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Skeletal muscle ischemia-reperfusion (IR) injury poses significant challenges due to its local and systemic complications. Traditional studies relying on two-dimensional (2D) cell culture or animal models often fall short of faithfully replicating the human in vivo environment, thereby impeding the translational process from animal research to clinical applications. Three-dimensional (3D) constructs, such as skeletal muscle spheroids with enhanced cell-cell interactions from human pluripotent stem cells (hPSCs) offer a promising alternative by partially mimicking human physiological cellular environment in vivo processes. This study aims to establish an innovative in vitro model, human skeletal muscle spheroids based on sphere differentiation from hPSCs, to investigate human skeletal muscle developmental processes and IR mechanisms within a controlled laboratory setting. By eticulously recapitulating embryonic myogenesis through paraxial mesodermal differentiation of neuro-mesodermal progenitors, we successfully established 3D skeletal muscle spheroids that mirror the dynamic colonization observed during human skeletal muscle development. Co-culturing human skeletal muscle spheroids with spinal cord spheroids facilitated the formation of neuromuscular junctions, providing functional relevance to skeletal muscle spheroids. Furthermore, through oxygen-glucose deprivation/re-oxygenation treatment, 3D skeletal muscle spheroids provide insights into the molecular events and pathogenesis of IR injury. The findings presented in this study significantly contribute to our understanding of skeletal muscle development and offer a robust platform for in vitro studies on skeletal muscle IR injury, holding potential applications in drug testing, therapeutic development, and personalized medicine within the realm of skeletal muscle-related pathologies., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Age-related reduction in muscle stem cell (MuSC) regenerative capacity is associated with cell-autonomous and non-cell-autonomous changes caused by alterations in systemic and skeletal muscle environments, ultimately leading to a decline in MuSC number and function. Previous studies demonstrated that STAT3 plays a key role in driving MuSC expansion and differentiation after injury-activated regeneration, by regulating autophagy in activated MuSCs. However, autophagy gradually declines in MuSCs during lifespan and contributes to the impairment of MuSC-mediated regeneration of aged muscles. Here, we show that STAT3 inhibition restores the autophagic process in aged MuSCs, thereby recovering MuSC ability to promote muscle regeneration in geriatric mice. We show that STAT3 inhibition could activate autophagy at the nuclear level, by promoting transcription of autophagy-related genes, and at the cytoplasmic level, by targeting STAT3/PKR phosphorylation of eIF2α. These results point to STAT3 inhibition as a potential intervention to reverse the age-related autophagic block that impairs MuSC ability to regenerate aged muscles. They also reveal that STAT3 regulates MuSC function by both transcription-dependent and transcription-independent regulation of autophagy., (© 2024 Catarinella et al.)

Excessive accumulation of intermuscular adipose tissue (IMAT) is a common pathological feature in various metabolic and health conditions and can cause muscle atrophy, reduced function, inflammation, insulin resistance, cardiovascular issues, and unhealthy aging. Although IMAT results from fat accumulation in muscle, the mechanisms underlying its onset, development, cellular components, and functions remain unclear. IMAT levels are influenced by several factors, such as changes in the tissue environment, muscle type and origin, extent and duration of trauma, and persistent activation of fibro-adipogenic progenitors (FAPs). FAPs are a diverse and transcriptionally heterogeneous population of stromal cells essential for tissue maintenance, neuromuscular stability, and tissue regeneration. However, in cases of chronic inflammation and pathological conditions, FAPs expand and differentiate into adipocytes, resulting in the development of abnormal and ectopic IMAT. This review discusses the role of FAPs in adipogenesis and how they remodel IMAT. It highlights evidence supporting FAPs and FAP-derived adipocytes as constituents of IMAT, emphasizing their significance in adipose tissue maintenance and development, as well as their involvement in metabolic disorders, chronic pathologies and diseases. We also investigated the intricate molecular pathways and cell interactions governing FAP behavior, adipogenesis, and IMAT accumulation in chronic diseases and muscle deconditioning. Finally, we hypothesize that impaired cellular metabolic flexibility in dysfunctional muscles impacts FAPs, leading to IMAT. A deeper understanding of the biology of IMAT accumulation and the mechanisms regulating FAP behavior and fate are essential for the development of new therapeutic strategies for several debilitating conditions., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Adult stem cells occupy a niche that contributes to their function, but how stem cells rebuild their microenvironment after injury remains an open-ended question. Herein, biomaterial-based systems and metabolic labeling are utilized to evaluate how skeletal muscle stem cells deposit extracellular matrix. Muscle stem cells and committed myoblasts are observed to generate less nascent matrix than muscle resident fibro-adipogenic progenitors. When cultured on substrates that matched the stiffness of physiological uninjured and injured muscles, muscle stem cells increased nascent matrix deposition with activation kinetics. Reducing the ability to deposit nascent matrix by an inhibitor of vesicle trafficking (Exo-1) attenuated muscle stem cell function and mimicked impairments observed from muscle stem cells isolated from old muscles. Old muscle stem cells are observed to deposit less nascent matrix than young muscle stem cells, which is rescued with therapeutic supplementation of insulin-like growth factors. These results highlight the role of nascent matrix production with muscle stem cell activation., (© 2024 The Authors. Advanced Biology published by Wiley‐VCH GmbH.)

Cellular Communication Network Factor 2, CCN2, is a profibrotic cytokine implicated in physiological and pathological processes in mammals. The expression of CCN2 is markedly increased in dystrophic muscles. Interestingly, diminishing CCN2 genetically or inhibiting its function improves the phenotypes of chronic muscular fibrosis in rodent models. Elucidating the cell-specific mechanisms behind the induction of CCN2 is a fundamental step in understanding its relevance in muscular dystrophies. Here, we show that the small lipids LPA and 2S-OMPT induce CCN2 expression in fibro/adipogenic progenitors (FAPs) through the activation of the LPA 1 receptor and, to a lower extent, by also the LPA 6 receptor. These cells show a stronger induction than myoblasts or myotubes. We show that the LPA/LPARs axis requires ROCK kinase activity and organized actin cytoskeleton upstream of YAP/TAZ signaling effectors to upregulate CCN2 levels, suggesting that mechanical signals are part of the mechanism behind this process. In conclusion, we explored the role of the LPA/LPAR axis on CCN2 expression, showing a strong cytoskeletal-dependent response in muscular FAPs., Competing Interests: Declaration of competing interest Dr. Chun has an employment relationship with Neurocrine Biosciences, Inc., a company that may potentially benefit from the research results. Dr. Chun's relationship with Neurocrine Biosciences, Inc. has been reviewed and approved by Sanford Burnham Prebys Medical Discovery Institute in accordance with its Conflict-of-Interest Policies. The other authors declare no competing or financial interests., (Copyright © 2024. Published by Elsevier B.V.)

