Differentiation Of Myogenic Progenitor Cells Research Articles

FLII Modulates the Myogenic Differentiation of Progenitor Cells via Actin Remodeling-Mediated YAP1 Regulation.

The dynamic rearrangement of the actin cytoskeleton plays an essential role in myogenesis, which is regulated by diverse mechanisms, such as mechanotransduction, modulation of the Hippo signaling pathway, control of cell proliferation, and the influence of morphological changes. Despite the recognized importance of actin-binding protein Flightless-1 (FLII) during actin remodeling, the role played by FLII in the differentiation of myogenic progenitor cells has not been explored. Here, we investigated the roles of FLII in the proliferation and differentiation of myoblasts. FLII was found to be enriched in C2C12 myoblasts, and its expression was stable during the early stages of differentiation but down-regulated in fully differentiated myotubes. Knockdown of FLII in C2C12 myoblasts resulted in filamentous actin (F-actin) accumulation and inhibited Yes-associated protein 1 (YAP1) phosphorylation, which triggers its nuclear translocation from the cytoplasm. Consequently, the expressions of YAP1 target genes, including PCNA, CCNB1, and CCND1, were induced, and the cell cycle and proliferation of myoblasts were promoted. Moreover, FLII knockdown significantly inhibited the expression of myogenic regulatory factors, i.e., MyoD and MyoG, thereby impairing myoblast differentiation, fusion, and myotube formation. Thus, our findings demonstrate that FLII is crucial for the differentiation of myoblasts via modulation of the F-actin/YAP1 axis and suggest that FLII is a putative novel therapeutic target for muscle wasting.

Induction of miR-665-3p Impairs the Differentiation of Myogenic Progenitor Cells by Regulating the TWF1-YAP1 Axis.

Actin dynamics are known to orchestrate various myogenic processes in progenitor cells. Twinfilin-1 (TWF1) is an actin-depolymerizing factor that plays a crucial role in the differentiation of myogenic progenitor cells. However, little is known about the mechanisms underlying the epigenetic regulation of TWF1 expression and impaired myogenic differentiation in the background of muscle wasting. This study investigated how miR-665-3p affects TWF1 expression, actin filaments' modulation, proliferation, and myogenic differentiation in progenitor cells. Palmitic acid, the most prevalent saturated fatty acid (SFA) in food, suppressed TWF1 expression and inhibited the myogenic differentiation of C2C12 cells while increasing the level of miR-665-3p expression. Interestingly, miR-665-3p inhibited TWF1 expression by targeting TWF1 3'UTR directly. In addition, miR-665-3p accumulated filamentous actin (F-actin) and enhanced the nuclear translocation of Yes-associated protein 1 (YAP1), consequently promoting cell cycle progression and proliferation. Furthermore, miR-665-3p suppressed the expressions of myogenic factors, i.e., MyoD, MyoG, and MyHC, and consequently impaired myoblast differentiation. In conclusion, this study suggests that SFA-inducible miR-665-3p suppresses TWF1 expression epigenetically and inhibits myogenic differentiation by facilitating myoblast proliferation via the F-actin/YAP1 axis.

Structural and ultrastructural changes in the skeletal muscles of dysferlin-deficient mice during postnatal ontogenesis

ABSTRACT A number of sarcolemma proteins are responsible for muscle fiber repair. Dysferlin encoded by the DYSF gene is one of these proteins. Dysferlin promotes membrane repair in striated muscle fibers (MFs). Mutations in DYSF lead to loss of or decreased dysferlin expression, impaired membrane repair in MF, and its destruction, clinically manifesting as dysferlinopathy. Preclinical studies of cell and gene therapies aimed at restoring impaired muscle regeneration require well-characterized small animal models. Our investigation aimed to distinguish the histopathological features of a mouse strain lacking dysferlin expression (Bla/J strain). Ultrastructural changes in the sarcolemma, mitochondria and contractile apparatus were observed. It was shown that postnatal histogenesis of skeletal muscles in genetically determined dysferlin deficiency is characterized by a higher proportion of necrotic muscle fibers, compensatory hypertrophy of muscle fibers with their subsequent atrophy, and decreases in proliferative activity and the level of myogenic differentiation of myogenic progenitor cells compared to wild-type mice (C57Bl/6).

