NADPH Oxidase 4 Contributes to Myoblast Fusion and Skeletal Muscle Regeneration

Myoblast fusion is an essential step in skeletal muscle development and regeneration. NADPH oxidase 4 (Nox4) regulates cellular processes such as proliferation, differentiation, and survival by producing reactive oxygen species (ROS). Insulin-like growth factor 1 induces muscle hypertrophy via Nox4, but its function in myoblast fusion remains elusive. Here, we report a ROS-dependent role of Nox4 in myoblast differentiation. Regenerating muscle fibers after injury by cardiotoxin had a lower cross-sectional area in Nox4-knockout (KO) mice than myofibers in wild-type (WT) mice. Diameters and fusion index values of myotubes differentiated from Nox4-KO primary myoblasts were significantly lower than those of myotubes derived from WT myoblasts. However, no difference was observed in the differentiation index and expression of MyoD, myogenin, and myosin heavy chain 3 (MHC) between KO and WT myotubes. The decreased fusion index was also observed during differentiation of primary myoblasts and C2C12 cells with suppressed Nox4 expression. In contrast, in C2C12 cells overexpressing Nox4, the fusion index was increased, whereas the differentiation index and MHC and myogenin protein expression were not affected compared to control. Interestingly, the expression of myomaker (Tmem8c), a fusogenic protein that controls myoblast fusion, was reduced in Nox4-knockdown C2C12 cells. The myomaker expression level was proportional to the cellular ROS level, which was regulated by of Nox4 expression level. These results suggests that Nox4 contributes to myoblast fusion, possibly through the regulation of myomaker expression via ROS production, and that Nox4-dependent ROS may promote skeletal muscle regeneration and growth.

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Tenogenic Contribution to Skeletal Muscle Regeneration: The Secretome of Scleraxis Overexpressing Mesenchymal Stem Cells Enhances Myogenic Differentiation In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms21061965 ◽

2020 ◽

Vol 21 (6) ◽

pp. 1965

Author(s):

Maximilian Strenzke ◽

Paolo Alberton ◽

Attila Aszodi ◽

Denitsa Docheva ◽

Elisabeth Haas ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Regeneration ◽

Skeletal Muscles ◽

Myogenic Differentiation ◽

Muscular Contraction ◽

Myoblast Fusion ◽

Skeletal Muscle Regeneration ◽

Potential Candidate ◽

Musculoskeletal Tissue

Integrity of the musculoskeletal system is essential for the transfer of muscular contraction force to the associated bones. Tendons and skeletal muscles intertwine, but on a cellular level, the myotendinous junctions (MTJs) display a sharp transition zone with a highly specific molecular adaption. The function of MTJs could go beyond a mere structural role and might include homeostasis of this musculoskeletal tissue compound, thus also being involved in skeletal muscle regeneration. Repair processes recapitulate several developmental mechanisms, and as myotendinous interaction does occur already during development, MTJs could likewise contribute to muscle regeneration. Recent studies identified tendon-related, scleraxis-expressing cells that reside in close proximity to the MTJs and the muscle belly. As the muscle-specific function of these scleraxis positive cells is unknown, we compared the influence of two immortalized mesenchymal stem cell (MSC) lines—differing only by the overexpression of scleraxis—on myoblasts morphology, metabolism, migration, fusion, and alignment. Our results revealed a significant increase in myoblast fusion and metabolic activity when exposed to the secretome derived from scleraxis-overexpressing MSCs. However, we found no significant changes in myoblast migration and myofiber alignment. Further analysis of differentially expressed genes between native MSCs and scleraxis-overexpressing MSCs by RNA sequencing unraveled potential candidate genes, i.e., extracellular matrix (ECM) proteins, transmembrane receptors, or proteases that might enhance myoblast fusion. Our results suggest that musculotendinous interaction is essential for the development and healing of skeletal muscles.

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Plasmin activity is required for myogenesis in vitro and skeletal muscle regeneration in vivo

Blood ◽

10.1182/blood.v99.8.2835 ◽

2002 ◽

Vol 99 (8) ◽

pp. 2835-2844 ◽

Cited By ~ 62

Author(s):

Mònica Suelves ◽

Roser López-Alemany ◽

Frederic Lluı́s ◽

Gloria Aniorte ◽

Erika Serrano ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Regeneration ◽