Background: Different experiments require different sample storage methods. The commonly used preservation methods in biobank practice cannot fully meet the multifarious requirements of experimental techniques. Programmable controlled slow freezing (PCSF) can maintain the viability of tissue. In this study, we hypothesized that PCSF-preserved samples have potential advantages in matching subsequent experiments compared with existing methods. Methods: We compared the differences on skeletal muscle tissue RNA integrity, protein integrity, microstructure integrity, and cell viability between four existing cryopreservation methods: liquid nitrogen (LN 2 ) snap-freezing, LN 2 -cooled isopentane snap-freezing, RNAlater ® -based freezing, and PCSF. RNA integrity was evaluated using agarose gel electrophoresis and RNA integrity number. Freezing-related microstructural damage in the muscle tissue was evaluated using ice crystal diameter and muscle fiber cross-sectional area. Protein integrity was evaluated using immunofluorescence staining. Cell viability was evaluated using trypan blue staining after primary muscle cell isolation. Results: PCSF preserved RNA integrity better than LN 2 and isopentane, with a statistically significant difference. RNAlater preserved RNA integrity best. PCSF best controlled ice crystal size in myofibers, with a significant difference compared with LN 2 . The PCSF method best preserved the integrity of protein epitopes according to the mean fluorescence intensity results, with a significant difference. Cell viability was best preserved in the PCSF method compared with the other three methods, with a significant difference. Conclusion: PCSF protected the RNA integrity, microstructural integrity, protein integrity, and cell viability of skeletal muscle tissue. The application of PCSF in biobank practice is recommended as a multi-experiment-compatible cryopreservation method.

Current studies on cultured meat mainly focus on the muscle tissue reconstruction in vitro, but lack the formation of intramuscular fat, which is a crucial factor in determining taste, texture, and nutritional contents. Therefore, incorporating fat into cultured meat is of superior value. In this study, we employed the myogenic/lipogenic transdifferentiation of chicken fibroblasts in 3D to produce muscle mass and deposit fat into the same cells without the co-culture or mixture of different cells or fat substances. The immortalized chicken embryonic fibroblasts were implanted into the hydrogel scaffold, and the cell proliferation and myogenic transdifferentiation were conducted in 3D to produce the whole-cut meat mimics. Compared to 2D, cells grown in 3D matrix showed elevated myogenesis and collagen production. We further induced fat deposition in the transdifferentiated muscle cells and the triglyceride content could be manipulated to match and exceed the levels of chicken meat. The gene expression analysis indicated that both lineage-specific and multifunctional signalings could contribute to the generation of muscle/fat matrix. Overall, we were able to precisely modulate muscle, fat, and extracellular matrix contents according to balanced or specialized meat preferences. These findings provide new avenues for customized cultured meat production with desired intramuscular fat contents that can be tailored to meet the diverse demands of consumers., Competing Interests: TM, RR, JL, RY, XZ, YH, GZ, HW No competing interests declared, (© 2024, Ma, Ren, Lv et al.)

Maintenance of mitochondrial function plays a crucial role in the regulation of muscle stem cell (MuSC), but the underlying mechanisms remain ill defined. In this study, we monitored mitophagy in MuSCS under various myogenic states and examined the role of PINK1 in maintaining regenerative capacity. Results indicate that quiescent MuSCs actively express mitophagy genes and exhibit a measurable mitophagy flux and prominent mitochondrial localization to autophagolysosomes, which become rapidly decreased during activation. Genetic disruption of Pink1 in mice reduces PARKIN recruitment to mitochondria and mitophagy in quiescent MuSCs, which is accompanied by premature activation/commitment at the expense of self-renewal and progressive loss of muscle regeneration, but unhindered proliferation and differentiation capacity. Results also show that impaired fate decisions in PINK1-deficient MuSCs can be restored by scavenging excess mitochondrial ROS. These data shed light on the regulation of mitophagy in MuSCs and position PINK1 as an important regulator of their mitochondrial properties and fate decisions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cultured meat is a meat analogue produced by in vitro cell culture, which can replace the conventional animal production system. Tissue engineering using myogenic cells and biomaterials is a core technology for cultured meat production. In this study, we provide an efficient and economical method to produce skeletal muscle tissue-like structures by culturing chicken myoblasts in a fetal bovine serum (FBS)-free medium and plant-derived scaffolds. An FBS-free medium supplemented with 10% horse serum (HS) and 5% chick embryo extract (CEE) was suitable for the proliferation and differentiation of chicken myoblasts. Decellularized celery scaffolds (Decelery), manufactured using 1% sodium dodecyl sulfate (SDS), were nontoxic to cells and supported myoblast proliferation and differentiation. Decelery could support the 3D culture of chicken myoblasts, which could adhere and coagulate to the surface of the Decelery and form MYH1E + and F-actin + myotubes. After 2 weeks of culture on Decelery, fully grown myoblasts completely covered the surface of the scaffolds and formed fiber-like myotube structures. They further differentiated to form spontaneously contracting myofiber-like myotubes on the scaffold surface, indicating that the Decelery scaffold system could support the formation of a functional mature myofiber structure. In addition, as the spontaneously contracting myofibers did not detach from the surface of the Decelery, the Decelery system is a suitable biomaterial for the long-term culture and maintenance of the myofiber structures.

Glutathione peroxidase 2 (GPX2) is a selenium-dependent enzyme and protects cells against oxidative damage. Recently, GPX2 has been identified as a candidate gene for backfat and feed efficiency in pigs. However, it is unclear whether GPX2 regulates the development of porcine preadipocytes and skeletal muscle cells. In this study, adenoviral gene transfer was used to overexpress GPX2. Our findings suggest that overexpression of GPX2 gene inhibited proliferation of porcine preadipocytes. And the process is accompanied by the reduction of the p-p38. GPX2 inhibited adipogenic differentiation and promoted lipid degradation, while ERK1/2 was reduced and p-p38 was increased. Proliferation of porcine skeletal muscle cells was induced after GPX2 overexpression, was accompanied by activation in JNK, ERK1/2, and p-p38. Overexpression methods confirmed that GPX2 has a promoting function in myoblastic differentiation. ERK1/2 pathway was activated and p38 was suppressed during the process. This study lays a foundation for the functional study of GPX2 and provides theoretical support for promoting subcutaneous fat reduction and muscle growth., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Intermuscular adipose tissue (IMAT) is a relatively understudied adipose depot located between muscle fibers. IMAT content increases with age and BMI and is associated with metabolic and muscle degenerative diseases; however, an understanding of the biological properties of IMAT and its interplay with the surrounding muscle fibers is severely lacking. In recent years, single-cell and nuclei RNA sequencing have provided us with cell type-specific atlases of several human tissues. However, the cellular composition of human IMAT remains largely unexplored due to the inherent challenges of its accessibility from biopsy collection in humans. In addition to the limited amount of tissue collected, the processing of human IMAT is complicated due to its proximity to skeletal muscle tissue and fascia. The lipid-laden nature of the adipocytes makes it incompatible with single-cell isolation. Hence, single nuclei RNA sequencing is optimal for obtaining high-dimensional transcriptomics at single-cell resolution and provides the potential to uncover the biology of this depot, including the exact cellular composition of IMAT. Here, we present a detailed protocol for nuclei isolation and library preparation of frozen human IMAT for single nuclei RNA sequencing. This protocol allows for the profiling of thousands of nuclei using a droplet-based approach, thus providing the capacity to detect rare and low-abundant cell types.

