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Autophagy Sep 2019Macroautophagy/autophagy is a degradative process essential for various cellular processes. We previously demonstrated that autophagy-deficiency causes myoblast...
Macroautophagy/autophagy is a degradative process essential for various cellular processes. We previously demonstrated that autophagy-deficiency causes myoblast apoptosis and impairs myotube formation. In this study, we continued this work with particular emphasis on mitochondrial remodelling and stress/apoptotic signaling. We found increased (p < 0.05) autophagic (e.g., altered LC3B levels, increased ATG7, decreased SQSTM1) and mitophagic (e.g., BNIP3 upregulation, mitochondrial localized GFP-LC3 puncta, and elevated mitochondrial LC3B-II) signaling during myoblast differentiation. shRNA-mediated knockdown of ATG7 (sh) decreased these autophagic and mitophagic responses, while increasing CASP3 activity and ANXA5/annexin V staining in differentiating myoblasts; ultimately resulting in dramatically impaired myogenesis. Further confirming the importance of mitophagy in these responses, CRISPR-Cas9-mediated knockout of () resulted in increased CASP3 activity and DNA fragmentation as well as impaired myoblast differentiation. In addition, sh myoblasts displayed greater endoplasmic reticulum (e.g., increased CAPN activity and HSPA) and mitochondrial (e.g., mPTP formation, reduced mitochondrial membrane potential, elevated mitochondrial 4-HNE) stress. sh and myoblasts also displayed altered mitochondria-associated signaling (e.g., PPARGC1A, DNM1L, OPA1) and protein content (e.g., SLC25A4, VDAC1, CYCS). Moreover, sh myoblasts displayed CYCS and AIFM1 release from mitochondria, and CASP9 activation. Similarly, myoblasts had significantly higher CASP9 activation during differentiation. Importantly, administration of a chemical inhibitor of CASP9 (Ac-LEHD-CHO) or dominant-negative CASP9 (ad-DNCASP9) partially recovered differentiation and myogenesis in sh myoblasts. Together, these data demonstrate an essential role for autophagy in protecting myoblasts from mitochondrial oxidative stress and apoptotic signaling during differentiation, as well as in the regulation of mitochondrial network remodelling and myogenesis. : 3MA: 3-methyladenine; 4-HNE: 4-hydroxynonenal; ACT: actin; AIFM1/AIF: apoptosis-inducing factor, mitochondrion-associated 1; ANXA5: annexin V; ATG7: autophagy related 7; AU: arbitrary units; BAX: BCL2-associated X protein; BCL2: B cell leukemia/lymphoma 2; BECN1: beclin 1, autophagy related; BNIP3: BCL2/adenovirus E1B interacting protein 3; CAPN: calpain; CASP: caspase; CASP3: caspase 3; CASP8: caspase 8; CASP9: caspase 9; CASP12: caspase 12; CAT: catalase; CQ: chloroquine; CYCS: cytochrome c, somatic; DCF; 2',7'-dichlorofluorescein; DNM1L/DRP1: dynamin 1-like; DM: differentiation media; DMEM: Dulbecco's modified Eagle's medium; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GM: growth media; p-H2AFX: phosphorylated H2A histone family, member X; H2BFM: H2B histone family, member M; HBSS: Hanks balanced salt solution; HSPA/HSP70: heat shock protein family A; JC-1: tetraethylbenzimidazolylcarbocyanine iodide; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; mPTP: mitochondrial permeability transition pore; MYH: myosin heavy chain; MYOG: myogenin; OPA1: OPA1, mitochondrial dynamin like GTPase; PI: propidium iodide; PINK1: PTEN induced putative kinase 1; PPARGC1A/PGC1α: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; ROS: reactive oxygen species; SLC25A4/ANT1: solute carrier family 25 (mitochondrial carrier, adenine nucleotide translocator), member 4; SOD1: superoxide dismutase 1, soluble; SOD2: superoxide dismutase 2, mitochondrial; SQSTM1/p62: sequestosome 1; VDAC1: voltage-dependent anion channel 1.
