Multinucleated Myofibers Research Articles

Skeletal muscle: molecular structure, myogenesis, biological functions, and diseases.

Skeletal muscle is an important motor organ with multinucleated myofibers as its smallest cellular units. Myofibers are formed after undergoing cell differentiation, cell-cell fusion, myonuclei migration, and myofibril crosslinking among other processes and undergo morphological and functional changes or lesions after being stimulated by internal or external factors. The above processes are collectively referred to as myogenesis. After myofibers mature, the function and behavior of skeletal muscle are closely related to the voluntary movement of the body. In this review, we systematically and comprehensively discuss the physiological and pathological processes associated with skeletal muscles from five perspectives: molecule basis, myogenesis, biological function, adaptive changes, and myopathy. In the molecular structure and myogenesis sections, we gave a brief overview, focusing on skeletal muscle-specific fusogens and nuclei-related behaviors including cell-cell fusion and myonuclei localization. Subsequently, we discussed the three biological functions of skeletal muscle (muscle contraction, thermogenesis, and myokines secretion) and its response to stimulation (atrophy, hypertrophy, and regeneration), and finally settled on myopathy. In general, the integration of these contents provides a holistic perspective, which helps to further elucidate the structure, characteristics, and functions of skeletal muscle.

Development of porcine skeletal muscle extracellular matrix–derived hydrogels with improved properties and low immunogenicity

Hydrogels derived from decellularized extracellular matrices (ECM) of animal origin show immense potential for regenerative applications due to their excellent cytocompatibility and biomimetic properties. Despite these benefits, the impact of decellularization protocols on the properties and immunogenicity of these hydrogels remains relatively unexplored. In this study, porcine skeletal muscle ECM (smECM) underwent decellularization using mechanical disruption (MD) and two commonly employed decellularization detergents, sodium deoxycholate (SDC) or Triton X-100. To mitigate immunogenicity associated with animal-derived ECM, all decellularized tissues were enzymatically treated with α-galactosidase to cleave the primary xenoantigen-the α-Gal antigen. Subsequently, the impact of the different decellularization protocols on the resultant hydrogels was thoroughly investigated. All methods significantly reduced total DNA content in hydrogels. Moreover, α-galactosidase treatment was crucial for cleaving α-Gal antigens, suggesting that conventional decellularization methods alone are insufficient. MD preserved total protein, collagen, sulfated glycosaminoglycan, laminin, fibronectin, and growth factors more efficiently than other protocols. The decellularization method impacted hydrogel gelation kinetics and ultrastructure, as confirmed by turbidimetric and scanning electron microscopy analyses. MD hydrogels demonstrated high cytocompatibility, supporting satellite stem cell recruitment, growth, and differentiation into multinucleated myofibers. In contrast, the SDC and Triton X-100 protocols exhibited cytotoxicity. Comprehensive in vivo immunogenicity assessments in a subcutaneous xenotransplantation model revealed MD hydrogels' biocompatibility and low immunogenicity. These findings highlight the significant influence of the decellularization protocol on hydrogel properties. Our results suggest that combining MD with α-galactosidase treatment is an efficient method for preparing low-immunogenic smECM-derived hydrogels with enhanced properties for skeletal muscle regenerative engineering and clinical applications.

Chromatin profiling reveals TFAP4 as a critical transcriptional regulator of bovine satellite cell differentiation