Decellularization is a novel technique employed for scaffold manufacturing, as a strategy for skeletal muscle (SM) tissue engineering applications. However, poor decellularization efficacy is still a problem for the use of decellularized scaffolds as truly biocompatible biomaterials. For recellularization, adipose-derived stem cells (ASCs) are a good option, due to their immunomodulatory and pro-regenerative capacity, but few studies have described their combination with muscle-decellularized matrices (mDMs). This work aimed to evaluate the efficiency of four multi-step decellularization protocols to produce mDMs and to investigate in vitro biocompatibility with ASCs. Here, we described the different efficacies of muscle decellularization methods, suggesting the need for stricter standardization of the method, considering the large range of applications in SM tissue engineering, which is also a promising platform for preclinical studies with rat disease models using autologous cells., Competing Interests: Declaration of competing interest None., (Copyright © 2024 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Muscle atrophy and functional decline (sarcopenia) are common manifestations of frailty and are critical contributors to morbidity and mortality in older people 1 . Deciphering the molecular mechanisms underlying sarcopenia has major implications for understanding human ageing 2 . Yet, progress has been slow, partly due to the difficulties of characterizing skeletal muscle niche heterogeneity (whereby myofibres are the most abundant) and obtaining well-characterized human samples 3,4 . Here we generate a single-cell/single-nucleus transcriptomic and chromatin accessibility map of human limb skeletal muscles encompassing over 387,000 cells/nuclei from individuals aged 15 to 99 years with distinct fitness and frailty levels. We describe how cell populations change during ageing, including the emergence of new populations in older people, and the cell-specific and multicellular network features (at the transcriptomic and epigenetic levels) associated with these changes. On the basis of cross-comparison with genetic data, we also identify key elements of chromatin architecture that mark susceptibility to sarcopenia. Our study provides a basis for identifying targets in the skeletal muscle that are amenable to medical, pharmacological and lifestyle interventions in late life., (© 2024. The Author(s).)

The current geopolitical context has brought the radiological nuclear risk to the forefront of concerns. High-dose localized radiation exposure leads to the development of a musculocutaneous radiation syndrome affecting the skin and subcutaneous muscles. Despite the implementation of a gold standard treatment based on an invasive surgical procedure coupled with autologous cell therapy, a muscular defect frequently persists. Targeting the modulation of the Hedgehog (Hh) signaling pathway appears to be a promising therapeutic approach. Activation of this pathway enhances cell survival and promotes proliferation after irradiation, while inhibition by Cyclopamine facilitates differentiation. In this study, we compared the effects of three antagonists of Hh, Cyclopamine (CA), Vismodegib (VDG) and Sonidegib (SDG) on differentiation. A stable cell line of murine myoblasts, C2C12, was exposed to X-ray radiation (5 Gy) and treated with CA, VDG or SDG. Analysis of proliferation, survival (apoptosis), morphology, myogenesis genes expression and proteins production were performed. According to the results, VDG does not have a significant impact on C2C12 cells. SDG increases the expression/production of differentiation markers to a similar extent as CA, while morphologically, SDG proves to be more effective than CA. To conclude, SDG can be used in the same way as CA but already has a marketing authorization with an indication against basal cell cancers, facilitating their use in vivo. This proof of concept demonstrates that SDG represents a promising alternative to CA to promotes differentiation of murine myoblasts. Future studies on isolated and cultured satellite cells and in vivo will test this proof of concept., (©2024 by Radiation Research Society. All rights of reproduction in any form reserved.)

Mammalian skeletal myogenesis is a complex process that allows precise control of myogenic cells' proliferation, differentiation, and fusion to form multinucleated, contractile, and functional muscle fibers. Typically, myogenic progenitors continue growth and division until acquiring a differentiated state, which then permanently leaves the cell cycle and enters terminal differentiation. These processes have been intensively studied using the skeletal muscle developing models in vitro and in vivo, uncovering a complex cellular intrinsic network during mammalian skeletal myogenesis containing transcription factors, translation factors, extracellular matrix, metabolites, and mechano-sensors. Examining the events and how they are knitted together will better understand skeletal myogenesis's molecular basis. This review describes various regulatory mechanisms and recent advances in myogenic cell proliferation and differentiation during mammalian skeletal myogenesis. We focus on significant cell cycle regulators, myogenic factors, and chromatin regulators impacting the coordination of the cell proliferation versus differentiation decision, which will better clarify the complex signaling underlying skeletal myogenesis., Competing Interests: Declaration of Competing Interest The authors declared that they have no conflicts of interest to this work., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The myotendinous junction (MTJ) is a vulnerable region at the interface of skeletal muscle and tendon that forms an integrated mechanical unit. This study presents a technique for the spatially restrictive co-culture of human embryonic stem cell (hESC)-derived skeletal myocytes and primary tenocytes for two-dimensional modeling of the MTJ. Micropatterned lanes of extracellular matrix and a 2-well culture chamber define the initial regions of occupation. On day 1, both lines occupy less than 20 % of the initially vacant interstitial zone, referred to henceforth as the junction. Myocyte-tenocyte interdigitations are observed by day 7. Immunocytochemistry reveals enhanced organization and alignment of patterned myocyte and tenocyte features, as well as differential expression of multiple MTJ markers. On day 24, electrically stimulated junction myocytes demonstrate negative contractile strains, while positive tensile strains are exhibited by mechanically passive tenocytes at the junction. Unpatterned tenocytes distal to the junction experience significantly decreased strains in comparison to cells at the interface. Unpatterned myocytes have impaired organization and uncoordinated contractile behavior. These findings suggest that this platform is capable of inducing myocyte-tenocyte junction formation and mechanical coupling similar to the native MTJ, showing transduction of force across the cell-cell interface. STATEMENT OF SIGNIFICANCE: The myotendinous junction (MTJ) is an integrated structure that transduces force across the muscle-tendon boundary, making the region vulnerable to strain injury. Despite the clinical relevance, previous in vitro models of the MTJ lack the structure and mechanical accuracy of the native tissue and have difficulty transmitting force across the cell-cell interface. This study demonstrates an in vitro model of the MTJ, using spatially restrictive cues to inform human myocyte-tenocyte interactions and architecture. The model expressed MTJ markers and developed anisotropic myocyte-tenocyte integrations that resemble the native tissue and allow for force transduction from contracting myocytes to passive tenocyte regions. As such, this study presents a system capable of investigating development, injury, and pathology in the human MTJ., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Ubiquitin-conjugation enzyme E2C (UBE2C) is a crucial component of the ubiquitin-proteasome system that is involved in numerous cancers. In this study, we find that UBE2C expression is significantly increased in mouse embryos, a critical stage during skeletal muscle development. We further investigate the function of UBE2C in myogenesis. Knockdown of UBE2C inhibits C2C12 cell differentiation and decreases the expressions of MyoG and MyHC, while overexpression of UBE2C promotes C2C12 cell differentiation. Additionally, knockdown of UBE2C , specifically in the tibialis anterior muscle (TA), severely impedes muscle regeneration in vivo . Mechanistically, we show that UBE2C knockdown reduces the level of phosphorylated protein kinase B (p-Akt) and promotes the degradation of Akt. These findings suggest that UBE2C plays a critical role in myoblast differentiation and muscle regeneration and that UBE2C regulates myogenesis through the Akt signaling pathway.

