Multinucleated Muscle Fibers Research Articles

Lineage tracing of nuclei in skeletal myofibers uncovers distinct transcripts and interplay between myonuclear populations

Multinucleated skeletal muscle cells need to acquire additional nuclei through fusion with activated skeletal muscle stem cells when responding to both developmental and adaptive growth stimuli. A fundamental question in skeletal muscle biology has been the reason underlying this need for new nuclei in cells that already harbor hundreds of nuclei. Here we utilize nuclear RNA-sequencing approaches and develop a lineage tracing strategy capable of defining the transcriptional state of recently fused nuclei and distinguishing this state from that of pre-existing nuclei. Our findings reveal the presence of conserved markers of newly fused nuclei both during development and after a hypertrophic stimulus in the adult. However, newly fused nuclei also exhibit divergent gene expression that is determined by the myogenic environment to which they fuse. Moreover, accrual of new nuclei through fusion is required for nuclei already resident in adult myofibers to mount a normal transcriptional response to a load-inducing stimulus. We propose a model of mutual regulation in the control of skeletal muscle development and adaptations, where newly fused and pre-existing myonuclear populations influence each other to maintain optimal functional growth.

Precise quantification of skeletal muscle fibers reveals the physiological basis for growth rate discrepancies in broilers1

Skeletal muscle is composed of multinucleated muscle fibers, which play a crucial role in determining the quality of meat products in livestock. Quantifying the total number of muscle fibers (TNM) is essential for understanding muscle composition, yet remains challenging in poultry, particularly due to the size of the livestock that complicates the preparation of tissue sections for analysis and renders the counting process laborious. Our previous study developed an automatic muscle fiber quantification tool powered by deep learning, named MyoV, which has addressed this bottleneck. This study aimed to employ the tool for the accurate quantification of the TNM in the pectoral muscles of slow-growing (SL), medium-growing (ML), and fast-growing (FL) broilers. Results showed that FL exhibited higher growth performance compared to ML and SL from embryonic to rearing stages. Processing of whole slide images of pectoral muscle revealed significantly higher TNM in FL and ML than in SL (P < 0.01). The TNM of FL, ML and SL were 693,568.00 ± 54,169.80, 652,122.00 ± 65,822.60 and 539,778.57±40,722.94 at 7 days of age (D7), respectively. And the TNM at D35 were 663,014.93±58,801.11, 645,784.76±80,204.34 and 507,280.29±98,092.16 of FL, ML and SL. Differences in cross-sectional area (CSA) of muscle fibers among the three groups were consistent with TNM results. Correlation analysis showed a correlation coefficient of 0.73-0.89 between body weight (BW) and TNM and a correlation coefficient of 0.78-0.87 between BW and CSA. These findings directly indicate that the number of muscle fibers in broilers is an important foundation for their rapid growth and development. This study precisely quantifies the muscle fiber number of important skeletal muscle in poultry for the first time, providing the direct evidence for the physiological basis of rapid development in broilers and offering important data support for further in-depth researches on muscle fiber development.

Establishment and Characterization of a Chicken Myoblast Cell Line.

Skeletal muscle, which is predominantly constituted by multinucleated muscle fibers, plays a pivotal role in sustaining bodily movements and energy metabolism. Myoblasts, which serve as precursor cells for differentiation and fusion into muscle fibers, are of critical importance in the exploration of the functional genes associated with embryonic muscle development. However, the in vitro proliferation of primary myoblasts is inherently constrained. In this study, we achieved a significant breakthrough by successfully establishing a chicken myoblast cell line through the introduction of the exogenous chicken telomerase reverse transcriptase (chTERT) gene, followed by rigorous G418-mediated pressure screening. This newly developed cell line, which was designated as chTERT-myoblasts, closely resembled primary myoblasts in terms of morphology and exhibited remarkable stability in culture for at least 20 generations of population doublings without undergoing malignant transformation. In addition, we conducted an exhaustive analysis that encompassed cellular proliferation, differentiation, and transfection characteristics. Our findings revealed that the chTERT-myoblasts had the ability to proliferate, differentiate, and transfect after multiple rounds of population doublings. This achievement not only furnished a valuable source of homogeneous avian cell material for investigating embryonic muscle development, but also provided valuable insights and methodologies for establishing primary cell lines.