BackgroundSatellite cells are myogenic precursor cells in adult skeletal muscle and play a crucial role in skeletal muscle regeneration, maintenance, and growth. Like embryonic myoblasts, satellite cells have the ability to proliferate, differentiate, and fuse to form multinucleated myofibers. In this study, we aimed to identify additional transcription factors that control gene expression during bovine satellite cell proliferation and differentiation.ResultsUsing chromatin immunoprecipitation followed by sequencing, we identified 56,973 and 54,470 genomic regions marked with both the histone modifications H3K4me1 and H3K27ac, which were considered active enhancers, and 50,956 and 59,174 genomic regions marked with H3K27me3, which were considered repressed enhancers, in proliferating and differentiating bovine satellite cells, respectively. In addition, we identified 1,216 and 1,171 super-enhancers in proliferating and differentiating bovine satellite cells, respectively. Analyzing these enhancers showed that in proliferating bovine satellite cells, active enhancers were associated with genes stimulating cell proliferation or inhibiting myoblast differentiation whereas repressed enhancers were associated with genes essential for myoblast differentiation, and that in differentiating satellite cells, active enhancers were associated with genes essential for myoblast differentiation or muscle contraction whereas repressed enhancers were associated with genes stimulating cell proliferation or inhibiting myoblast differentiation. Active enhancers in proliferating bovine satellite cells were enriched with binding sites for many transcription factors such as MYF5 and the AP-1 family transcription factors; active enhancers in differentiating bovine satellite cells were enriched with binding sites for many transcription factors such as MYOG and TFAP4; and repressed enhancers in both proliferating and differentiating bovine satellite cells were enriched with binding sites for NF-kB, ZEB-1, and several other transcription factors. The role of TFAP4 in satellite cell or myoblast differentiation was previously unknown, and through gene knockdown and overexpression, we experimentally validated a critical role for TFAP4 in the differentiation and fusion of bovine satellite cells into myofibers.ConclusionsSatellite cell proliferation and differentiation are controlled by many transcription factors such as AP-1, TFAP4, NF-kB, and ZEB-1 whose roles in these processes were previously unknown in addition to those transcription factors such as MYF5 and MYOG whose roles in these processes are widely known.

Molecular mechanisms underlying the impact of muscle fiber types on meat quality in livestock and poultry

In the past, the primary emphasis of livestock and poultry breeding was mainly on improving the growth rate, meat production efficiency and disease resistance. However, the improvement of meat quality has become a major industrial focus due to the ongoing advancements in livestock and poultry breeding. Skeletal muscles consist of multinucleated myofibers formed through the processes of myoblast proliferation, differentiation and fusion. Muscle fibers can be broadly classified into two main types: slow-twitch (Type I) and fast-twitch (Type II). Fast-twitch fibers can be further categorized into Type IIa, Type IIx, and Type IIb. The proportion of Type I and Type IIa muscle fibers is positively associated with meat quality, while the presence of Type IIb muscle fibers in skeletal muscle tissue is inversely related to meat quality. Consequently, muscle fiber composition directly influences meat quality. The distribution of these fiber types within skeletal muscle is governed by a complex network, which encompasses numerous pivotal regulators and intricate signaling pathways. This article aims to succinctly outline the parameters utilized for assessing meat quality, elucidate the relationship between muscle fiber composition and meat quality as well as elaborate on the relevant genetic factors and their molecular mechanisms that regulate muscle fiber types in livestock and poultry. This summary will enrich our comprehension of how to improve meat quality in livestock and poultry, providing valuable insights for future improvements.

Secreted ADAMTS-like 2 promotes myoblast differentiation by potentiating WNT signaling

Myogenesis is the process that generates multinucleated contractile myofibers from muscle stem cells during skeletal muscle development and regeneration. Myogenesis is governed by myogenic regulatory transcription factors, including MYOD1. Here, we identified the secreted matricellular protein ADAMTS-like 2 (ADAMTSL2) as part of a Wnt-dependent positive feedback loop, which augmented or sustained MYOD1 expression and thus promoted myoblast differentiation. ADAMTSL2 depletion resulted in severe retardation of myoblast differentiation in vitro and its ablation in myogenic precursor cells resulted in aberrant skeletal muscle architecture. Mechanistically, ADAMTSL2 potentiated WNT signaling by binding to WNT ligands and WNT receptors. We identified the WNT-binding ADAMTSL2 peptide, which was sufficient to promote myogenesis in vitro. Since ADAMTSL2 was previously described as a negative regulator of TGFβ signaling in fibroblasts, ADAMTSL2 now emerges as a signaling hub that could integrate WNT, TGFβ and potentially other signaling pathways within the dynamic microenvironment of differentiating myoblasts during skeletal muscle development and regeneration.