Culture of muscle cells from livestock species has typically involved laborious enzyme-based approaches that yield heterogeneous populations with limited proliferative and myogenic differentiation capacity, thus limiting their use in physiologically-meaningful studies. This study reports the use of a simple explant culture technique to derive progenitor cell populations from porcine muscle that could be maintained and differentiated long-term in culture. Fragments of semitendinosus muscle from 4 to 8 week-old piglets (n = 4) were seeded on matrigel coated culture dishes to stimulate migration of muscle-derived progenitor cells (MDPCs). Cell outgrowths appeared within a few days and were serially passaged and characterised using RT-qPCR, immunostaining and flow cytometry. MDPCs had an initial mean doubling time of 1.4 days which increased to 2.5 days by passage 14. MDPC populations displayed steady levels of the lineage-specific markers, PAX7 and MYOD, up until at least passage 2 (positive immunostaining in about 40% cells for each gene), after which the expression of myogenic markers decreased gradually. Remarkably, MDPCs were able to readily generate myotubes in culture up until passage 8. Moreover, a decrease in myogenic capacity during serial passaging was concomitant with a gradual increase in the expression of the pre-adipocyte markers, CD105 and PDGFRA, and an increase in the ability of MDPCs to differentiate into adipocytes. In conclusion, explant culture provided a simple and efficient method to harvest enriched myogenic progenitors from pig skeletal muscle which could be maintained long-term and differentiated in vitro, thus providing a suitable system for studies on porcine muscle biology and applications in the expanding field of cultured meat., (© 2024. The Author(s).)

Skeletal muscle degeneration is responsible for major mobility complications, and this muscle type has little regenerative capacity. Several biomaterials have been proposed to induce muscle regeneration and function restoration. Decellularized scaffolds present biological properties that allow efficient cell culture, providing a suitable microenvironment for artificial construct development and being an alternative for in vitro muscle culture. For translational purposes, biomaterials derived from large animals are an interesting and unexplored source for muscle scaffold production. Therefore, this study aimed to produce and characterize bovine muscle scaffolds to be applied to muscle cell 3D cultures. Bovine muscle fragments were immersed in decellularizing solutions for 7 days. Decellularization efficiency, structure, composition, and three-dimensionality were evaluated. Bovine fetal myoblasts were cultured on the scaffolds for 10 days to attest cytocompatibility. Decellularization was confirmed by DAPI staining and DNA quantification. Histological and immunohistochemical analysis attested to the preservation of main ECM components. SEM analysis demonstrated that the 3D structure was maintained. In addition, after 10 days, fetal myoblasts were able to adhere and proliferate on the scaffolds, attesting to their cytocompatibility. These data, even preliminary, infer that generated bovine muscular scaffolds were well structured, with preserved composition and allowed cell culture. This study demonstrated that biomaterials derived from bovine muscle could be used in tissue engineering.

During embryogenesis, basic fibroblast growth factor (bFGF) is released from neural tube and myotome to promote myogenic fate in the somite, and is routinely used for the culture of adult skeletal muscle (SKM) stem cells (MuSC, called satellite cells). However, the mechanism employed by bFGF to promote SKM lineage and MuSC proliferation has not been analyzed in detail. Furthermore, the question of if the post-translational modification (PTM) of bFGF is important to its stemness-promoting effect has not been answered. In this study, GST-bFGF was expressed and purified from E.coli , which lacks the PTM system in eukaryotes. We found that both GST-bFGF and commercially available bFGF activated the Akt-Erk pathway and had strong cell proliferation effect on C2C12 myoblasts and MuSC. GST-bFGF reversibly compromised the myogenesis of C2C12 myoblasts and MuSC, and it increased the expression of Myf5 , Pax3/7 , and Cyclin D1 but strongly repressed that of MyoD , suggesting the maintenance of myogenic stemness amid repressed MyoD expression. The proliferation effect of GST-bFGF was conserved in C2C12 over-expressed with MyoD (C2C12-tTA-MyoD), implying its independence of the down-regulation of MyoD . In addition, the repressive effect of GST-bFGF on myogenic differentiation was almost totally rescued by the over-expression of MyoD . Together, these evidences suggest that (1) GST-bFGF and bFGF have similar effects on myogenic cell proliferation and differentiation, and (2) GST-bFGF can promote MuSC stemness and proliferation by differentially regulating MRFs and Pax3/7, (3) MyoD repression by GST-bFGF is reversible and independent of the proliferation effect, and (4) GST-bFGF can be a good substitute for bFGF in sustaining MuSC stemness and proliferation.

Sarcopenia is a common complication of chronic kidney disease (CKD) and has a detrimental effect on prognosis. Previous studies have explored the role of secondary calciprotein particles (CPP2) in determining the progression of complications and poor outcomes in patients with CKD. However, no study has demonstrated that CPP2 impairs skeletal myogenesis. Our study revealed that CPP2 exposure inhibits skeletal myogenesis by suppressing myotube formation and expression of skeletal muscle-specific myosin heavy chain and actin in human primary myoblasts. Moreover, CPP2 exposure altered the expression patterns of lineage-determinative transcription factors responsible for regulating myotube differentiation marker genes. This study first demonstrated that CPP2 interferes with myoblast differentiation and myotube formation in vitro.

Skeletal muscle is one of the largest tissues in human body. Besides enabling voluntary movements and maintaining body's metabolic homeostasis, skeletal muscle is also a target of many pathological conditions. Mitochondria occupy 10-15% volume of a muscle myofiber and regulate many cellular processes, which often determine the fate of the cell. Isolation of mitochondria from skeletal muscle provides opportunities for various multi-omics studies with a focus on mitochondria in biomedical research field. Here we describe a protocol to efficiently isolate mitochondria with high quality and purity from skeletal muscle of mice using Nycodenz density gradient ultracentrifugation., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Embryonic skeletal muscle growth is contingent upon a population of somite derived satellite cells, however, the contribution of these cells to early postnatal skeletal muscle growth remains relatively high. As prepubertal postnatal development proceeds, the activity and contribution of satellite cells to skeletal muscle growth diminishes. Eventually, at around puberty, a population of satellite cells escapes terminal commitment, continues to express the paired box transcription factor Pax7, and reside in a quiescent state orbiting the myofiber periphery adjacent to the basal lamina. After adolescence, some satellite cell contributions to muscle maintenance and adaptation occur, however, their necessity is reduced relative to embryonic, early postnatal, and prepubertal growth., (Copyright © 2024. Published by Elsevier Inc.)

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., (Copyright © 2024. Published by Elsevier Inc.)