Decoding the muscle transcriptome of patients with late onset Pompe disease reveals markers of disease progression.

Late-onset Pompe Disease (LOPD) is a rare genetic disorder caused by the deficiency of acid alpha-glucosidase leading to progressive cellular dysfunction due to the accumulation of glycogen in the lysosome. The mechanism of relentless muscle damage - a classic manifestation of the disease - has been extensively studied by analysing the whole muscle tissue; however, little, if any, is known about transcriptional heterogeneity among nuclei within the multinucleated skeletal muscle cells. This is the first report of application of single nuclei RNA sequencing to uncover changes in the gene expression profile in muscle biopsies from eight patients with LOPD and four muscle samples from age and gender matched healthy controls. We matched these changes with histology findings using GeoMx Spatial Transcriptomics to compare the transcriptome of control myofibers from healthy individuals with non-vacuolated (histologically unaffected) and vacuolated (histologically affected) myofibers of LODP patients. We observed an increase in the proportion of slow and regenerative muscle fibers and macrophages in LOPD muscles. The expression of the genes involved in glycolysis was reduced, whereas the expression of the genes involved in the metabolism of lipids and amino acids was increased in non-vacuolated fibers, indicating early metabolic abnormalities. Additionally, we detected upregulation of autophagy genes, and downregulation of the genes involved in ribosomal and mitochondrial function leading to defective oxidative phosphorylation. The upregulation of the genes associated with inflammation, apoptosis and muscle regeneration was observed only in vacuolated fibers. Notably, enzyme replacement therapy - the only available therapy for the disease - showed a tendency to restore metabolism dysregulation, particularly within slow fibers. A combination of single nuclei RNA sequencing and spatial transcriptomics revealed the landscape of normal and the diseased muscle, and highlighted the early abnormalities associated with the disease progression. Thus, the application of these two new cutting-edge technologies provided insight into the molecular pathophysiology of muscle damage in LOPD and identified potential avenues for therapeutic intervention.

Temporal regulation of the Mediator complex during muscle proliferation, differentiation, regeneration, aging, and disease.

Genesis of skeletal muscle relies on the differentiation and fusion of mono-nucleated muscle progenitor cells into the multi-nucleated muscle fiber syncytium. The temporally-controlled cellular and morphogenetic changes underlying this process are initiated by a series of highly coordinated transcription programs. At the core, the myogenic differentiation cascade is driven by muscle-specific transcription factors, i.e., the Myogenic Regulatory Factors (MRFs). Despite extensive knowledge on the function of individual MRFs, very little is known about how they are coordinated. Ultimately, highly specific coordination of these transcription programs is critical for their masterfully timed transitions, which in turn facilitates the intricate generation of skeletal muscle fibers from a naïve pool of progenitor cells. The Mediator complex links basal transcriptional machinery and transcription factors to regulate transcription and could be the integral component that coordinates transcription factor function during muscle differentiation, growth, and maturation. In this study, we systematically deciphered the changes in Mediator complex subunit expression in skeletal muscle development, regeneration, aging, and disease. We incorporated our in vitro and in vivo experimental results with analysis of publicly available RNA-seq and single nuclei RNA-seq datasets and uncovered the regulation of Mediator subunits in different physiological and temporal contexts. Our experimental results revealed that Mediator subunit expression during myogenesis is highly dynamic. We also discovered unique temporal patterns of Mediator expression in muscle stem cells after injury and during the early regeneration period, suggesting that Mediator subunits may have unique contributions to directing muscle stem cell fate. Although we observed few changes in Mediator subunit expression in aging muscles compared to younger muscles, we uncovered extensive heterogeneity of Mediator subunit expression in dystrophic muscle nuclei, characteristic of chronic muscle degeneration and regeneration cycles. Taken together, our study provides a glimpse of the complex regulation of Mediator subunit expression in the skeletal muscle cell lineage and serves as a springboard for mechanistic studies into the function of individual Mediator subunits in skeletal muscle.