Twinfilin-1 is an essential regulator of myogenic differentiation through the modulation of YAP in C2C12 myoblasts

Actin cytoskeletal dynamics play a critical role in the regulation of myogenesis through mechanotransduction, Hippo signaling modulation, cell proliferation, and morphological changes. Although Twinfilin-1 (TWF1), a highly conserved actin-depolymerizing factor, is known to regulate actin filament assembly by sequestering actin monomer and capping barbed ends, the biological significance of TWF1 during the differentiation of myogenic progenitor cells has not been investigated. In this study, we unveiled the roles played by TWF1 in the proliferation and differentiation of C2C12 myoblasts. TWF1 was the predominant isoform in myoblasts, and its expression was induced during the early stage of differentiation. Knockdown of TWF1 by siRNA (siTWF1) induced the accumulation of actin filaments (F-actin) and promoted the nuclear translocation of Yes-associated protein (YAP) in the Hippo signaling pathway. TWF1 depletion activated transcription of YAP target genes and induced cell cycle and proliferation in myoblasts. Furthermore, TWF1 knockdown markedly reduced the expressions of myogenic regulatory factors, such as MyoD and MyoG, and drastically hindered myoblast differentiation, fusion, and myotube formation. Collectively, this study highlights the essential role of TWF1 in the myogenic differentiation of progenitor cells via modulation of F-actin and YAP, and suggests TWF1 as a potential therapeutic target for muscle wasting and myopathies.

MiR-320-3p Regulates the Proliferation and Differentiation of Myogenic Progenitor Cells by Modulating Actin Remodeling.

Skeletal myogenesis is essential for the maintenance of muscle quality and quantity, and impaired myogenesis is intimately associated with muscle wasting diseases. Although microRNA (miRNA) plays a crucial role in myogenesis and relates to muscle wasting in obesity, the molecular targets and roles of miRNAs modulated by saturated fatty acids (SFA) are largely unknown. In the present study, we investigated the role of miR-320-3p on the differentiation of myogenic progenitor cells. Palmitic acid (PA), the most abundant dietary SFA, suppressed myogenic factors expression and impaired differentiation in C2C12 myoblasts, and these effects were accompanied by CFL2 downregulation and miR-320-3p upregulation. In particular, miR-320-3p appeared to target CFL2 mRNA directly and suppress the expression of CFL2, an essential factor for filamentous actin (F-actin) depolymerization. Transfection of myoblasts with miR-320-3p mimic increased F-actin formation and nuclear translocation of Yes-associated protein 1 (YAP1), a key component of mechanotransduction. Furthermore, miR-320-3p mimic increased myoblast proliferation and markedly impeded the expression of MyoD and MyoG, consequently inhibiting myoblast differentiation. In conclusion, our current study highlights the role of miR-320-3p on CFL2 expression, YAP1 activation, and myoblast differentiation and suggests that PA-inducible miR-320-3p is a significant mediator of muscle wasting in obesity.

Complement C3 of the innate immune system secreted by muscle adipogenic cells promotes myogenic differentiation

Myogenic differentiation results in different cell type cooperation, but the molecules involved in the myogenic cell activation remain elusive. Here, we show that muscle-resident pre-adipocytes promote myogenic differentiation through the secretion of factors. Using proteomic and transcriptomic analyses, we identified that proliferative adipogenic lineage cells produce and secrete a key factor of the innate immune system, the complement C3. Cell culture experiments revealed that C3 promotes the differentiation of myogenic progenitors following internalisation of the immune molecule. These data demonstrate that the third component of the complement system, which is a pivotal factor in the immune response to pathogens, is also involved in the differentiation of myogenic progenitor cells.

Pask integrates hormonal signaling with histone modification via Wdr5 phosphorylation to drive myogenesis.