Myoblast Fusion ◽

Skeletal Muscle Regeneration ◽

Wild Type ◽

Plasmin Activity ◽

Type Plasminogen Activator ◽

Deficient Mice

Abstract Plasmin, the primary fibrinolytic enzyme, has a broad substrate spectrum and is implicated in biologic processes dependent upon proteolytic activity, such as tissue remodeling and cell migration. Active plasmin is generated from proteolytic cleavage of the zymogen plasminogen (Plg) by urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Here, we have investigated the role of plasmin in C2C12 myoblast fusion and differentiation in vitro, as well as in skeletal muscle regeneration in vivo, in wild-type and Plg-deficient mice. Wild-type mice completely repaired experimentally damaged skeletal muscle. In contrast, Plg−/− mice presented a severe regeneration defect with decreased recruitment of blood-derived monocytes and lymphocytes to the site of injury and persistent myotube degeneration. In addition, Plg-deficient mice accumulated fibrin in the degenerating muscle fibers; however, fibrinogen depletion of Plg-deficient mice resulted in a correction of the muscular regeneration defect. Because we found that uPA, but not tPA, was induced in skeletal muscle regeneration, and persistent fibrin deposition was also reproducible in uPA-deficient mice following injury, we propose that fibrinolysis by uPA-dependent plasmin activity plays a fundamental role in skeletal muscle regeneration. In summary, we identify plasmin as a critical component of the mammalian skeletal muscle regeneration process, possibly by preventing intramuscular fibrin accumulation and by contributing to the adequate inflammatory response after injury. Finally, we found that inhibition of plasmin activity with α2-antiplasmin resulted in decreased myoblast fusion and differentiation in vitro. Altogether, these studies demonstrate the requirement of plasmin during myogenesis in vitro and muscle regeneration in vivo.

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Heparan Sulfate 6-O-endosulfatases (Sulfs) Coordinate the Wnt Signaling Pathways to Regulate Myoblast Fusion during Skeletal Muscle Regeneration

Journal of Biological Chemistry ◽

10.1074/jbc.m112.353243 ◽

2012 ◽

Vol 287 (39) ◽

pp. 32651-32664 ◽

Cited By ~ 39

Author(s):

Thanh H. Tran ◽

Xiaofeng Shi ◽

Joseph Zaia ◽

Xingbin Ai

Keyword(s):

Skeletal Muscle ◽

Heparan Sulfate ◽

Wnt Signaling ◽

Signaling Pathways ◽

Muscle Regeneration ◽

Myoblast Fusion ◽

Skeletal Muscle Regeneration

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Dexamethasone accelerates muscle regeneration by modulating kinesin-1-mediated focal adhesion signals

Cell Death Discovery ◽

10.1038/s41420-021-00412-4 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Jong-Wei Lin ◽

Yi-Man Huang ◽

Yin-Quan Chen ◽

Ting-Yun Chuang ◽

Tien-Yun Lan ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscular Dystrophy ◽

Motor Activity ◽

Focal Adhesion ◽

Muscle Regeneration ◽

Focal Adhesions ◽

Myoblast Fusion ◽

Skeletal Muscle Regeneration ◽

Integrin Β1 ◽

Muscle Myosin

AbstractDuring differentiation, skeletal muscle develops mature multinucleated muscle fibers, which could contract to exert force on a substrate. Muscle dysfunction occurs progressively in patients with muscular dystrophy, leading to a loss of the ability to walk and eventually to death. The synthetic glucocorticoid dexamethasone (Dex) has been used therapeutically to treat muscular dystrophy by an inhibition of inflammation, followed by slowing muscle degeneration and stabilizing muscle strength. Here, in mice with muscle injury, we found that Dex significantly promotes muscle regeneration via promoting kinesin-1 motor activity. Nevertheless, how Dex promotes myogenesis through kinesin-1 motors remains unclear. We found that Dex directly increases kinesin-1 motor activity, which is required for the expression of a myogenic marker (muscle myosin heavy chain 1/2), and also for the process of myoblast fusion and the formation of polarized myotubes. Upon differentiation, kinesin-1 mediates the recruitment of integrin β1 onto microtubules allowing delivery of the protein into focal adhesions. Integrin β1-mediated focal adhesion signaling then guides myoblast fusion towards a polarized morphology. By imposing geometric constrains via micropatterns, we have proved that cell adhesion is able to rescue the defects caused by kinesin-1 inhibition during the process of myogenesis. These discoveries reveal a mechanism by which Dex is able to promote myogenesis, and lead us towards approaches that are more efficient in improving skeletal muscle regeneration.

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Matrix metalloproteinase 13 is a new contributor to skeletal muscle regeneration and critical for myoblast migration

AJP Cell Physiology ◽

10.1152/ajpcell.00051.2013 ◽

2013 ◽

Vol 305 (5) ◽

pp. C529-C538 ◽

Cited By ~ 26

Author(s):

Hanqin Lei ◽

Dephne Leong ◽

Lucas R. Smith ◽

Elisabeth R. Barton

Keyword(s):