Yap/Taz activity is associated with increased expression of phosphoglycerate dehydrogenase that supports myoblast proliferation

In skeletal muscle, the Hippo effector Yap promotes satellite cell, myoblast, and rhabdomyoblast proliferation but prevents myogenic differentiation into multinucleated muscle fibres. We previously noted that Yap drives expression of the first enzyme of the serine biosynthesis pathway, phosphoglycerate dehydrogenase (Phgdh). Here, we examined the regulation and function of Phgdh in satellite cells and myoblasts and found that Phgdh protein increased during satellite cell activation. Analysis of published data reveal that Phgdh mRNA in mouse tibialis anterior muscle was highly expressed at day 3 of regeneration after cardiotoxin injection, when markers of proliferation are also robustly expressed and in the first week of synergist-ablated muscle. Finally, siRNA-mediated knockdown of PHGDH significantly reduced myoblast numbers and the proliferation rate. Collectively, our data suggest that Phgdh is a proliferation-enhancing metabolic enzyme that is induced when quiescent satellite cells become activated.

Fast, multiplexable and efficient somatic gene deletions in adult mouse skeletal muscle fibers using AAV-CRISPR/Cas9

Molecular screens comparing different disease states to identify candidate genes rely on the availability of fast, reliable and multiplexable systems to interrogate genes of interest. CRISPR/Cas9-based reverse genetics is a promising method to eventually achieve this. However, such methods are sorely lacking for multi-nucleated muscle fibers, since highly efficient nuclei editing is a requisite to robustly inactive candidate genes. Here, we couple Cre-mediated skeletal muscle fiber-specific Cas9 expression with myotropic adeno-associated virus-mediated sgRNA delivery to establish a system for highly effective somatic gene deletions in mice. Using well-characterized genes, we show that local or systemic inactivation of these genes copy the phenotype of traditional gene-knockout mouse models. Thus, this proof-of-principle study establishes a method to unravel the function of individual genes or entire signaling pathways in adult skeletal muscle fibers without the cumbersome requirement of generating knockout mice.

Cryo-EM structures of Myomaker reveal a molecular basis for myoblast fusion.

The fusion of mononucleated myoblasts produces multinucleated muscle fibers leading to the formation of skeletal muscle. Myomaker, a skeletal muscle-specific membrane protein, is essential for myoblast fusion. Here we report the cryo-EM structures of mouse Myomaker (mMymk) and Ciona robusta Myomaker (cMymk). Myomaker contains seven transmembrane helices (TMs) that adopt a G-protein-coupled receptor-like fold. TMs 2-4 form a dimeric interface, while TMs 3 and 5-7 create a lipid-binding site that holds the polar head of a phospholipid and allows the alkyl tails to insert into Myomaker. The similarity of cMymk and mMymk suggests a conserved Myomaker-mediated cell fusion mechanism across evolutionarily distant species. Functional analyses demonstrate the essentiality of the dimeric interface and the lipid-binding site for fusogenic activity, and heterologous cell-cell fusion assays show the importance of transcellular interactions of Myomaker protomers for myoblast fusion. Together, our findings provide structural and functional insights into the process of myoblast fusion.

Systematic Myostatin Expression Screening Platform for Identification and Evaluation of Myogenesis-Related Phytogenic in Pigs.