Expression of Myomaker and Myomerger in myofibers causes muscle pathology

BackgroundSkeletal muscle development and regeneration depend on cellular fusion of myogenic progenitors to generate multinucleated myofibers. These progenitors utilize two muscle-specific fusogens, Myomaker and Myomerger, which function by remodeling cell membranes to fuse to each other or to existing myofibers. Myomaker and Myomerger expression is restricted to differentiating progenitor cells as they are not detected in adult myofibers. However, Myomaker remains expressed in myofibers from mice with muscular dystrophy. Ablation of Myomaker from dystrophic myofibers results in reduced membrane damage, leading to a model where persistent fusogen expression in myofibers, in contrast to myoblasts, is harmful.MethodsDox-inducible transgenic mice were developed to ectopically express Myomaker or Myomerger in the myofiber compartment of skeletal muscle. We quantified indices of myofiber membrane damage, such as serum creatine kinase and IgM+ myofibers, and assessed general muscle histology, including central nucleation, myofiber size, and fibrosis.ResultsMyomaker or Myomerger expression in myofibers independently caused membrane damage at acute time points. This damage led to muscle pathology, manifesting with centrally nucleated myofibers and muscle atrophy. Dual expression of both Myomaker and Myomerger in myofibers exacerbated several aspects of muscle pathology compared to expression of either fusogen by itself.ConclusionsThese data reveal that while myofibers can tolerate some level of Myomaker and Myomerger, expression of a single fusogen above a threshold or co-expression of both fusogens is damaging to myofibers. These results explain the paradigm that their expression in myofibers can have deleterious consequences in muscle pathologies and highlight the need for their highly restricted expression during myogenesis and fusion.

Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery

Entry of enveloped viruses into cells is mediated by viral fusogenic proteins that drive membrane rearrangements needed for fusion between viral and target membranes. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens but do not structurally or functionally resemble classical viral fusogens. We asked whether the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver μDystrophin to skeletal muscle of a mouse model of Duchenne muscular dystrophy and alleviate pathology. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.

3D human induced pluripotent stem cell-derived bioengineered skeletal muscles for tissue, disease and therapy modeling.

Skeletal muscle is a complex tissue composed of multinucleated myofibers responsible for force generation that are supported by multiple cell types. Many severe and lethal disorders affect skeletal muscle; therefore, engineering models to reproduce such cellular complexity and function are instrumental for investigating muscle pathophysiology and developing therapies. Here, we detail the modular 3D bioengineering of multilineage skeletal muscles from human induced pluripotent stem cells, which are first differentiated into myogenic, neural and vascular progenitor cells and then combined within 3D hydrogels under tension to generate an aligned myofiber scaffold containing vascular networks and motor neurons. 3D bioengineered muscles recapitulate morphological and functional features of human skeletal muscle, including establishment of a pool of cells expressing muscle stem cell markers. Importantly, bioengineered muscles provide a high-fidelity platform to study muscle pathology, such as emergence of dysmorphic nuclei in muscular dystrophies caused by mutant lamins. The protocol is easy to follow for operators with cell culture experience and takes between 9 and 30 d, depending on the number of cell lineages in the construct. We also provide examples of applications of this advanced platform for testing gene and cell therapies in vitro, as well as for in vivo studies, providing proof of principle of its potential as a tool to develop next-generation neuromuscular or musculoskeletal therapies.