Skeletal Muscle ◽

Muscle Regeneration ◽

Repair Process ◽

C2c12 Cells ◽

Skeletal Muscle Regeneration ◽

Mdx Mice ◽

Primary Role ◽

Muscle Repair ◽

Chronic Injury ◽

Ecm Remodeling

Efficient skeletal muscle repair and regeneration require coordinated remodeling of the extracellular matrix (ECM). Previous reports have indicated that matrix metalloproteinases (MMPs) play the pivotal role in ECM remodeling during muscle regeneration. The goal of the current study was to determine if the interstitial collagenase MMP-13 was involved in the muscle repair process. Using intramuscular cardiotoxin injections to induce acute muscle injury, we found that MMP-13 expression and activity transiently increased during the regeneration process. In addition, in muscles from mdx mice, which exhibit chronic injury, MMP-13 expression and protein levels were elevated. In differentiating C2C12 cells, a murine myoblast cell line, Mmp13 expression was most pronounced after myoblast fusion and during myotube formation. Using pharmacological inhibition of MMP-13 to test whether MMP-13 activity is necessary for the proliferation, differentiation, migration, and fusion of C2C12 cells, we found a dramatic blockade of myoblast migration, as well as a delay in differentiation. In contrast, C2C12 cells with stable overexpression of MMP-13 showed enhanced migration, without affecting myoblast maturation. Taken together, these results support a primary role for MMP-13 in myoblast migration that leads to secondary effects on differentiation.

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Phospholipase D1 facilitates second-phase myoblast fusion and skeletal muscle regeneration

Molecular Biology of the Cell ◽

10.1091/mbc.e14-03-0802 ◽

2015 ◽

Vol 26 (3) ◽

pp. 506-517 ◽

Cited By ~ 12

Author(s):

Shuzhi Teng ◽

David Stegner ◽

Qin Chen ◽

Tsunaki Hongu ◽

Hiroshi Hasegawa ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Regeneration ◽

Muscle Development ◽

Myoblast Fusion ◽

Skeletal Muscle Regeneration ◽

Fusion Pore ◽

Myoblast Differentiation ◽

Second Phase ◽

Genetic Ablation ◽

Phospholipase D1

Myoblast differentiation and fusion is a well-orchestrated multistep process that is essential for skeletal muscle development and regeneration. Phospholipase D1 (PLD1) has been implicated in the initiation of myoblast differentiation in vitro. However, whether PLD1 plays additional roles in myoblast fusion and exerts a function in myogenesis in vivo remains unknown. Here we show that PLD1 expression is up-regulated in myogenic cells during muscle regeneration after cardiotoxin injury and that genetic ablation of PLD1 results in delayed myofiber regeneration. Myoblasts derived from PLD1-null mice or treated with PLD1-specific inhibitor are unable to form mature myotubes, indicating defects in second-phase myoblast fusion. Concomitantly, the PLD1 product phosphatidic acid is transiently detected on the plasma membrane of differentiating myocytes, and its production is inhibited by PLD1 knockdown. Exogenous lysophosphatidylcholine, a key membrane lipid for fusion pore formation, partially rescues fusion defect resulting from PLD1 inhibition. Thus these studies demonstrate a role for PLD1 in myoblast fusion during myogenesis in which PLD1 facilitates the fusion of mononuclear myocytes with nascent myotubes.

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Impaired Skeletal Muscle Development and Regeneration in Transglutaminase 2 Knockout Mice

Cells ◽

10.3390/cells10113089 ◽

2021 ◽

Vol 10 (11) ◽

pp. 3089

Author(s):

Zsófia Budai ◽

Nour Al-Zaeed ◽

Péter Szentesi ◽

Hajnalka Halász ◽

László Csernoch ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Regeneration ◽

Muscle Development ◽

Tibialis Anterior Muscle ◽

Transglutaminase 2 ◽

Myoblast Fusion ◽

Skeletal Muscle Regeneration ◽

Cell Proliferation And Differentiation ◽

Proliferation And Differentiation ◽

Inflammatory Phenotype

Skeletal muscle regeneration is triggered by local inflammation and is accompanied by phagocytosis of dead cells at the injury site. Efferocytosis regulates the inflammatory program in macrophages by initiating the conversion of their inflammatory phenotype into the healing one. While pro-inflammatory cytokines induce satellite cell proliferation and differentiation into myoblasts, growth factors, such as GDF3, released by healing macrophages drive myoblast fusion and myotube growth. Therefore, improper efferocytosis may lead to impaired muscle regeneration. Transglutaminase 2 (TG2) is a versatile enzyme participating in efferocytosis. Here, we show that TG2 ablation did not alter the skeletal muscle weights or sizes but led to the generation of small size myofibers and to decreased grip force in TG2 null mice. Following cardiotoxin-induced injury, the size of regenerating fibers was smaller, and the myoblast fusion was delayed in the tibialis anterior muscle of TG2 null mice. Loss of TG2 did not affect the efferocytic capacity of muscle macrophages but delayed their conversion to Ly6C−CD206+, GDF3 expressing cells. Finally, TG2 promoted myoblast fusion in differentiating C2C12 myoblasts. These results indicate that TG2 expressed by both macrophages and myoblasts contributes to proper myoblast fusion, and its ablation leads to impaired muscle development and regeneration in mice.

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