Topics: Animals; Apoptosis; Autophagy-Related Protein 7; Caspase 3; Caspase 9; Cell Differentiation; Cell Line; Endoplasmic Reticulum; Membrane Proteins; Mice; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Proteins; Mitophagy; Muscle Development; Muscle, Skeletal; Myoblasts; Oxidative Stress; Reactive Oxygen Species
PubMed: 30859901
DOI: 10.1080/15548627.2019.1591672 -
Experimental Neurology Jan 2020Muscular dystrophies are a group of genetic muscle disorders that cause progressive muscle weakness and degeneration. Within this group, Duchenne muscular dystrophy... (Review)
Review
Muscular dystrophies are a group of genetic muscle disorders that cause progressive muscle weakness and degeneration. Within this group, Duchenne muscular dystrophy (DMD) is the most common and one of the most severe. DMD is an X chromosome linked disease that occurs to 1 in 3500 to 1 in 5000 boys. The cause of DMD is a mutation in the dystrophin gene, whose encoded protein provides both structural support and cell signaling capabilities. So far, there are very limited therapeutic options available and there is no cure for this disease. In this review, we discuss the existing cell therapy research, especially stem cell-based, which utilize myoblasts, satellite cells, bone marrow cells, mesoangioblasts and CD133+ cells. Finally, we focus on human pluripotent stem cells (hPSCs) which hold great potential in treating DMD. hPSCs can be used for autologous transplantation after being specified to a myogenic lineage. Over the last few years, there has been a rapid development of isolation, as well as differentiation, techniques in order to achieve effective transplantation results of myogenic cells specified from hPSCs. In this review, we summarize the current methods of hPSCs myogenic commitment/differentiation, and describe the current status of hPSC-derived myogenic cell transplantation.
Topics: Cell Differentiation; Humans; Muscular Dystrophy, Duchenne; Myoblasts; Pluripotent Stem Cells; Stem Cell Transplantation
PubMed: 31639376
DOI: 10.1016/j.expneurol.2019.113086 -
Journal of Cachexia, Sarcopenia and... Dec 2021Transmembrane proteins are vital for intercellular signalling and play important roles in the control of cell fate. However, their physiological functions and mechanisms...
BACKGROUND
Transmembrane proteins are vital for intercellular signalling and play important roles in the control of cell fate. However, their physiological functions and mechanisms of action in myogenesis and muscle disorders remain largely unexplored. It has been found that transmembrane protein 182 (TMEM182) is dramatically up-regulated during myogenesis, but its detailed functions remain unclear. This study aimed to analyse the function of TMEM182 during myogenesis and muscle regeneration.
METHODS
RNA sequencing, quantitative real-time polymerase chain reaction, and immunofluorescence approaches were used to analyse TMEM182 expression during myoblast differentiation. A dual-luciferase reporter assay was used to identify the promoter region of the TMEM182 gene, and a chromatin immunoprecipitation assay was used to investigate the regulation TMEM182 transcription by MyoD. We used chickens and TMEM182-knockout mice as in vivo models to examine the function of TMEM182 in muscle growth and muscle regeneration. Chickens and mouse primary myoblasts were used to extend the findings to in vitro effects on myoblast differentiation and fusion. Co-immunoprecipitation and mass spectrometry were used to identify the interaction between TMEM182 and integrin beta 1 (ITGB1). The molecular mechanism by which TMEM182 regulates myogenesis and muscle regeneration was examined by Transwell migration, cell wound healing, adhesion, glutathione-S-transferse pull down, protein purification, and RNA immunoprecipitation assays.
RESULTS
TMEM182 was specifically expressed in skeletal muscle and adipose tissue and was regulated at the transcriptional level by the myogenic regulatory factor MyoD1. Functionally, TMEM182 inhibited myoblast differentiation and fusion. The in vivo studies indicated that TMEM182 induced muscle fibre atrophy and delayed muscle regeneration. TMEM182 knockout in mice led to significant increases in body weight, muscle mass, muscle fibre number, and muscle fibre diameter. Skeletal muscle regeneration was accelerated in TMEM182-knockout mice. Furthermore, we revealed that the inhibitory roles of TMEM182 in skeletal muscle depend on ITGB1, an essential membrane receptor involved in cell adhesion and muscle formation. TMEM182 directly interacted with ITGB1, and this interaction required an extracellular hybrid domain of ITGB1 (aa 387-470) and a conserved region (aa 52-62) within the large extracellular loop of TMEM182. Mechanistically, TMEM182 modulated ITGB1 activation by coordinating the association between ITGB1 and laminin and regulating the intracellular signalling of ITGB1. Myogenic deletion of TMEM182 increased the binding activity of ITGB1 to laminin and induced the activation of the FAK-ERK and FAK-Akt signalling axes during myogenesis.
