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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 -
In Vivo (Athens, Greece) 2018Geranylgeraniol (GGOH) is a C20 isoprenoid found in fruits, vegetables, and grains, including rice. As a food substance, GGOH is categorized as 'Generally Recognized as...
BACKGROUND
Geranylgeraniol (GGOH) is a C20 isoprenoid found in fruits, vegetables, and grains, including rice. As a food substance, GGOH is categorized as 'Generally Recognized as Safe'. GGOH is an intermediate product in the mevalonate pathway and acts as a precursor to geranylgeranyl pyrophosphate.
MATERIALS AND METHODS
C2C12 mouse myoblasts derived from muscle satellite cells were used. Quantitative reverse-transcriptase polymerase chain reaction, western blotting analysis, and immunocytochemical analysis were performed to respectively assess mRNA expression, protein levels, and the number of myofibers.
RESULTS
GGOH reduced the expression levels of skeletal muscle atrophy-related ubiquitin ligases in myofibers derived from C2C12 cells. GGOH induced myogenic differentiation of C2C12 cells via geranylgeranylation. GGOH did not adversely affect the proliferation of C2C12 cells.
CONCLUSION
GGOH induces myoblast differentiation in C2C12 cells.
Topics: Animals; Cell Differentiation; Cell Line; Cell Proliferation; Diterpenes; Immunohistochemistry; Metabolic Networks and Pathways; Mice; Myoblasts
PubMed: 30348697
DOI: 10.21873/invivo.11395 -
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 -
Skeletal Muscle Jan 2018The fusion of muscle precursor cells is a required event for proper skeletal muscle development and regeneration. Numerous proteins have been implicated to function in... (Review)
Review
The fusion of muscle precursor cells is a required event for proper skeletal muscle development and regeneration. Numerous proteins have been implicated to function in myoblast fusion; however, the majority are expressed in diverse tissues and regulate numerous cellular processes. How myoblast fusion is triggered and coordinated in a muscle-specific manner has remained a mystery for decades. Through the discovery of two muscle-specific fusion proteins, Myomaker and Myomerger-Minion, we are now primed to make significant advances in our knowledge of myoblast fusion. This article reviews the latest findings regarding the biology of Myomaker and Minion-Myomerger, places these findings in the context of known pathways in mammalian myoblast fusion, and highlights areas that require further investigation. As our understanding of myoblast fusion matures so does our potential ability to manipulate cell fusion for therapeutic purposes.
Topics: Animals; Cell Fusion; Membrane Proteins; Mice; Muscle Development; Muscle Proteins; Muscle, Skeletal; Mutagenesis; Myoblasts; Signal Transduction
PubMed: 29386054
DOI: 10.1186/s13395-017-0149-3 -
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 FEBS Journal Nov 2017Xk-related protein 8 (Xkr8) is a scramblase and responsible for phosphatidylserine (PS) exposure on the cell surface in a caspase-dependent manner. Although PS exposure...
Xk-related protein 8 (Xkr8) is a scramblase and responsible for phosphatidylserine (PS) exposure on the cell surface in a caspase-dependent manner. Although PS exposure is found to be important for myotube formation during myoblast differentiation, the role of Xkr8 during myogenesis has not been elucidated. Here we show that Xkr8 contributes to myoblast differentiation. Xkr8 overexpression induced the formation of large myotubes during early differentiation, but this phenotype was not related to caspase-dependent cleavage of Xkr8. Furthermore, forced Xkr8 expression accelerated myoblast differentiation and conferred cell-death resistance after the induction of differentiation. Consistent with these results, Xkr8-knocked-down myoblasts exhibited impaired differentiation and more apoptotic cells during differentiation, implying the involvements of Xkr8 in the survival and proliferation of myoblasts. Taken together, the study shows Xkr8 influences myogenesis by acting as a positive regulator of terminal differentiation and myoblast survival.
Topics: Animals; Apoptosis Regulatory Proteins; Cell Differentiation; Cell Survival; Humans; Membrane Proteins; Mice; Myoblasts; RNA, Messenger
PubMed: 28881496
DOI: 10.1111/febs.14261 -
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 -
BioMed Research International 2016MicroRNAs are a class of 18-22-nucleotide noncoding RNAs that posttranscriptionally regulate gene expression and have been shown to play an important role during...
MicroRNAs are a class of 18-22-nucleotide noncoding RNAs that posttranscriptionally regulate gene expression and have been shown to play an important role during myoblast differentiation. In this study, we found that the expression of miR-145a-5p was gradually increased during C2C12 myoblast differentiation, and miR-145a-5p inhibitors or mimics significantly suppressed or promoted the relative expression of specific myogenesis related marker genes. Moreover, overexpression or inhibition of miR-145a-5p enhanced or repressed the expression of some special genes involved in the endogenous Wnt signaling pathway during C2C12 myoblast differentiation, including Wnt5a, LRP5, Axin2, and β-catenin. These results indicated that miR-145a-5p might be considered as a new myogenic differentiation-associated microRNA that can promote C2C12 myoblast differentiation by enhancing genes related to myoblasts differentiation.
Topics: Animals; Cell Differentiation; Cell Line; Gene Expression Regulation, Developmental; Mice; MicroRNAs; Myoblasts; Wnt Signaling Pathway
PubMed: 27239472
DOI: 10.1155/2016/5276271