Adult muscle tissue largely comprised of differentiated myofibers also harbors quiescent muscle-resident stem cells (MuSCs) that are responsible for its maintenance, repair and regeneration. Emerging evidence suggests that quiescent MuSCs exhibit a specific metabolic state, which is regulated during physiological and pathological alterations. However, a detailed understanding of the metabolic state of quiescent MuSCs and its alteration during activation and repair is lacking. Direct profiling of MuSCs in vivo is challenging because the cells are rare and dispersed, while isolation and enrichment leads to their activation and loss of quiescence. In this study, we employed 1H-nuclear magnetic resonance (NMR) spectroscopy to profile metabolites in an established culture model of quiescent MuSC-derived myoblasts and compared with activated, proliferative and differentiated muscle cells to determine the state-specific metabolome. We report that the proliferating and differentiated cells are highly enriched in metabolites involved in energy generation, the quiescent state is enriched in metabolites related to phospholipid catabolism (glycerophosphocholine and choline) and depleted for phosphocholine which is enriched in proliferating cells. We propose that the ratio of these metabolites may be useful as a biomarker of MuSC quiescence.

Skeletal muscle is composed of a variety of tissue and non-tissue resident cells that participate in homeostasis. In particular, the muscle stem cell niche is a dynamic system, requiring direct and indirect communications between cells, involving local and remote cues. Interactions within the niche must happen in a timely manner for the maintenance or recovery of the homeostatic niche. For instance, after an injury, pro-myogenic cues delivered too early will impact on muscle stem cell proliferation, delaying the repair process. Within the niche, myofibers, endothelial cells, perivascular cells (pericytes, smooth muscle cells), fibro-adipogenic progenitors, fibroblasts, and immune cells are in close proximity with each other. Each cell behavior, membrane profile, and secretome can interfere with muscle stem cell fate and skeletal muscle regeneration. On top of that, the muscle stem cell niche can also be modified by extra-muscle (remote) cues, as other tissues may act on muscle regeneration via the production of circulating factors or the delivery of cells. In this review, we highlight recent publications evidencing both local and remote effectors of the muscle stem cell niche., (Copyright © 2024. Published by Elsevier Inc.)

Preserving the potency of stem cells in adult tissues is very demanding and relies on the concerted action of various cellular and non-cellular elements in a precise stoichiometry. This balanced microenvironment is found in specific anatomical "pockets" within the tissue, known as the stem cell niche. In this review, we explore the interplay between stem cells and their niches, with a primary focus on skeletal muscle stem cells and the extracellular matrix (ECM). Quiescent muscle stem cells, known as satellite cells are active producers of a diverse array of ECM molecules, encompassing major constituents like collagens, laminins, and integrins, some of which are explored in this review. The conventional perception of ECM as merely a structural scaffold is evolving. Collagens can directly interact as ligands with receptors on satellite cells, while other ECM proteins have the capacity to sequester growth factors and regulate their release, especially relevant during satellite cell turnover in homeostasis or activation upon injury. Additionally, we explore an evolutionary perspective on the ECM across a range of multicellular organisms and discuss a model wherein satellite cells are self-sustained by generating their own niche. Considering the prevalence of ECM proteins in the connective tissue of various organs it is not surprising that mutations in ECM genes have pathological implications, including in muscle, where they can lead to myopathies. However, the particular role of certain disease-related ECM proteins in stem cell maintenance highlights the potential contribution of stem cell deregulation to the progression of these disorders., (Copyright © 2024. Published by Elsevier Inc.)

Skeletal muscle is a postmitotic tissue composed of contractile myofibers that are oriented and connected to different layers of connective tissue. Nevertheless, adult muscle fibers retain the capacity to regenerate in response to damage, activating the classical muscle stem cell compartment, namely, satellite cells (SCs), which are mitotically quiescent cells until required for growth or repair and are localized between the basal lamina and sarcolemma of myofibers. The transition of SCs from the quiescent state toward activation, commitment, and differentiation involves the genetic and epigenetic adaptation to novel biological functions, entailing dynamic changes in the protein expression profile. Interestingly, some of the activities and signaling regulating proliferation, commitment, differentiation, and survival/apoptosis of satellite cells have been also partially recapitulated in vitro, taking advantage of robust markers, reliable techniques, and reproducible protocols. Over the years, different techniques of muscular cell culture have been designed including primary cultures from embryonic or postnatal muscle, myogenic cell line, and three-dimensional (3D) skeletal muscle construct. Typical two-dimensional (2D) muscle cell culture cannot fully recapitulate the complexity of living muscle tissues, restricting their usefulness for physiological studies. The development of functional 3D culture models represents a valid alternative to overcome the limitations of already available in vitro model, increasing our understanding of the roles played by the various cell types and how they interact. In this chapter, the development of bidimensional and three-dimensional cell cultures have been described, improving the technical aspect of satellite cell isolation, the best culture-based conditions for muscle cell growth and differentiation, and the procedures required to develop a three-dimensional skeletal muscle construct., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Skeletal muscle has an extraordinary capacity to regenerate itself after injury due to the presence of tissue-resident muscle stem cells. While these muscle stem cells are the primary contributor to the regenerated myofibers, the process occurs in a regenerative microenvironment where multiple different cell types act in a coordinated manner to clear the damaged myofibers and restore tissue homeostasis. In this regenerative environment, immune cells play a well-characterized role in initiating repair by establishing an inflammatory state that permits the removal of dead cells and necrotic muscle tissue at the injury site. More recently, it has come to be appreciated that the immune cells also play a crucial role in communicating with the stem cells within the regenerative environment to help coordinate the timing of repair events through the secretion of cytokines, chemokines, and growth factors. Evidence also suggests that stem cells can help modulate the extent of the inflammatory response by signaling to the immune cells, demonstrating a cross-talk between the different cells in the regenerative environment. Here, we review the current knowledge on the innate immune response to sterile muscle injury and provide insight into the epigenetic mechanisms used by the cells in the regenerative niche to integrate the cellular cross-talk required for efficient muscle repair., (Copyright © 2024. Published by Elsevier Inc.)

Skeletal muscle is a force-producing organ composed of muscle tissues, connective tissues, blood vessels, and nerves, all working in synergy to enable movement and provide support to the body. While robust biomechanical descriptions of skeletal muscle force production at the body or tissue level exist, little is known about force application on microstructures within the muscles, such as cells. Among various cell types, skeletal muscle stem cells reside in the muscle tissue environment and play a crucial role in driving the self-repair process when muscle damage occurs. Early evidence indicates that the fate and function of skeletal muscle stem cells are controlled by both biophysical and biochemical factors in their microenvironments, but much remains to accomplish in quantitatively describing the biophysical muscle stem cell microenvironment. This book chapter aims to review current knowledge on the influence of biophysical stresses and landscape properties on muscle stem cells in heath, aging, and diseases., (Copyright © 2024. Published by Elsevier Inc.)