CONCLUSIONS
Our data reveal that TMEM182 is a novel negative regulator of myogenic differentiation and muscle regeneration.
Topics: Animals; Cell Differentiation; Chickens; Integrin beta1; Integrins; Mice; Muscle, Skeletal; Myoblasts; Regeneration
PubMed: 34427057
DOI: 10.1002/jcsm.12767 -
Biomedicine & Pharmacotherapy =... Dec 2019A high glucose level is usually considered to be the factor that induces tissue and cell dysfunction and damage, commonly known as "glucose toxicity".
BACKGROUND
A high glucose level is usually considered to be the factor that induces tissue and cell dysfunction and damage, commonly known as "glucose toxicity".
OBJECTIVE
This study aimed to explore the effects and the potential molecular mechanisms of high glucose on myoblast differentiation and insulin sensitivity.
MATERIALS AND METHODS
C2C12 cells were cultured in differentiation medium containing 25, 40, or 60 mM glucose for 1, 3, or 5 days. E-MHC positive area and GLUT4 fluorescence were evaluated through Immunofluorescence. The expression of Myf5, MyoD, myogenin were measured by performing western blot and qRT-PCR. The protein expression of GLUT4 on cell membrane and glucose uptake in C2C12 myotubes were measured through western blot and 2-NBDG assay. AKT activator SC79 and inhibitor MK2206 was utilized to reveal the important role of AKT signaling in myogenesis and insulin sensitivity inhibited by high glucose.
RESULTS
60 mM glucose inhibits myogenesis by decreasing the expression of MyoD and myogenin, and induces insulin resistance by reducing both basal and insulin-stimulated GLUT4 expressions and glucose uptakes. The influences of high glucose on myogenesis and IR was related to decreased AKT activation. SC79 rescued the inhibition of high glucose on myogenesis and attenuated IR. MK2206 inhibits the myogenic differentiation and induces IR.
CONCLUSION
The present study reveals that high glucose inhibited myogenisis accompanied by inducing IR, through AKT signaling inhibition, which may help to further research for resisting degenerative muscular diseases caused by glucose metabolism disorders.
Topics: Animals; Cell Differentiation; Cell Line; Cell Survival; Down-Regulation; Gene Expression Regulation; Glucose; Insulin Resistance; Mice; Muscle Development; Myoblasts; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt
PubMed: 31634780
DOI: 10.1016/j.biopha.2019.109498 -
Developmental Cell Dec 2021Myoblast fusion is essential for muscle development and regeneration. Yet, it remains poorly understood how mononucleated myoblasts fuse with preexisting fibers. We...
Myoblast fusion is essential for muscle development and regeneration. Yet, it remains poorly understood how mononucleated myoblasts fuse with preexisting fibers. We demonstrate that ERK1/2 inhibition (ERKi) induces robust differentiation and fusion of primary mouse myoblasts through a linear pathway involving RXR, ryanodine receptors, and calcium-dependent activation of CaMKII in nascent myotubes. CaMKII activation results in myotube growth via fusion with mononucleated myoblasts at a fusogenic synapse. Mechanistically, CaMKII interacts with and regulates MYMK and Rac1, and CaMKIIδ/γ knockout mice exhibit smaller regenerated myofibers following injury. In addition, the expression of a dominant negative CaMKII inhibits the formation of large multinucleated myotubes. Finally, we demonstrate the evolutionary conservation of the pathway in chicken myoblasts. We conclude that ERK1/2 represses a signaling cascade leading to CaMKII-mediated fusion of myoblasts to myotubes, providing an attractive target for the cultivated meat industry and regenerative medicine.