Skeletal muscle growth in livestock impacts meat quantity and quality. Concerns arise because certain feed additives, like beta-agonists, may affect food safety. Skeletal muscle is a specialized tissue consisting of nondividing and multinucleated muscle fibers. Myostatin (MSTN), a protein specific to skeletal muscle, is secreted and functions as a negative regulator of muscle mass by inhibiting the proliferation and differentiation of myoblasts. To enhance livestock muscle growth, phytogenic feed additives could be an alternative as they inhibit MSTN activity. The objective of this study was to establish a systematic screening platform using MSTN activity to evaluate phytogenics, providing scientific evidence of their assessment and potency. In this study, we established a screening platform to monitor myostatin promoter activity in rat L8 myoblasts. Extract of Glycyrrhiza uralensis (GUE), an oriental herbal medicine, was identified through this screening platform, and the active fractions of GUE were identified using a process-scale liquid column chromatography system. For in vivo study, GUE as a feed additive was investigated in growth-finishing pigs. The results showed that GUE significantly increased body weight, carcass weight, and lean content in pigs. Microbiota analysis indicated that GUE did not affect the composition of gut microbiota in pigs. In summary, this established rodent myoblast screening platform was used to identify a myogenesis-related phytogenic, GUE, and further demonstrated that the active fractions and compounds inhibited MSTN expression. These findings suggest a novel application for GUE in growth performance enhancement through modulation of MSTN expression. Moreover, this well-established screening platform holds significant potential for identifying and assessing a diverse range of phytogenics that contribute to the process of myogenesis.

Induced Pluripotent Stem Cells for Modeling Physiological and Pathological Striated Muscle Complexity.

Neuromuscular disorders (NMDs) are a large group of diseases associated with either alterations of skeletal muscle fibers, motor neurons or neuromuscular junctions. Most of these diseases is characterized with muscle weakness or wasting and greatly alter the life of patients. Animal models do not always recapitulate the phenotype of patients. The development of innovative and representative human preclinical models is thus strongly needed for modeling the wide diversity of NMDs, characterization of disease-associated variants, investigation of novel genes function, or the development of therapies. Over the last decade, the use of patient's derived induced pluripotent stem cells (hiPSC) has resulted in tremendous progress in biomedical research, including for NMDs. Skeletal muscle is a complex tissue with multinucleated muscle fibers supported by a dense extracellular matrix and multiple cell types including motor neurons required for the contractile activity. Major challenges need now to be tackled by the scientific community to increase maturation of muscle fibers in vitro, in particular for modeling adult-onset diseases affecting this tissue (neuromuscular disorders, cachexia, sarcopenia) and the evaluation of therapeutic strategies. In the near future, rapidly evolving bioengineering approaches applied to hiPSC will undoubtedly become highly instrumental for investigating muscle pathophysiology and the development of therapeutic strategies.

Autophagy modulation in primary culture of porcine satellite cells

Autophagy is a lysosomal degradation pathway with a role in the turnover of cell components via self-digestion. Over the past decade, it has been recognised as an essential process to maintain cellular and energy homeostasis. Nevertheless, little attention has been paid to this process in farm animals. In pigs, the role of autophagy in skeletal muscle homeostasis and more specifically on the formation of multinucleated muscle fibres needs to be determined. Primary culture of satellite cells, the resident muscle stem cells, is an appropriate model to investigate macroautophagy (hereafter autophagy), the main autophagy process. The objective of the current study was to evaluate tools to monitor autophagy in this cell model and to specify the role of autophagy on cell differentiation. Samples of longissimus muscle were collected from 3- to 4-day-old piglets. After isolation, satellite cells were plated in growth medium, allowed to proliferate up to 80% confluence and then placed in an appropriate culture medium to differentiate into myotubes. Cells were explored from day 0 to day 3 of differentiation. Autophagy-related proteins and Adenosine Mono Phosphate-activated protein kinase (AMPK), a major sensor for cell energy, were detected by Western blotting. Expression of genes related to autophagy were also quantified by qPCR. The Microtubule-associated protein 1 light-chain 3β forms ratio increased during cell differentiation whereas phosphatidylinositol 3-kinase and sequestosome 1 proteins decreased significantly. Mitochondrial protein expression also decreased significantly with satellite cell differentiation. Then, cell treatment with an inhibitor of autophagy flux, Bafilomycin A1, confirmed that autophagy was activated during the conversion of myoblasts into myotubes along with AMPK activation in our satellite cell culture model. In conclusion, we provided tools for porcine autophagy investigation in tissues or cells and demonstrated that basal autophagy and energy metabolism are concomitantly modulated during porcine myogenesis in vitro.