Myomaker and Myomixer Characterization in Gilthead Sea Bream under Different Myogenesis Conditions

Skeletal muscle is formed by multinucleated myofibers originated by waves of hyperplasia and hypertrophy during myogenesis. Tissue damage triggers a regeneration process including new myogenesis and muscular remodeling. During myogenesis, the fusion of myoblasts is a key step that requires different genes' expression, including the fusogens myomaker and myomixer. The present work aimed to characterize these proteins in gilthead sea bream and their possible role in in vitro myogenesis, at different fish ages and during muscle regeneration after induced tissue injury. Myomaker is a transmembrane protein highly conserved among vertebrates, whereas Myomixer is a micropeptide that is moderately conserved. myomaker expression is restricted to skeletal muscle, while the expression of myomixer is more ubiquitous. In primary myocytes culture, myomaker and myomixer expression peaked at day 6 and day 8, respectively. During regeneration, the expression of both fusogens and all the myogenic regulatory factors showed a peak after 16 days post-injury. Moreover, myomaker and myomixer were present at different ages, but in fingerlings there were significantly higher transcript levels than in juveniles or adult fish. Overall, Myomaker and Myomixer are valuable markers of muscle growth that together with other regulatory molecules can provide a deeper understanding of myogenesis regulation in fish.

Biasing the conformation of ELMO2 reveals that myoblast fusion can be exploited to improve muscle regeneration

Myoblast fusion is fundamental for the development of multinucleated myofibers. Evolutionarily conserved proteins required for myoblast fusion include RAC1 and its activator DOCK1. In the current study we analyzed the contribution of the DOCK1-interacting ELMO scaffold proteins to myoblast fusion. When Elmo1−/− mice underwent muscle-specific Elmo2 genetic ablation, they exhibited severe myoblast fusion defects. A mutation in the Elmo2 gene that reduced signaling resulted in a decrease in myoblast fusion. Conversely, a mutation in Elmo2 coding for a protein with an open conformation increased myoblast fusion during development and in muscle regeneration. Finally, we showed that the dystrophic features of the Dysferlin-null mice, a model of limb-girdle muscular dystrophy type 2B, were reversed when expressing ELMO2 in an open conformation. These data provide direct evidence that the myoblast fusion process could be exploited for regenerative purposes and improve the outcome of muscle diseases.

Pax7+ Satellite Cells in Human Skeletal Muscle After Exercise: A Systematic Review and Meta-analysis.

Skeletal muscle has extraordinaryregenerativecapabilities against challenge, mainly owing to its resident muscle stemcells, commonly identified by Pax7+, which expediently donate nuclei to the regenerating multinucleated myofibers. This local reserve of stem cells in damaged muscle tissues is replenished by undifferentiated bone marrow stem cells (CD34+) permeating into the surrounding vascular system. The purpose of the study was to provide a quantitative estimate for the changes in Pax7+ muscle stem cells (satellite cells) in humans following an acute bout of exercise until 96h, in temporal relation to circulating CD34+ bone marrow stem cells. A subgroup analysis of age was also performed. Four databases (Web of Science, PubMed, Scopus, and BASE) were used for the literature search until February 2022. Pax7+ cells in human skeletal muscle were the primary outcome. Circulating CD34+ cells were the secondary outcome. The standardized mean difference (SMD) was calculated using a random-effects meta-analysis. Subgroup analyses were conducted to examine the influence of age, training status, type of exercise, and follow-up time after exercise. The final search identified 20 studies for Pax7+ cells comprising a total of 370 participants between the average age of 21 and 74years and 26 studies for circulating CD34+ bone marrow stem cells comprising 494 participants between the average age of 21 and 67years. Only one study assessed Pax7+ cells immediately after aerobic exercise and showed a 32% reduction in exercising muscle followed by a fast repletion to pre-exercise level within 3h. A large effect on increasing Pax7+ cell content in skeletal muscles was observed 24h after resistance exercise (SMD = 0.89, p < 0.001). Pax7+ cells increased to ~ 50% above pre-exercise level 24-72h after resistance exercise. For a subgroup analysis of age, a large effect (SMD = 0.81, p < 0.001) was observed on increasing Pax7+ cells in exercised muscle among adults aged > 50years, whereas adults at younger age presented a medium effect (SMD = 0.64, p < 0.001). Both resistance exercise and aerobic exercise showed a medium overall effect in increasing circulating CD34+ cells (SMD = 0.53, p < 0.001), which declined quickly to the pre-exercise baseline level after exercise within 6h. An immediate depletion of Pax7+ cells in exercising skeletal muscle concurrent with a transient release of CD34+ cells suggest a replenishment of the local stem cell reserve from bone marrow. A protracted Pax7+ cell expansion in the muscle can be observed during 24-72h after resistance exercise. This result provides a scientific basis for exercise recommendations on weekly cycles allowing for adequate recovery time. Exercise-induced Pax7+ cell expansion in muscle remains significant at higher age, despite a lower stem cell reserve after age 50years. More studies are required to confirm whether Pax7+ cell increment can occur after aerobic exercise. Registered at the International Prospective Register of Systematic Reviews (PROSPERO) [identification code CRD42021265457].