PAS domain containing protein kinase (Pask) is an evolutionarily conserved protein kinase implicated in energy homeostasis and metabolic regulation across eukaryotic species. We now describe an unexpected role of Pask in promoting the differentiation of myogenic progenitor cells, embryonic stem cells and adipogenic progenitor cells. This function of Pask is dependent upon its ability to phosphorylate Wdr5, a member of several protein complexes including those that catalyze histone H3 Lysine 4 trimethylation (H3K4me3) during transcriptional activation. Our findings suggest that, during myoblast differentiation, Pask stimulates the conversion of repressive H3K4me1 to activating H3K4me3 marks on the promoter of the differentiation gene myogenin (Myog) via Wdr5 phosphorylation. This enhances accessibility of the MyoD transcription factor and enables transcriptional activation of the Myog promoter to initiate muscle differentiation. Thus, as an upstream kinase of Wdr5, Pask integrates signaling cues with the transcriptional network to regulate the differentiation of progenitor cells.

Increased Stiffness in Aged Skeletal Muscle Impairs Muscle Progenitor Cell Proliferative Activity

BackgroundSkeletal muscle aging is associated with a decreased regenerative potential due to the loss of function of endogenous stem cells or myogenic progenitor cells (MPCs). Aged skeletal muscle is characterized by the deposition of extracellular matrix (ECM), which in turn influences the biomechanical properties of myofibers by increasing their stiffness. Since the stiffness of the MPC microenvironment directly impacts MPC function, we hypothesized that the increase in muscle stiffness that occurs with aging impairs the behavior of MPCs, ultimately leading to a decrease in regenerative potential.ResultsWe showed that freshly isolated individual myofibers from aged mouse muscles contain fewer MPCs overall than myofibers from adult muscles, with fewer quiescent MPCs and more proliferative and differentiating MPCs. We observed alterations in cultured MPC behavior in aged animals, where the proliferation and differentiation of MPCs were lower and higher, respectively. These alterations were not linked to the intrinsic properties of aged myofibers, as shown by the similar values for the cumulative population-doubling values and fusion indexes. However, atomic force microscopy (AFM) indentation experiments revealed a nearly 4-fold increase in the stiffness of the MPC microenvironment. We further showed that the increase in stiffness is associated with alterations to muscle ECM, including the accumulation of collagen, which was correlated with higher hydroxyproline and advanced glycation end-product content. Lastly, we recapitulated the impaired MPC behavior observed in aging using a hydrogel substrate that mimics the stiffness of myofibers.ConclusionsThese findings provide novel evidence that the low regenerative potential of aged skeletal muscle is independent of intrinsic MPC properties but is related to the increase in the stiffness of the MPC microenvironment.

Notch Signalling Is Required for the Formation of Structurally Stable Muscle Fibres in Zebrafish

BackgroundAccurate regulation of Notch signalling is central for developmental processes in a variety of tissues, but its function in pectoral fin development in zebrafish is still unknown.Methodology/Principal FindingsHere we show that core elements necessary for a functional Notch pathway are expressed in developing pectoral fins in or near prospective muscle territories. Blocking Notch signalling at different levels of the pathway consistently leads to the formation of thin, wavy, fragmented and mechanically weak muscles fibres and loss of stress fibres in endoskeletal disc cells in pectoral fins. Although the structural muscle genes encoding Desmin and Vinculin are normally transcribed in Notch-disrupted pectoral fins, their proteins levels are severely reduced, suggesting that weak mechanical forces produced by the muscle fibres are unable to stabilize/localize these proteins. Moreover, in Notch signalling disrupted pectoral fins there is a decrease in the number of Pax7-positive cells indicative of a defect in myogenesis.Conclusions/SignificanceWe propose that by controlling the differentiation of myogenic progenitor cells, Notch signalling might secure the formation of structurally stable muscle fibres in the zebrafish pectoral fin.

Community effect triggers terminal differentiation of myogenic cells derived from muscle satellite cells by quenching Smad signaling

A high concentration of bone morphogenetic proteins (BMPs) stimulates myogenic progenitor cells to undergo heterotopic osteogenic differentiation. However, the physiological role of the Smad signaling pathway during terminal muscle differentiation has not been resolved. We report here that Smad1/5/8 was phosphorylated and activated in undifferentiated growing mouse myogenic progenitor Ric10 cells without exposure to any exogenous BMPs. The amount of phosphorylated Smad1/5/8 was severely reduced during precocious myogenic differentiation under the high cell density culture condition even in growth medium supplemented with a high concentration of serum. Inhibition of the Smad signaling pathway by dorsomorphin, an inhibitor of Smad activation, or noggin, a specific antagonist of BMP, induced precocious terminal differentiation of myogenic progenitor cells in a cell density-dependent fashion even in growth medium. In addition, Smad1/5/8 was transiently activated in proliferating myogenic progenitor cells during muscle regeneration in rats. The present results indicate that the Smad signaling pathway is involved in a critical switch between growth and differentiation of myogenic progenitor cells both in vitro and in vivo. Furthermore, precocious cell density-dependent myogenic differentiation suggests that a community effect triggers the terminal muscle differentiation of myogenic cells by quenching the Smad signaling.