I.09 Induced pluripotent stem cells for modeling neuromuscular disorders: development of disease-specific assays, live cells functional testing and drug design

Induced pluripotent stem cells (iPSC) represent an innovative, easily obtained and renewable stem cell source. This somatic cell-derived model that can be derived without considerable ethical issues has generated significant enthusiasm for its potential application in basic and translational research or development of new therapeutics. In particular, patient-specific iPS-derived tissues and latterly, organoids offer attractive platform to model a wide range of diseases but also personalized or regenerative medicine. Compared to other tissues, only a limited number of large-scale protocols describe the production of mature skeletal muscle fibers from human iPSCs (hiPSCs). We developed a procedure for simultaneous differentiation of hiPSC into muscle cells and motor neurons, that generates innervated and contractile multinucleated skeletal muscle fibers with sarcomeric organization and formation of neuromuscular junction. Our protocol permits the production of expandable skeletal muscle progenitor cells and mature skeletal muscle fibers that can be maintained and regenerates on the long-term. These in vitro muscle constructs can be used for the exploration of skeletal muscle differentiation for basic research, disease modeling and drug discovery. Our “muscle in a dish” model has been used to successfully model a number of neuromuscular disorders. By combining transcriptomics with artificial intelligence, we were able to design specific pipelines for a comprehensive understanding of the molecular, cell biological and functional properties of hiPSC-derived muscle tissue in diseases. Overall, this strategy opens a wide rage of opportunities for disease modeling, personalized medicine for patients in diagnosis deadlocks, drug design or tissue bioengineering and bioprinting. Induced pluripotent stem cells (iPSC) represent an innovative, easily obtained and renewable stem cell source. This somatic cell-derived model that can be derived without considerable ethical issues has generated significant enthusiasm for its potential application in basic and translational research or development of new therapeutics. In particular, patient-specific iPS-derived tissues and latterly, organoids offer attractive platform to model a wide range of diseases but also personalized or regenerative medicine. Compared to other tissues, only a limited number of large-scale protocols describe the production of mature skeletal muscle fibers from human iPSCs (hiPSCs). We developed a procedure for simultaneous differentiation of hiPSC into muscle cells and motor neurons, that generates innervated and contractile multinucleated skeletal muscle fibers with sarcomeric organization and formation of neuromuscular junction. Our protocol permits the production of expandable skeletal muscle progenitor cells and mature skeletal muscle fibers that can be maintained and regenerates on the long-term. These in vitro muscle constructs can be used for the exploration of skeletal muscle differentiation for basic research, disease modeling and drug discovery. Our “muscle in a dish” model has been used to successfully model a number of neuromuscular disorders. By combining transcriptomics with artificial intelligence, we were able to design specific pipelines for a comprehensive understanding of the molecular, cell biological and functional properties of hiPSC-derived muscle tissue in diseases. Overall, this strategy opens a wide rage of opportunities for disease modeling, personalized medicine for patients in diagnosis deadlocks, drug design or tissue bioengineering and bioprinting.

Evolution of a chordate-specific mechanism for myoblast fusion.

Vertebrate myoblast fusion allows for multinucleated muscle fibers to compound the size and strength of mononucleated cells, but the evolution of this important process is unknown. We investigated the evolutionary origins and function of membrane-coalescing agents Myomaker and Myomixer in various groups of chordates. Here, we report that Myomaker likely arose through gene duplication in the last common ancestor of tunicates and vertebrates, while Myomixer appears to have evolved de novo in early vertebrates. Functional tests revealed a complex evolutionary history of myoblast fusion. A prevertebrate phase of muscle multinucleation driven by Myomaker was followed by the later emergence of Myomixer that enables the highly efficient fusion system of vertebrates. Evolutionary comparisons between vertebrate and nonvertebrate Myomaker revealed key structural and mechanistic insights into myoblast fusion. Thus, our findings suggest an evolutionary model of chordate fusogens and illustrate how new genes shape the emergence of novel morphogenetic traits and mechanisms.

Epigenetic Control of Muscle Stem Cells: Focus on Histone Lysine Demethylases.