Background: Microand nanoplastics pollution can cause substantial damage to ecosystems. Since scientists have focused mainly on their impact on aquatic environments, less attention has been paid to the accumulation of polymer particles in terrestrial organisms., Objectives: We checked if submicron (<5 mm) polystyrene (PS) particles, which can accumulate in living organisms, lead to changes in the physicochemical properties of mammalian cell membranes., Material and Methods: The influence of submicron PS particles on the properties of rat-derived L6 myocytes and H9c2 cardiomyocytes was analyzed. Non-functionalized and amine-functionalized PS particles of 100 nm and 200 nm in diameter were used. The MTT assay was performed to evaluate the viability of the polymers-treated cells. The effect of short (6 h) and prolonged (48 h) incubation with different concentrations of PS particles on the cell's zeta (ζ) potential was examined with the electrophoretic light scattering technique (ELS). Polystyrene particles' physicochemical characteristics (size and stability) were performed using dynamic light scattering (DLS) and electrophoretic light scattering methods., Results: The results show that submicron PS particles affect cell viability and cause changes in the physiochemical parameters of rat cell membranes. Differences were observed depending on the origin of the cells. We observed doseand time-dependent alterations in the studied parameters after submicron PS particle incubation in L6 myotubes and H9c2 cardiomyocytes., Conclusions: The size and modification of PS particle surfaces determine the extent to which they affect the analyzed properties of rat cardiomyocytes and myocytes membranes.

The skeletal muscle is well known for its remarkable ability to regenerate after injuries. The regeneration is a complex and dynamic process that involves muscle stem cells (also called muscle satellite cells, MuSCs), fibro-adipogenic progenitors (FAPs), immune cells, and other muscle-resident cell populations. The MuSCs are the myogenic cell populaiton that contribute nuclei directly to the regenerated myofibers, while the other cell types collaboratively establish a microenvironment that facilitates myogenesis of MuSCs. The myogenic process includes activation, proliferation and differentiationof MuSCs, and subsequent fusion their descendent mononuclear myocytes into multinuclear myotubes. While the contributions of FAPs and immune cells to this microenvironment have been well studied, the influence of MuSCs on other cell types remains poorly understood. This review explores recent evidence supporting the potential role of MuSCs as immunomodulators during muscle regeneration, either through cytokine production or ligand-receptor interactions., (Copyright © 2024. Published by Elsevier Inc.)

Skeletal muscle is a highly represented tissue in mammals and is composed of fibers that are extremely adaptable and capable of regeneration. This characteristic of muscle fibers is made possible by a cell type called satellite cells. Adjacent to the fibers, satellite cells are found in a quiescent state and located between the muscle fibers membrane and the basal lamina. These cells are required for the growth and regeneration of skeletal muscle through myogenesis. This process is known to be tightly sequenced from the activation to the differentiation/fusion of myofibers. However, for the past fifteen years, researchers have been interested in examining satellite cell heterogeneity and have identified different subpopulations displaying distinct characteristics based on localization, quiescence state, stemness capacity, cell-cycle progression or gene expression. A small subset of satellite cells appears to represent multipotent long-term self-renewing muscle stem cells (MuSC). All these distinctions led us to the hypothesis that the characteristics of myogenesis might not be linear and therefore may be more permissive based on the evidence that satellite cells are a heterogeneous population. In this review, we discuss the different subpopulations that exist within the satellite cell pool to highlight the heterogeneity and to gain further understanding of the myogenesis progress. Finally, we discuss the long term self-renewing MuSC subpopulation that is capable of dividing asymmetrically and discuss the molecular mechanisms regulating MuSC polarization during health and disease., (Copyright © 2024. Published by Elsevier Inc.)

The role of the cellular microenvironment has recently gained attention in the context of muscle health, adaption, and disease. Emerging evidence supports major roles for the extracellular matrix (ECM) in regeneration and the dynamic regulation of the satellite cell niche. Satellite cells normally reside in a quiescent state in healthy muscle, but upon muscle injury, they activate, proliferate, and fuse to the damaged fibers to restore muscle function and architecture. This chapter reviews the composition and mechanical properties of skeletal muscle ECM and the role of these factors in contributing to the satellite cell niche that impact muscle regeneration. In addition, the chapter details the effects of satellite cell-matrix interactions and provides evidence that there is bidirectional regulation affecting both the cellular and extracellular microenvironment within skeletal muscle. Lastly, emerging methods to investigate satellite cell-matrix interactions will be presented., (Copyright © 2024. Published by Elsevier Inc.)

Cultivated meat, an alternative to conventional meat, holds great promise in alleviating environmental and ethical concerns. Skeletal muscle stem cell isolation is a critical phase in cultivated meat production, and efficiency is a major determinant in the final differentiated muscle cell yield. The conventional enzymatic dissociation method for cell isolation presents drawbacks, including added costs and the destruction of vital extracellular matrix components. We developed an alternative cell isolation technique, explant cell isolation, to isolate muscle stem cells from muscle tissue. The present protocol yields myogenic cell populations, mainly composed of skeletal muscle stem cells without the use of enzymes, and through a simplified process. Overall, the explant method allows for propagation of cells in their natural environment, preserving intricate cell-cell and cell-matrix interactions, resulting in both economic efficiency and consistent generation of high-quality cells., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The proper functioning of skeletal muscles is essential throughout life. A crucial crosstalk between the environment and several cellular mechanisms allows striated muscles to perform successfully. Notably, the skeletal muscle tissue reacts to an injury producing a completely functioning tissue. The muscle's robust regenerative capacity relies on the fine coordination between muscle stem cells (MuSCs or "satellite cells") and their specific microenvironment that dictates stem cells' activation, differentiation, and self-renewal. Critical for the muscle stem cell pool is a fine regulation of chromatin organization and gene expression. Acquiring a lineage-specific 3D genome architecture constitutes a crucial modulator of muscle stem cell function during development, in the adult stage, in physiological and pathological conditions. The context-dependent relationship between genome structure, such as accessibility and chromatin compartmentalization, and their functional effects will be analysed considering the improved 3D epigenome knowledge, underlining the intimate liaison between environmental encounters and epigenetics., (Copyright © 2024. Published by Elsevier Inc.)

Myocyte fusion is a pivotal process in the development and regeneration of skeletal muscle. Failure during fusion can lead to a range of developmental as well as pathological consequences. This review aims to comprehensively explore the intricate processes underlying myocyte fusion, from the molecular to tissue scale. We shed light on key players, such as the muscle-specific fusogens - Myomaker and Myomixer, in addition to some lesser studied molecules contributing to myocyte fusion. Conserved across vertebrates, Myomaker and Myomixer play a crucial role in driving the merger of plasma membranes of fusing myocytes, ensuring the formation of functional muscle syncytia. Our multiscale approach also delves into broader cell and tissue dynamics that orchestrate the timing and positioning of fusion events. In addition, we explore the relevance of muscle fusogens to human health and disease. Mutations in fusogen genes have been linked to congenital myopathies, providing unique insights into the molecular basis of muscle diseases. We conclude with a discussion on potential therapeutic avenues that may emerge from manipulating the myocyte fusion process to remediate skeletal muscle disorders., (Copyright © 2024. Published by Elsevier Inc.)

