and Pax7 CreERT2. To induce Cdon ablation, mice were intraperitoneally injected with tamoxifen (tmx). Using cardiotoxin‐induced muscle injury, the effect of Cdon depletion on satellite cell function was examined by histochemistry, immunostaining, and 5‐ethynyl‐2'‐deoxyuridine (EdU) incorporation assay. Isolated myofibers or myoblasts were utilized to determine stem cell function and senescence. To determine pathways related to Cdon deletion, injured muscles were subjected to RNA sequencing analysis. Results Satellite cell‐specific Cdon ablation causes impaired muscle regeneration with fibrosis, likely attributable to decreased proliferation, and senescence, of satellite cells. Cultured Cdon‐depleted myofibers exhibited 32 ± 9.6% of EdU‐positive satellite cells compared with 58 ± 4.4% satellite cells in control myofibers (P < 0.05). About 32.5 ± 3.7% Cdon‐ablated myoblasts were positive for senescence‐associated β‐galactosidase (SA‐β‐gal) while only 3.6 ± 0.5% of control satellite cells were positive (P < 0.001). Transcriptome analysis of muscles at post‐injury Day 4 revealed alterations in genes related to mitogen‐activated protein kinase signalling (P < 8.29 e−5) and extracellular matrix (P < 2.65 e−24). Consistent with this, Cdon‐depleted tibialis anterior muscles had reduced phosphorylated extracellular signal‐regulated kinase (p‐ERK) protein levels and expression of ERK targets, such as Fos (0.23‐fold) and Egr1 (0.31‐fold), relative to mock‐treated control muscles (P < 0.001). Cdon‐depleted myoblasts exhibited impaired ERK activation in response to basic fibroblast growth factor. Cdon ablation resulted in decreased and/or mislocalized integrin β1 activation in satellite cells (weak or mislocalized integrin1 in tmx = 38.7 ± 1.9%, mock = 21.5 ± 6%, P < 0.05), previously linked with reduced fibroblast growth factor (FGF) responsiveness in aged satellite cells. In mechanistic studies, Cdon interacted with and regulated cell surface localization of FGFR1 and FGFR4, likely contributing to FGF responsiveness of satellite cells. Satellite cells from a progeria model, Zmpste24 −/− myofibers, showed decreased Cdon levels (Cdon‐positive cells in Zmpste24−/− = 63.3 ± 11%, wild type = 90 ± 7.7%, P < 0.05) and integrin β1 activation (weak or mislocalized integrin β1 in Zmpste24−/− = 64 ± 6.9%, wild type = 17.4 ± 5.9%, P < 0.01). Conclusions Cdon deficiency in satellite cells causes impaired proliferation of satellite cells and muscle regeneration via aberrant integrin and FGFR signalling.

BACKGROUND Impaired muscle regeneration and increased muscle fibrosis are observed in aged muscle accompanied by progressive loss of muscle mass (sarcopenia). However, the underlying mechanism is still unclear. METHODS The differentiated expressed genes in young and aged muscles after acute injury by cardiotoxin were identified by RNA-sequence analysis. Single-cell RNA-sequence analysis was used to identify cell clusters and functions in young muscle after acute injury, and flow cytometry analysis and sorting were used to validate the function. The proliferation and differentiation functions of satellite cells were accessed by immunostaining with 5-ethynyl-2'-deoxyuridine and embryonic myosin heavy chain (eMyHC), respectively. Muscle regeneration ability was accessed by histopathological and molecular biological methods. RESULTS Gene expression patterns associated with responses to interferon-gamma (IFN-γ) (15 genes; false discovery rate

To improve muscle healing upon injury, it is of importance to understand the interplay of key signaling pathways during muscle regeneration. To study this, mice were injected with cardiotoxin (CTX) or PBS in the Tibialis Anterior muscle and were sacrificed 2, 5 and 12 days upon injection. The time points represent different phases of the regeneration process, i.e. destruction, repair and remodeling, respectively. Two days upon CTX-injection, p-mTORC1 signaling and stress markers such as BiP and p-ERK1/2 were upregulated. Phospho-ERK1/2 and p-mTORC1 peaked at d5, while BiP expression decreased towards PBS levels. Phospho-FOXO decreased 2 and 5 days following CTX-injection, indicative of an increase in catabolic signaling. Furthermore, CTX-injection induced a shift in the fiber type composition, characterized by an initial loss in type IIa fibers at d2 and at d5. At d5, new type IIb fibers appeared, whereas type IIa fibers were recovered at d12. To conclude, CTX-injection severely affected key modulators of muscle metabolism and histology. These data provide useful information for the development of strategies that aim to improve muscle molecular signaling and thereby recovery. ispartof: Journal Of Muscle Research And Cell Motility vol:41 issue:4 pages:375-387 ispartof: location:Netherlands status: published