Adult skeletal muscle is mainly composed of post-mitotic, multinucleated muscle fibers. Upon injury, it has the unique ability to regenerate thanks to the activation of a subset of quiescent muscle stem cells (MuSCs). Activated MuSCs either differentiate to repair muscle, or self-renew to maintain the pool of MuSC. MuSC fate determination is regulated by an intricate network of intrinsic and extrinsic factors that control the expression of specific subsets of genes. Among these, the myogenic regulatory factors (MRFs) are key for muscle development, cell identity and regeneration. More globally, cell fate determination involves important changes in the epigenetic landscape of the genome. Such epigenetic changes, which include DNA methylation and post-translational modifications of histone proteins, are able to alter chromatin organization by controlling the accessibility of specific gene loci for the transcriptional machinery. Among the numerous epigenetic modifications of chromatin, extensive studies have pointed out the key role of histone methylation in cell fate control. Particularly, since the discovery of the first histone demethylase in 2004, the role of histone demethylation in the regulation of skeletal muscle differentiation and muscle stem cell fate has emerged to be essential. In this review, we highlight the current knowledge regarding the role of histone demethylases in the regulation of muscle stem cell fate choice.

Myonuclei Can Replicate DNA

Skeletal muscle is a heterogenous tissue composed of multinucleated muscle fibers and accompanying supporting cells like satellite cells (SCs). Myonuclei are believed to be post‐mitotic, arrested in G0 phase of the cell cycle and thus unable to proliferate. Consequently, it is hypothesized that growth of muscle is dependent on the donation of nuclei from SCs. Although some studies suggest that under certain conditions myonuclei can replicate, it has been difficult to address whether this replicative potential exists in vivo. We hypothesized that skeletal muscle resident myonuclei possess the ability to replicate in vivo. To test this hypothesis, we used the HSArtTA;Tet‐O‐H2B‐GFP (HSA‐H2B) mouse model, which uses doxycycline (DOX) treatment for temporal labeling (Tet‐ON) of residential myonuclei. After removing DOX, and thus turning off GFP‐labeling, we provided the stable isotope deuterium oxide (D2O) for 8 weeks to measure DNA synthesis. At the end of D2O labeling, muscles were harvested and GFP‐positive myonuclei were sorted using an antibody‐independent approach and a restrictive gating strategy. Both GFP+ (myonuclei) and GFP‐ (non‐myonuclei) fractions were then analyzed for D2O incorporation into DNA by GC‐QQQ. By our approach, DNA from GFP‐positive myonuclei was deuterium‐enriched and therefore deemed to be replicating. DNA replication occurred in at least one muscle of the 7 mice studied. We observed deuterium enrichment into DNA in 6 out of 7 plantaris (PLA), 4 out of 7 extensor digitorum longus (EDL), 4 out of 7 tibialis anterior (TA), 6 out of 7 gastrocnemius (GAS) and 7 out of 7 quadriceps (QUAD). The average fractional synthesis rates (FSR, %/day) of replicating myonuclei were: 0.0351 ± 0.017 in PLA, 0.0156 ± 0.015 in EDL, 0.0220 ± 0.018 in TA, 0.0223 ± 0.018 in GAS, and 0.02692 ± 0.043 in QUAD. These data are the first to unambiguously demonstrate that myonuclei replicate DNA in vivo. The proposed project challenges current dogma and might significantly impact skeletal muscle treatment to maintain muscle mass with age and disease.

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.

Changes in Myonuclear Number During Postnatal Growth - Implications for AAV Gene Therapy for Muscular Dystrophy.