Temporal Expression of Myogenic Regulatory Genes in Different Chicken Breeds during Embryonic Development

The basic units of skeletal muscle in all vertebrates are multinucleate myofibers, which are formed from the fusion of mononuclear myoblasts during the embryonic period. In order to understand the regulation of embryonic muscle development, we selected four chicken breeds, namely, Cornish (CN), White Plymouth Rock (WPR), White Leghorn (WL), and Beijing-You Chicken (BYC), for evaluation of their temporal expression patterns of known key regulatory genes (Myomaker, MYOD, and MSTN) during pectoral muscle (PM) and thigh muscle (TM) development. The highest expression level of Myomaker occurred from embryonic days E13 to E15 for all breeds, indicating that it was the crucial stage of myoblast fusion. Interestingly, the fast-growing CN showed the highest gene expression level of Myomaker during the crucial stage. The MYOD gene expression at D1 was much higher, implying that MYOD might have an important role after hatching. Histomorphology of PM and TM suggested that the myofibers was largely complete at E17, which was speculated to have occurred because of the expression increase in MSTN and the expression decrease in Myomaker. Our research contributes to lay a foundation for the study of myofiber development during the embryonic period in different chicken breeds.

Motoneurons innervation determines the distinct gene expressions in multinucleated myofibers

BackgroundNeuromuscular junctions (NMJs) are peripheral synapses connecting motoneurons and skeletal myofibers. At the postsynaptic side in myofibers, acetylcholine receptor (AChR) proteins are clustered by the neuronal agrin signal. Meanwhile, several nuclei in each myofiber are specially enriched around the NMJ for postsynaptic gene transcription. It remains mysterious that how gene expressions in these synaptic nuclei are systematically regulated, especially by motoneurons.ResultsWe found that synaptic nuclei have a distinctive chromatin structure and gene expression profiling. Synaptic nuclei are formed during NMJ development and maintained by motoneuron innervation. Transcriptome analysis revealed that motoneuron innervation determines the distinct expression patterns in the synaptic region and non-synaptic region in each multinucleated myofiber, probably through epigenetic regulation. Myonuclei in synaptic and non-synaptic regions have different responses to denervation. Weighted gene co-expression network analysis revealed that the histone lysine demethylases Kdm1a is a negative regulator of synaptic gene expression. Inhibition of Kdm1a promotes AChR expression but impairs motor functions.ConclusionThese results demonstrate that motoneurons innervation determines the distinct gene expressions in multinucleated myofibers. Thus, dysregulation of nerve-controlled chromatin structure and muscle gene expression might cause muscle weakness and atrophy in motoneuron degenerative disorders.

CRISPR/Cas9 editing of directly reprogrammed myogenic progenitors restores dystrophin expression in a mouse model of muscular dystrophy.