Macroautophagy/autophagy induction, i.e., the formation of autophagosomes, is robust following many forms of muscle injury. Autophagy inhibition studies strongly indicate that autophagy is necessary for successful muscle fiber recovery. Now, there are accumulating pieces of evidence indicating that autophagosome clearance, i.e., autophagy flux, does not increase to match the burden of accumulating damaged proteins and organelles after muscle fiber damage, creating a bottleneck effect. Some potential consequences of the bottleneck effect are reduced regenerative capacity marked by the inadequate activation of muscle stem cells (i.e., satellite cells) and a lesser commitment toward differentiation due to a deficiency in energetic substrates and/or molecular signaling pathways. These findings highlight an emerging area of investigation for both autophagy and muscle regeneration fields. The identification of the molecular mechanisms governing autophagy and autophagy flux may serve as targets for future therapies to enhance the recovery of its function in healthy and diseased muscle. ABBREVIATIONS: BNIP3: BCL2/adenovirus E1B interacting protein 3; CQ: chloroquine; DMD: Duchenne muscular dystrophy; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; ULK1: unc-51 like kinase 1.

Trichinella spiralis infection is capable of manipulating the human immunological system through modulation of cells and molecules of the host`s immune system. There are different mechanisms of T. spiralis for changing the balance of immunological response to be benefit for the parasite and host. Moreover, there is similarity in-between the response of infected muscle cells at early stage of infection and muscle regeneration. As a result, T. spiralis may have therapeutic effects in many autoimmune and degenerative diseases.

BACKGROUND: Anorexia, body wasting, inflammation, muscle, and adipose tissue loss are hallmarks of cancer cachexia, a syndrome that affects the majority of cancer patients, impairing their ability to endure chemotherapeutic therapies and reducing their lifespan. In the last 10 years, alterations of protein turnover and impairment of adult myogenesis have been proposed as major contributing factors. METHODS: Muscle stem cells, including satellite cells, mesoangioblasts, and fibroadipogenic progenitors, were isolated and characterized from C26 colon carcinoma-bearing (C26) mice. Circulating levels of interleukin-4/13 (IL4/IL13) were analysed by ELISA, and the effects of IL4 on muscle mass and function, protein synthesis, muscle regeneration, and myogenic progenitor cell number were analysed at both functional (treadmill and grip test) and molecular levels (qRT-PCR, immunofluorescence analysis, surface sensing of translation, and western blot). The Kaplan-Meier test was used to analyse the survival curve of IL4-treated and IL4-untreated C26 mice. RESULTS: The administration of IL4 to C26 mice rescued muscle mass by increasing protein synthesis. The IL4 treatment improved performances and prolonged survival of C26 mice. IL4 administration re-established both number and function of satellite cells and fibroadipogenic progenitors without affecting mesoangioblasts in C26 mice, rescuing myogenesis. Upon IL4 treatment, a high number of cytotoxic lymphocytes and type II macrophages were observed with a subsequent increase in necrotic areas of C26 tumours. CONCLUSIONS: The results here presented shed new light on IL4 signalling during muscle wasting and early stages of muscle regeneration that explain the beneficial effect observed in IL4-treated C26 mice. These findings might aid to develop therapeutic approaches to improve mobility and quality of life in cachectic patients. ispartof: Journal of Cachexia Sarcopenia and Muscle vol:11 issue:3 pages:783-801 ispartof: location:Germany status: Published online

The loss of skeletal muscle mass and function is a serious consequence of chronic diseases and aging. BST204 is a purified ginseng (the root of Panax ginseng) extract that has been processed using ginsenoside-[Formula: see text]-glucosidase and acid hydrolysis to enrich ginsenosides Rg3 and Rh2 from the crude ginseng. BST204 has a broad range of health benefits, but its effects and mechanism on muscle atrophy are currently unknown. In this study, we have examined the effects and underlying mechanisms of BST204 on myotube formation and myotube atrophy induced by tumor necrosis factor-[Formula: see text] (TNF-[Formula: see text]). BST204 promotes myogenic differentiation and multinucleated myotube formation through Akt activation. BST204 prevents myotube atrophy induced by TNF-[Formula: see text] through the activation of Akt/mTOR signaling and down-regulation of muscle-specific ubiquitin ligases, MuRF1, and Atrogin-1. Furthermore, BST204 treatment in atrophic myotubes suppresses mitochondrial reactive oxygen species (ROS) production and regulates mitochondrial transcription factors such as NRF1 and Tfam, through enhancing the activity and expression of peroxisome proliferator-activated receptor-[Formula: see text] coactivator1[Formula: see text] (PGC1[Formula: see text]). Collectively, our findings indicate that BST204 improves myotube formation and PGC1[Formula: see text]-mediated mitochondrial function, suggesting that BST204 is a potential therapeutic or neutraceutical remedy to intervene muscle weakness and atrophy.