Topics: Actins; Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Differentiation; Cell Fusion; Cell Proliferation; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Mice, Inbred C57BL; Models, Biological; Muscle Fibers, Skeletal; Muscle Proteins; Myoblasts; Protein Binding; Protein Kinase Inhibitors; Receptors, Retinoic Acid; Signal Transduction; rac1 GTP-Binding Protein; Mice
PubMed: 34932950
DOI: 10.1016/j.devcel.2021.11.022 -
Nature Communications Nov 2020Sarcopenia is characterized by decreased skeletal muscle mass and function with age. Aged muscles have altered lipid compositions; however, the role and regulation of...
Sarcopenia is characterized by decreased skeletal muscle mass and function with age. Aged muscles have altered lipid compositions; however, the role and regulation of lipids are unknown. Here we report that FABP3 is upregulated in aged skeletal muscles, disrupting homeostasis via lipid remodeling. Lipidomic analyses reveal that FABP3 overexpression in young muscles alters the membrane lipid composition to that of aged muscle by decreasing polyunsaturated phospholipid acyl chains, while increasing sphingomyelin and lysophosphatidylcholine. FABP3-dependent membrane lipid remodeling causes ER stress via the PERK-eIF2α pathway and inhibits protein synthesis, limiting muscle recovery after immobilization. FABP3 knockdown induces a young-like lipid composition in aged muscles, reduces ER stress, and improves protein synthesis and muscle recovery. Further, FABP3 reduces membrane fluidity and knockdown increases fluidity in vitro, potentially causing ER stress. Therefore, FABP3 drives membrane lipid composition-mediated ER stress to regulate muscle homeostasis during aging and is a valuable target for sarcopenia.
Topics: Aging; Animals; Cell Line; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factor-2; Fatty Acid Binding Protein 3; Female; Gene Knockdown Techniques; Lipidomics; Membrane Fluidity; Membrane Lipids; Mice, Inbred C57BL; Mice, Knockout; Muscle, Skeletal; Myoblasts; Phospholipids; Protein Serine-Threonine Kinases; Sarcopenia; Up-Regulation
PubMed: 33168829
DOI: 10.1038/s41467-020-19501-6 -
Journal of Cell Science Sep 2019Cell-cell fusion is a fundamental process underlying fertilization, development, regeneration and physiology of metazoans. It is a multi-step process involving cell... (Review)
Review
Cell-cell fusion is a fundamental process underlying fertilization, development, regeneration and physiology of metazoans. It is a multi-step process involving cell recognition and adhesion, actin cytoskeletal rearrangements, fusogen engagement, lipid mixing and fusion pore formation, ultimately resulting in the integration of two fusion partners. Here, we focus on the asymmetric actin cytoskeletal rearrangements at the site of fusion, known as the fusogenic synapse, which was first discovered during myoblast fusion in embryos and later also found in mammalian muscle and non-muscle cells. At the asymmetric fusogenic synapse, actin-propelled invasive membrane protrusions from an attacking fusion partner trigger actomyosin-based mechanosensory responses in the receiving cell. The interplay between the invasive and resisting forces generated by the two fusion partners puts the fusogenic synapse under high mechanical tension and brings the two cell membranes into close proximity, promoting the engagement of fusogens to initiate fusion pore formation. In this Cell Science at a Glance article and the accompanying poster, we highlight the molecular, cellular and biophysical events at the asymmetric fusogenic synapse using myoblast fusion as a model.
Topics: Actin Cytoskeleton; Animals; Cell Fusion; Drosophila; Drosophila Proteins; Embryo, Nonmammalian; Mechanotransduction, Cellular; Myoblasts
PubMed: 31527149
DOI: 10.1242/jcs.213124 -
The Journal of Clinical Investigation Nov 2020Muscular dystrophies are a heterogeneous group of genetic diseases, characterized by progressive degeneration of skeletal and cardiac muscle. Despite the intense... (Review)
Review
Muscular dystrophies are a heterogeneous group of genetic diseases, characterized by progressive degeneration of skeletal and cardiac muscle. Despite the intense investigation of different therapeutic options, a definitive treatment has not been developed for this debilitating class of pathologies. Cell-based therapies in muscular dystrophies have been pursued experimentally for the last three decades. Several cell types with different characteristics and tissues of origin, including myogenic stem and progenitor cells, stromal cells, and pluripotent stem cells, have been investigated over the years and have recently entered in the clinical arena with mixed results. In this Review, we do a roundup of the past attempts and describe the updated status of cell-based therapies aimed at counteracting the skeletal and cardiac myopathy present in dystrophic patients. We present current challenges, summarize recent progress, and make recommendations for future research and clinical trials.