SummaryGenetic mutations in dystrophin manifest in Duchenne muscular dystrophy (DMD), the most commonly inherited muscle disease. Here, we report on reprogramming of fibroblasts from two DMD mouse models into induced myogenic progenitor cells (iMPCs) by MyoD overexpression in concert with small molecule treatment. DMD iMPCs proliferate extensively, while expressing myogenic stem cell markers including Pax7 and Myf5. Additionally, DMD iMPCs readily give rise to multinucleated myofibers that express mature skeletal muscle markers; however, they lack DYSTROPHIN expression. Utilizing an exon skipping-based approach with CRISPR/Cas9, we report on genetic correction of the dystrophin mutation in DMD iMPCs and restoration of protein expression in vitro. Furthermore, engraftment of corrected DMD iMPCs into the muscles of dystrophic mice restored DYSTROPHIN expression and contributed to the muscle stem cell reservoir. Collectively, our findings report on a novel in vitro cellular model for DMD and utilize it in conjunction with gene editing to restore DYSTROPHIN expression in vivo.

It takes all kinds: heterogeneity among satellite cells and fibro-adipogenic progenitors during skeletal muscle regeneration.

Vertebrate skeletal muscle is composed of multinucleate myofibers that are surrounded by muscle connective tissue. Following injury, muscle is able to robustly regenerate because of tissue-resident muscle stem cells, called satellite cells. In addition, efficient and complete regeneration depends on other cells resident in muscle - including fibro-adipogenic progenitors (FAPs). Increasing evidence from single-cell analyses and genetic and transplantation experiments suggests that satellite cells and FAPs are heterogeneous cell populations. Here, we review our current understanding of the heterogeneity of satellite cells, their myogenic derivatives and FAPs in terms of gene expression, anatomical location, age and timing during the regenerative process - each of which have potentially important functional consequences.

RNA-Binding Proteins in the Post-transcriptional Control of Skeletal Muscle Development, Regeneration and Disease.

Embryonic myogenesis is a temporally and spatially regulated process that generates skeletal muscle of the trunk and limbs. During this process, mononucleated myoblasts derived from myogenic progenitor cells within the somites undergo proliferation, migration and differentiation to elongate and fuse into multinucleated functional myofibers. Skeletal muscle is the most abundant tissue of the body and has the remarkable ability to self-repair by re-activating the myogenic program in muscle stem cells, known as satellite cells. Post-transcriptional regulation of gene expression mediated by RNA-binding proteins is critically required for muscle development during embryogenesis and for muscle homeostasis in the adult. Differential subcellular localization and activity of RNA-binding proteins orchestrates target gene expression at multiple levels to regulate different steps of myogenesis. Dysfunctions of these post-transcriptional regulators impair muscle development and homeostasis, but also cause defects in motor neurons or the neuromuscular junction, resulting in muscle degeneration and neuromuscular disease. Many RNA-binding proteins, such as members of the muscle blind-like (MBNL) and CUG-BP and ETR-3-like factors (CELF) families, display both overlapping and distinct targets in muscle cells. Thus they function either cooperatively or antagonistically to coordinate myoblast proliferation and differentiation. Evidence is accumulating that the dynamic interplay of their regulatory activity may control the progression of myogenic program as well as stem cell quiescence and activation. Moreover, the role of RNA-binding proteins that regulate post-transcriptional modification in the myogenic program is far less understood as compared with transcription factors involved in myogenic specification and differentiation. Here we review past achievements and recent advances in understanding the functions of RNA-binding proteins during skeletal muscle development, regeneration and disease, with the aim to identify the fundamental questions that are still open for further investigations.

Can we talk about myoblast fusion?

Can we talk about myoblast fusion?

Defining Post‐transcriptional Regulation of the Copper Transporter ATP7A in Skeletal Muscle Cells