The influence of extracellular matrix derived from skeletal muscle tissue on the proliferation and differentiation of myogenic progenitor cells ex vivo

Skeletal muscle relies upon regeneration to maintain homeostasis and repair injury. This process involves the recruitment of the tissue's resident stem cell, the muscle progenitor cell, and a subsequent proliferative response by newly generated myoblasts, which must then align and fuse to generate new muscle fibers. During regeneration, cells rely on environmental input for direction. Extracellular matrix (ECM) represents a crucial component of a cell's microenvironment that aids in guiding muscle regeneration. We hypothesized that ECM extracted from skeletal muscle would provide muscle progenitor cells and myoblasts with an ideal substrate for growth and differentiation ex vivo. To test this hypothesis, we developed a method to extract ECM from the large thigh muscles of adult rats and present it to cells as a surface coating. Myogenic cells cultured on ECM extract experienced enhanced proliferation and differentiation relative to standard growth surfaces. As the methodology can be applied to any size muscle, these results demonstrate that bioactive ECM can be readily obtained from skeletal muscle and used to develop biomaterials that enhance muscle regeneration. Furthermore, the model system demonstrated here can be applied to the study of interactions between the ECM of a particular tissue and a cell population of interest.

Environment and plasticity of myogenesis in teleost fish

Embryonic development in teleosts is profoundly affected by environmental conditions, particularly temperature and dissolved oxygen concentrations. The environment determines the rate of myogenesis, the composition of sub-cellular organelles, patterns of gene expression, and the number and size distribution of muscle fibres. During the embryonic and larval stages, muscle plasticity to the environment is usually irreversible due to the rapid pace of ontogenetic change. In the early life stages, muscle can affect locomotory performance and behaviour, with potential consequences for larval survival. Postembryonic growth involves myogenic progenitor cells (MPCs) that originate in the embryo. The embryonic temperature regime can have long-term consequences for the growth of skeletal muscle in some species, including the duration and intensity of myotube formation in adult stages. In juvenile and adult fish, abiotic (temperature, day-length, water flow characteristics, hypoxia) and biotic factors (food availability, parasitic infection) have complex effects on the signalling pathways regulating the proliferation and differentiation of MPCs, protein synthesis and degradation, and patterns of gene expression. The phenotypic responses observed to the environment frequently vary during ontogeny and are integrated with endogenous physiological rhythms, particularly sexual maturation. Studies with model teleosts provide opportunities for investigating the underlying genetic mechanisms of muscle plasticity that can subsequently be applied to non-model species of more ecological or commercial interest.

Geometric control of myogenic cell fate

This work combines expertise in stem cell biology and bioengineering to define the system for geometric control of proliferation and differentiation of myogenic progenitor cells. We have created an artificial niche of myogenic progenitor cells, namely, modified extracellular matrix (ECM) substrates with spatially embedded growth or differentiation factors (GF, DF) that predictably direct muscle cell fate in a geometric pattern. Embedded GF and DF signal progenitor cells from specifically defined areas on the ECM successfully competed against culture media for myogenic cell fate determination at a clearly defined boundary. Differentiation of myoblasts into myotubes is induced in growth-promoting medium, myotube formation is delayed in differentiation-promoting medium, and myogenic cells, at different stages of proliferation and differentiation, can be induced to coexist adjacently in identical culture media. This method can be used to identify molecular interactions between cells in different stages of myogenic differentiation, which are likely to be important determinants of tissue repair. The designed ECM niches can be further developed into a vehicle for transplantation of myogenic progenitor cells maintaining their regenerative potential. Additionally, this work may also serve as a general model to engineer synthetic cellular niches to harness the regenerative potential of organ stem cells.