Adult skeletal muscle is a relatively stable tissue, as the multinucleated muscle fibres contain post-mitotic myonuclei. During early postnatal life, muscle growth occurs by the addition of skeletal muscle stem cells (satellite cells) or their progeny to growing muscle fibres. In Duchenne muscular dystrophy, which we shall use as an example of muscular dystrophies, the muscle fibres lack dystrophin and undergo necrosis. Satellite-cell mediated regeneration occurs, to repair and replace the necrotic muscle fibres, but as the regenerated muscle fibres still lack dystrophin, they undergo further cycles of degeneration and regeneration.AAV gene therapy is a promising approach for treating Duchenne muscular dystrophy. But for a single dose of, for example, AAV coding for microdystrophin, to be effective, the treated myonuclei must persist, produce sufficient dystrophin and a sufficient number of nuclei must be targeted. This latter point is crucial as AAV vector remains episomal and does not replicate in dividing cells. Here, we describe and compare the growth of skeletal muscle in rodents and in humans and discuss the evidence that myofibre necrosis and regeneration leads to the loss of viral genomes within skeletal muscle. In addition, muscle growth is expected to lead to the dilution of the transduced nuclei especially in case of very early intervention, but it is not clear if growth could result in insufficient dystrophin to prevent muscle fibre breakdown. This should be the focus of future studies.

Lipid droplets contribute myogenic differentiation in C2C12 by promoting the remodeling of the acstin-filament

Lipid droplet (LD), a multi-functional organelle, is found in most eukaryotic cells. LDs participate in the regulation of many cellular processes including proliferation, stress, and apoptosis. Previous studies showed the athlete’s paradox that trained athletes accumulate LDs in their skeletal muscle. However, the impact of LDs on skeletal muscle and myogenesis is not clear. We discovered that C2C12 myoblast cells containing more LDs formed more multinucleated muscle fibers. We also discovered that LDs promoted cell migration and fusion by promoting actin-filaments remodeling. Mechanistically, two LD-proteins, Acyl-CoA synthetase long chain family member 3 (ACSL3) and lysophosphatidylcholine acyltransferase 1 (LPCAT1), medicated the recruitment of actinin proteins which contributed to actin-filaments formation on the surface of LDs. During remodeling, the actinin proteins on LDs surface translocated to actin-filaments via ARF1/COPI vesicles. Our study demonstrate LDs contribute to cell differentiation, which lead to new insight into the LD function.

CDC50A is required for aminophospholipid transport and cell fusion in mouse C2C12 myoblasts.

Myoblast fusion is essential for the formation of multinucleated muscle fibers and is promoted by transient changes in the plasma membrane lipid distribution. However, little is known about the lipid transporters regulating these dynamic changes. Here, we show that proliferating myoblasts exhibit an aminophospholipid flippase activity that is downregulated during differentiation. Deletion of the P4-ATPase flippase subunit CDC50A (also known as TMEM30A) results in loss of the aminophospholipid flippase activity and compromises actin remodeling, RAC1 GTPase membrane targeting and cell fusion. In contrast, deletion of the P4-ATPase ATP11A affects aminophospholipid uptake without having a strong impact on cell fusion. Our results demonstrate that myoblast fusion depends on CDC50A and may involve multiple CDC50A-dependent P4-ATPases that help to regulate actin remodeling.

Ultrastructural pathology of bioprosthetic heart valves with infectious endocarditis

Aim. To study the ultrastructure of mitral bioprosthetic heart valves (BHVs) which failed due to infective endocarditis.Materials and Methods. Here we examined 7 ethylene glycol diglycidyl ether-treated xenopericardial BHVs excised during repeated BHV replacement because of prosthetic endocarditis. After being fixed in formalin and postfixed in osmium tetroxide, BHVs were dehydrated and stained in uranyl acetate with the subsequent embedding into epoxy resin, grinding, polishing, and lead citrate counterstaining. Upon the sputter coating with carbon, we visualised the BHV microanatomy by means of backscattered scanning electron microscopy at 15 kV voltage.Results. The extracellular matrix underwent degradation and disintegration resulting in loosening, fragmentation, and reduction in the electron density of collagen and elastin fibers. We observed a number of recipient cells (macrophages, multinucleated giant cells, neutrophils, endothelial cells and smooth muscle cells) within the BHVs. The highest number of cells was localized on the valve surfaces. The localization of the recipient cells on the ventricular and atrial surfaces was different. The central part of the valves was abundantly populated by macrophages.Conclusion. Prosthetic endocarditis is accompanied by the migration of recipient cells into the BHV structure, which is the consequence of surface and extracellular matrix disintegration.