Topics: Cell Differentiation; Humans; Muscle, Skeletal; Muscular Dystrophies; Myoblasts; Myocardium; Pluripotent Stem Cells; Regeneration; Stem Cell Transplantation
PubMed: 32946430
DOI: 10.1172/JCI142031 -
Annual Review of Genetics Dec 2019Cell-cell fusion is indispensable for creating life and building syncytial tissues and organs. Ever since the discovery of cell-cell fusion, how cells join together to... (Review)
Review
Cell-cell fusion is indispensable for creating life and building syncytial tissues and organs. Ever since the discovery of cell-cell fusion, how cells join together to form zygotes and multinucleated syncytia has remained a fundamental question in cell and developmental biology. In the past two decades, myoblast fusion has been used as a powerful genetic model to unravel mechanisms underlying cell-cell fusion in vivo. Many evolutionarily conserved fusion-promoting factors have been identified and so has a surprising and conserved cellular mechanism. In this review, we revisit key findings in myoblast fusion and highlight the critical roles of cellular invasion and resistance in driving cell membrane fusion.
Topics: Actins; Actomyosin; Animals; Cell Adhesion Molecules; Cell Fusion; Drosophila; Drosophila Proteins; Embryo, Nonmammalian; Lipid Bilayers; Muscles; Myoblasts; Pupa
PubMed: 31283358
DOI: 10.1146/annurev-genet-120116-024603 -
International Journal of Molecular... Sep 2021Primary cilia are non-motile, cell cycle-associated organelles that can be found on most vertebrate cell types. Comprised of microtubule bundles organised into an... (Review)
Review
Primary cilia are non-motile, cell cycle-associated organelles that can be found on most vertebrate cell types. Comprised of microtubule bundles organised into an axoneme and anchored by a mature centriole or basal body, primary cilia are dynamic signalling platforms that are intimately involved in cellular responses to their extracellular milieu. Defects in ciliogenesis or dysfunction in cilia signalling underlie a host of developmental disorders collectively referred to as ciliopathies, reinforcing important roles for cilia in human health. Whilst primary cilia have long been recognised to be present in striated muscle, their role in muscle is not well understood. However, recent studies indicate important contributions, particularly in skeletal muscle, that have to date remained underappreciated. Here, we explore recent revelations that the sensory and signalling functions of cilia on muscle progenitors regulate cell cycle progression, trigger differentiation and maintain a commitment to myogenesis. Cilia disassembly is initiated during myoblast fusion. However, the remnants of primary cilia persist in multi-nucleated myotubes, and we discuss their potential role in late-stage differentiation and myofiber formation. Reciprocal interactions between cilia and the extracellular matrix (ECM) microenvironment described for other tissues may also inform on parallel interactions in skeletal muscle. We also discuss emerging evidence that cilia on fibroblasts/fibro-adipogenic progenitors and myofibroblasts may influence cell fate in both a cell autonomous and non-autonomous manner with critical consequences for skeletal muscle ageing and repair in response to injury and disease. This review addresses the enigmatic but emerging role of primary cilia in satellite cells in myoblasts and myofibers during myogenesis, as well as the wider tissue microenvironment required for skeletal muscle formation and homeostasis.
Topics: Animals; Axoneme; Cell Cycle; Cell Differentiation; Centrosome; Cilia; Cytoskeleton; Extracellular Matrix; Humans; Muscle Development; Muscle Fibers, Skeletal; Muscle, Skeletal; Myoblasts; Organelles; Signal Transduction
PubMed: 34502512
DOI: 10.3390/ijms22179605