Objectives: Intermittent fasting (IF) activates autophagy in cardiac muscle and pancreatic islets. We examined the effect of IF on markers of autophagy in liver and skeletal muscle in mice and in humans., Methods: Ten-wk-old C57 BL/6 J male mice were ad libitum (AL) fed a high-fat diet (HFD) or chow diet for 8 wk, before randomization to AL or IF (24-h fast, 3 non-consecutive days per week) for 8 wk (8-16 per group). Tissue was collected in the fed or 22-h fasted state. Fifty women (51 ± 2 y, 31.8 ± 4.3 kg/m 2 ) were randomly assigned to one of two IF protocols (24-hfast, 3 non-consecutive days per week) and fed at 70% (IF70) or 100% (IF100) of energy requirements for 8 wk. Vastus lateralis muscle was collected at 0800 after 12- and 24-h fasts. Microtubule-associated protein light chain 1 (Map1 lc3 b), Beclin1 (Becn1), Sequestosome 1 (Sqstm1/p62), and Lysosomal associated membrane protein 2 (Lamp2) were assessed by quantitative polymerase chain reaction and LC3, BECLIN1 and LAMP1 protein content by immunoblotting., Results: Fasting increased hepatic LC3 I protein and Map1 lc3 b mRNA levels in IF mice fed chow or HFD. LAMP1 protein and Beclin1 mRNA levels in liver were also increased by fasting, but only in chow-fed mice. IF did not activate markers of autophagy in mouse muscle. In humans, a 24-h fast increased SQSTM1. BECLIN1, SQSTM1 and LAMP2 mRNA levels were decreased in IF70 after a 12-h overnight fast ., Conclusion: Markers of autophagy in liver, but not in muscle, were elevated in response to IF in mice. In humans, autophagy markers in muscle were reduced, likely in response to weight loss., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Numerous studies have established the critical roles of microRNAs in regulating post-transcriptional gene expression in diverse biological processes. Here, we report on the role and mechanism of miR-24-3p in skeletal muscle differentiation and regeneration. miR-24-3p promotes myoblast differentiation and skeletal muscle regeneration by directly targeting high mobility group AT-hook 1 (HMGA1) and regulating it and its direct downstream target, the inhibitor of differentiation 3 (ID3). miR-24-3p knockdown in neonatal mice increases PAX7-positive proliferating muscle stem cells (MuSCs) by derepressing Hmga1 and Id3. Similarly, inhibition of miR-24-3p in the tibialis anterior muscle prevents Hmga1 and Id3 downregulation and impairs regeneration. These findings provide evidence that the miR-24-3p/HMGA1/ID3 axis is required for MuSC differentiation and skeletal muscle regeneration in vivo., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Fluorescently labelling proteins such as insulin have wide ranging applications in a pharmaceutical research and drug delivery. Human insulin (Actrapid®) was labelled with fluorescein isothiocyanate (FITC) and the synthesised conjugate identified using reverse phase high performance liquid chromatography (RP-HPLC) on a C18 column and a gradient method with mobile phase A containing 0.1% trifluoroacetic acid (TFA) in Millipore water and mobile phase B containing 90% Acetonitrile, 10% Millipore water and 0.1% TFA. Syntheses were carried out at varying reaction times between 4 and 20 h. Mono-labelled FITC-insulin conjugate was successfully synthesised with labelling at the B1 position on the insulin chain using a molar ratio of 2:1 (FITC:insulin) at a reaction time of 18 h and confirmed by electrospray mass spectroscopy. Reactions were studied across a pH range of 7-9.8 and the quantities switch from mono-labelled to di-labelled FITC-insulin conjugates at a reaction time of 2 h (2:1 molar ratio) at pH > 8. The conjugates isolated from the studies had biological activities in comparison to native insulin of 99.5% monoB1, 78% monoA1, 51% diA1B1 and 0.06% triA1B1B29 in HUVEC cells by examining AKT phosphorylation levels. MonoB1 FITC-insulin conjugate was also compared to native insulin by examining cell surface GLUT4 in C2C12 skeletal muscle cells. No significant difference in the cellular response was observed for monoB1 produced in-house compared to native insulin. Therefore mono-labelled FITC-insulin at the B1 position showed similar biological activity as native insulin and can potentially be used for future biomedical applications., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Skeletal muscle cells can both gain and lose volume during periods of exercise and rest. Muscle cells do not behave as perfect osmometers because the cell volume changes are less than predicted from the change in extracellular osmolality. Therefore, there are mechanisms involved in regulating cell volume, and they are different for regulatory volume decreases and regulatory volume increases. Also, after an initial rapid change in cell volume, there is a gradual and partial recovery of cell volume that is effected by ion and water transport mechanisms. The mechanisms have been studied in non-contracting muscle cells, but remain to be fully elucidated in contracting muscle. Changes in muscle cell volume are known to affect the strength of contractile activity as well as anabolic/catabolic signaling, perhaps indicating that cell volume should be a regulated variable in skeletal muscle cells. Muscles contracting at moderate to high intensity gain intracellular volume because of increased intracellular osmolality. Concurrent increases in interstitial (extracellular) muscle volume occur from an increase in osmotically active molecules and increased vascular filtration pressure. At the same time, non-contracting muscles lose cell volume because of increased extracellular (blood) osmolality. This review provides the physiological foundations and highlights key concepts that underpin our current understanding of volume regulatory processes in skeletal muscle, beginning with consideration of osmosis more than 200 years ago and continuing through to the process of regulatory volume decrease and regulatory volume increase., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The in vitro generation of human cardiomyocytes derived from induced pluripotent stem cells (iPSC) is of great importance for cardiac disease modeling, drug-testing applications and for regenerative medicine. Despite the development of various cultivation strategies, a sufficiently high degree of maturation is still a decisive limiting factor for the successful application of these cardiac cells. The maturation process includes, among others, the proper formation of sarcomere structures, mediating the contraction of cardiomyocytes. To precisely monitor the maturation of the contractile machinery, we have established an imaging-based strategy that allows quantitative evaluation of important parameters, defining the quality of the sarcomere network. iPSC-derived cardiomyocytes were subjected to different culture conditions to improve sarcomere formation, including prolonged cultivation time and micro patterned surfaces. Fluorescent images of α-actinin were acquired using super-resolution microscopy. Subsequently, we determined cell morphology, sarcomere density, filament alignment, z-Disc thickness and sarcomere length of iPSC-derived cardiomyocytes. Cells from adult and neonatal heart tissue served as control. Our image analysis revealed a profound effect on sarcomere content and filament orientation when iPSC-derived cardiomyocytes were cultured on structured, line-shaped surfaces. Similarly, prolonged cultivation time had a beneficial effect on the structural maturation, leading to a more adult-like phenotype. Automatic evaluation of the sarcomere filaments by machine learning validated our data. Moreover, we successfully transferred this approach to skeletal muscle cells, showing an improved sarcomere formation cells over different differentiation periods. Overall, our image-based workflow can be used as a straight-forward tool to quantitatively estimate the structural maturation of contractile cells. As such, it can support the establishment of novel differentiation protocols to enhance sarcomere formation and maturity., (© 2022. The Author(s).)

