Evaluating the effect of nitric oxide on myoblast proliferation, migration and differentiation.
Sibisi, Ntethelelo Charles.
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Wound healing is the process of restoring tissue integrity in response to injury. This process involves four major phases namely; haemostasis, inflammation, regeneration and remodelling. These phases are regulated by various growth factors and cytokines that are released at the site of injury to facilitate wound repair. Cells involved in wound healing include neutrophils, macrophages, lymphocytes, fibroblasts and stem cells. Satellite cells are mesenchymal stem cells which facilitate skeletal muscle regeneration through a process known as myogenesis. These cells are quiescently located underneath the sarcolemma of the muscle fiber and are activated upon injury to enter the growth phase of cell cycle. They then proliferate and migrate to the injury site to differentiate and fuse with existing fibers to form multinucleated muscle cells.Growth factors and signalling molecules, such as hepatocyte growth factor (HGF) and nitric oxide (NO), induce satellite cell activation by altering the expression of transcription factors such as paired box transcription factor 7 (Pax7), myogenic regulatory factor 5 (Myf5), myogenic differentiation antigen (MyoD) and Myogenin. The role of NO in the subsequent process of myoblast proliferation, migration and differentiation is however unclear. The present study therefore evaluated the effect of nitric oxide on myoblast proliferation, migration and terminal differentiation. C2C12 myoblasts were cultured in standard growth media and subsequently plated for analysis in serum free media. Proliferation or differentiation was induced via the addition of either 2 ng/ml HGF or 2% horse serum respectively, while migration was stimulated using the standard in vitro wound healing assay. L-NAME (a NOS inhibitor; 100 µM and 200 µM) and SIN-1 (a NO donor; 10 µM or 25 µM) were utilized to modify NO levels in vitro, while NO levels were assessed using a nitric oxide colorimetric assay kit. Proliferation was assessed via cell counts, migration by assessing the percentage wound closure and differentiation determined by calculating myoblast alignment and subsequent fusion into multinucleated myotubes. There was no significant change in nitric oxide generated by myoblasts during proliferation and migration studies. However, NO levels increased significantly in response to differentiation, L-NAME significantly prevented this NO increase at day of differentiation. L-NAME also significantly decreased myoblast terminal differentiation by inhibiting myoblast alignment and fusion at day 5 of differentiation. L-NAME also significantly reduced the proliferative effect of HGF on myoblasts at 24 hours, and significantly reduced percentage wound closure at 16 hours post-injury. NO release by C2C12 myoblast was observed to increase in response to SIN-1 in a dose dependent manner. NO levels significantly increased from 0.58 nmol in a control up to 1.35 nmol and 1.9 nmol at 1 hour in response to 10 µM and 25 µM SIN-1 respectively. These levels increased until they reached 2.5 nmol in response to 25 µM SIN-1 at 16 hours. SIN-1 showed no significant effect on myoblast proliferation, however, it significantly promoted myoblast migration in a dose dependent manner by increasing the percentage wound closure to 42% and 45% at 7 hours for 10 µM and 25 µM respectively compared to 38% of the control. SIN-1 also significantly stimulated myoblast fusion with myofiber area of 26% as compared to 18.6% of the control at day 5 of differentiation. In conclusion, nitric oxide levels increase significantly during myoblast differentiation, but not during proliferation and migration. Despite this, inhibition of nitric oxide synthase significantly affects all these processes. In contrast NOS-independent elevation of NO (through incubation with SIN-1) significantly increased myoblast migration and fusion, but not proliferation. This suggests a central role for NO in regulating myogenesis; however, this role requires further investigation.