Copper is an important trace nutrient that functions as an enzyme cofactor and signaling molecule. Copper is also toxic, so intracellular copper must be tightly regulated by transporters and chaperones. In most cells, copper export is mediated by the trans-Golgi copper transporter, ATP7A, which provides copper to secreted cupro-enzymes. Loss of ATP7A causes Menkes disease, wherein dietary copper absorption is impaired, cellular copper homeostasis is disrupted, and secreted cupro-enzymes are not supplied with copper. One symptom of Menkes disease is muscle weakness, which is largely unexplained. Skeletal muscle is a post-mitotic tissue comprised of multi-nucleated myofibers and can be regenerated by a closely-associated pool of stem cells. Few studies have focused on ATP7A in skeletal muscle. Our previous work showed that total cellular copper, ATP7A levels, and Atp7a mRNA stability increase during muscle cell differentiation. Here, we used an immortalized muscle cell line, C2C12, to demonstrate that Atp7a mRNA is stabilized in higher copper concentrations in undifferentiated muscle cells. We used a candidate-based approach to identify RNA binding proteins that regulate the Atp7a mRNA. Candidates were chosen based on previously described interaction with Atp7a mRNA and other copper homeostasis gene transcripts, their potential ability to bind copper, and their role in muscle development. With these criteria, we identified PTBP1 as a candidate. Using RNA immunoprecipitation, we confirmed that PTBP1 binds Atp7a mRNA in C2C12 cells. We used siRNA to knock down Ptbp1 in C2C12 myoblasts and detected increased Atp7a mRNA and protein levels. Our work further defines the post-transcriptional regulation of Atp7a and identifies PTBP1 as a potential regulator of copper homeostasis.

MiR-96-5p Induced by Palmitic Acid Suppresses the Myogenic Differentiation of C2C12 Myoblasts by Targeting FHL1.

Skeletal myogenesis is a multi-stage process that includes the cell cycle exit, myogenic transcriptional activation, and morphological changes to form multinucleated myofibers. Recent studies have shown that saturated fatty acids (SFA) and miRNAs play crucial roles in myogenesis and muscle homeostasis. Nevertheless, the target molecules and myogenic regulatory mechanisms of miRNAs are largely unknown, particularly when myogenesis is dysregulated by SFA deposition. This study investigated the critical role played by miR-96-5p on the myogenic differentiation in C2C12 myoblasts. Long-chain SFA palmitic acid (PA) significantly reduced FHL1 expression and inhibited the myogenic differentiation of C2C12 myoblasts but induced miR-96-5p expression. The knockdown of FHL1 by siRNA stimulated cell proliferation and inhibited myogenic differentiation of myoblasts. Interestingly, miR-96-5p suppressed FHL1 expression by directly targeting the 3’UTR of FHL1 mRNA. The transfection of an miR-96-5p mimic upregulated the expressions of cell cycle-related genes, such as PCNA, CCNB1, and CCND1, and increased myoblast proliferation. Moreover, the miR-96-5p mimic inhibited the expressions of myogenic factors, such as myoblast determination protein (MyoD), myogenin (MyoG), myocyte enhancer factor 2C (MEF2C), and myosin heavy chain (MyHC), and dramatically impeded differentiation and fusion of myoblasts. Overall, this study highlights the role of miR-96-5p in myogenesis via FHL1 suppression and suggests a novel regulatory mechanism for myogenesis mediated by miRNA in a background of obesity.

Single-nucleus RNA-seq identifies transcriptional heterogeneity in multinucleated skeletal myofibers

While the majority of cells contain a single nucleus, cell types such as trophoblasts, osteoclasts, and skeletal myofibers require multinucleation. One advantage of multinucleation can be the assignment of distinct functions to different nuclei, but comprehensive interrogation of transcriptional heterogeneity within multinucleated tissues has been challenging due to the presence of a shared cytoplasm. Here, we utilized single-nucleus RNA-sequencing (snRNA-seq) to determine the extent of transcriptional diversity within multinucleated skeletal myofibers. Nuclei from mouse skeletal muscle were profiled across the lifespan, which revealed the presence of distinct myonuclear populations emerging in postnatal development as well as aging muscle. Our datasets also provided a platform for discovery of genes associated with rare specialized regions of the muscle cell, including markers of the myotendinous junction and functionally validated factors expressed at the neuromuscular junction. These findings reveal that myonuclei within syncytial muscle fibers possess distinct transcriptional profiles that regulate muscle biology.