Skeletal muscle stem cells, also called Satellite Cells (SCs), are actively maintained in quiescence but can activate quickly upon extrinsic stimuli. However, the mechanisms of how quiescent SCs (QSCs) activate swiftly remain elusive. Here, using a whole mouse perfusion fixation approach to obtain bona fide QSCs, we identify massive proteomic changes during the quiescence-to-activation transition in pathways such as chromatin maintenance, metabolism, transcription, and translation. Discordant correlation of transcriptomic and proteomic changes reveals potential translational regulation upon SC activation. Importantly, we show Cytoplasmic Polyadenylation Element Binding protein 1 (CPEB1), post-transcriptionally affects protein translation during SC activation by binding to the 3' UTRs of different transcripts. We demonstrate phosphorylation-dependent CPEB1 promoted Myod1 protein synthesis by binding to the cytoplasmic polyadenylation elements (CPEs) within its 3' UTRs to regulate SC activation and muscle regeneration. Our study characterizes CPEB1 as a key regulator to reprogram the translational landscape directing SC activation and subsequent proliferation., (© 2022. The Author(s).)

Human pluripotent stem cells (hPSCs) provide a human model for developmental myogenesis, disease modeling and development of therapeutics. Differentiation of hPSCs into muscle stem cells has the potential to provide a cell-based therapy for many skeletal muscle wasting diseases. This review describes the current state of hPSCs towards recapitulating human myogenesis ex vivo, considerations of stem cell and progenitor cell state as well as function for future use of hPSC-derived muscle cells in regenerative medicine., (Published by Elsevier Inc.)

The processes of myogenesis during both development and regeneration share a number of similarities across both amniotes and teleosts. In amniotes, the process of muscle formation is considered largely biphasic, with developmental myogenesis occurring through hyperplastic fibre deposition and postnatal muscle growth driven through hypertrophy of existing fibres. In contrast, teleosts continue generating new muscle fibres during adult myogenesis through a process of eternal hyperplasia using a dedicated stem cell system termed the external cell layer. During developmental and regenerative myogenesis alike, muscle progenitors interact with their niche to receive cues guiding their transition into myoblasts and ultimately mature myofibres. During development, muscle precursors receive input from neighbouring embryological tissues; however, during repair, this role is fulfilled by other injury resident cell types, such as those of the innate immune response. Recent work has focused on the role of macrophages as a pro-regenerative cell type which provides input to muscle satellite cells during regenerative myogenesis. As zebrafish harbour a satellite cell system analogous to that of mammals, the processes of regeneration can be interrogated in vivo with the imaging intensive approaches afforded in the zebrafish system. This review discusses the strengths of zebrafish with a focus on both the similarities and differences to amniote myogenesis during both development and repair., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Muscle stem (satellite) cells express Pax7, a key transcription factor essential for satellite cell maintenance and adult muscle regeneration. We identify the corepressor transducin-like enhancer of split-4 (TLE4) as a Pax7 interaction partner expressed in quiescent satellite cells under homeostasis. A subset of satellite cells transiently downregulate TLE4 during early time points following muscle injury. We identify these to be activated satellite cells, and that TLE4 downregulation is required for Myf5 activation and myogenic commitment. Our results indicate that TLE4 represses Pax7-mediated Myf5 transcriptional activation by occupying the -111 kb Myf5 enhancer to maintain quiescence. Loss of TLE4 function causes Myf5 upregulation, an increase in satellite cell numbers and altered differentiation dynamics during regeneration. Thus, we have uncovered a novel mechanism to maintain satellite cell quiescence and regulate muscle differentiation mediated by the corepressor TLE4., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Cardiac patch implantation helps maximize the paracrine function of grafted cells and serves as a reservoir of soluble proangiogenic factors required for the neovascularization of infarcted hearts. We have previously fabricated a cardiac patch, EF-HAM, composed of a human amniotic membrane (HAM) coated with aligned PLGA electrospun fibers (EF). In this study, we aimed to evaluate the biocompatibility and angiogenic effects of EF-HAM scaffolds with varying fiber thicknesses on the paracrine behavior of skeletal muscle cells (SkM). Conditioned media (CM) obtained from SkM-seeded HAM and EF-HAM scaffolds were subjected to multiplex analysis of angiogenic factors and tested on HUVECs for endothelial cell viability, migration, and tube formation analyses. All three different groups of EF-HAM scaffolds demonstrated excellent biocompatibility with SkM. CM derived from SkM-seeded EF-HAM 7 min scaffolds contained significantly elevated levels of proangiogenic factors, including angiopoietin-1, IL-8, and VEGF-C compared to plain CM, which was obtained from SkM cultured on the plain surface. CM obtained from all SkM-seeded EF-HAM scaffolds significantly increased the viability of HUVECs compared to plain CM after five days of culture. However, only EF-HAM 7 min CM induced a higher migration capacity in HUVECs and formed a longer and more elaborate capillary-like network on Matrigel compared with plain CM. Surface roughness and wettability of EF-HAM 7 min scaffolds might have influenced the proportion of skeletal myoblasts and fibroblasts growing on the scaffolds and subsequently potentiated the angiogenic paracrine function of SkM. This study demonstrated the angioinductive properties of EF-HAM composite scaffold and its potential applications in the repair and regeneration of ischemic tissues.

Mitochondria are energy-generating organelles and mitochondrial biogenesis is stimulated to meet energy requirements in response to extracellular stimuli, including exercise. However, the mechanisms underlying mitochondrial biogenesis remain unknown. Here, we demonstrate that transcriptional coactivator with PDZ-binding motif (TAZ) stimulates mitochondrial biogenesis in skeletal muscle. In muscle-specific TAZ-knockout (mKO) mice, mitochondrial biogenesis, respiratory metabolism, and exercise ability were decreased compared to wild-type mice. Mechanistically, TAZ stimulates the translation of mitochondrial transcription factor A via Ras homolog enriched in brain (Rheb)/Rheb like 1 (Rhebl1)-mTOR axis. TAZ stimulates Rhebl1 expression via TEA domain family transcription factor. Rhebl1 introduction by adeno-associated virus or mTOR activation recovered mitochondrial biogenesis in mKO muscle. Physiologically, mKO mice did not stimulate exercise-induced mitochondrial biogenesis. Collectively, our results suggested that TAZ is a novel stimulator for mitochondrial biogenesis and exercise-induced muscle adaptation., (© 2022. The Author(s).)

Endogenous skeletal muscle development, regeneration, and pathology are extremely complex processes, influenced by local and systemic factors. Unpinning how these mechanisms function is crucial for fundamental biology and to develop therapeutic interventions for genetic disorders, but also conditions like sarcopenia and volumetric muscle loss. Ex vivo skeletal muscle models range from two- and three-dimensional primary cultures of satellite stem cell-derived myoblasts grown alone or in co-culture, to single muscle myofibers, myobundles, and whole tissues. Together, these systems provide the opportunity to gain mechanistic insights of stem cell behavior, cell-cell interactions, and mature muscle function in simplified systems, without confounding variables. Here, we highlight recent advances (published in the last 5 years) using in vitro primary cells and ex vivo skeletal muscle models, and summarize the new insights, tools, datasets, and screening methods they have provided. Finally, we highlight the opportunity for exponential advance of skeletal muscle knowledge, with spatiotemporal resolution, that is offered by guiding the study of muscle biology and physiology with in silico modelling and implementing high-content cell biology systems and ex vivo physiology platforms., (Copyright © 2021 Elsevier Inc. All rights reserved.)