Xin, an actin binding protein, is expressed within muscle satellite cells and newly regenerated skeletal muscle fibers.
Xin is a muscle-specific actin binding protein of which its role and regulation within skeletal muscle is not well understood. Here we demonstrate that Xin mRNA is robustly upregulated (>16-fold) within 12 h of skeletal muscle injury and is localized to the muscle satellite cell population. RT-PCR confirmed the expression pattern of Xin during regeneration, as well as within primary muscle myoblast cultures, but not other known stem cell populations. Immunohistochemical staining of single myofibers demonstrate Xin expression colocalized with the satellite cell marker Syndecan-4 further supporting the mRNA expression of Xin in satellite cells. In situ hybridization of regenerating muscle 5-7 days postinjury illustrates Xin expression within newly regenerated myofibers. Promoter-reporter assays demonstrate that known myogenic transcription factors [myocyte enhancer factor-2 (MEF2), myogenic differentiation-1 (MyoD), and myogenic factor-5 (Myf-5)] transactivate Xin promoter constructs supporting the muscle-specific expression of Xin. To determine the role of Xin within muscle precursor cells, proliferation, migration, and differentiation analysis using Xin, short hairpin RNA (shRNA) were undertaken in C2C12 myoblasts. Reducing endogenous Xin expression resulted in a 26% increase (P < 0.05) in cell proliferation and a 20% increase (P < 0.05) in myoblast migratory capacity. Skeletal muscle myosin heavy chain protein levels were increased (P < 0.05) with Xin shRNA administration; however, this was not accompanied by changes in myoglobin protein (another marker of differentiation) nor overt morphological differences relative to differentiating control cells. Taken together, the present findings support the hypothesis that Xin is expressed within muscle satellite cells during skeletal muscle regeneration and is involved in the regulation of myoblast function.
Hawke
Thomas J
TJ
School of Kinesiology and Health Science, York Univ., 4700 Keele St., Toronto ON. Canada. thawke@yorku.ca
Atkinson
Daniel J
DJ
Kanatous
Shane B
SB
Van der Ven
Peter F M
PF
Goetsch
Sean C
SC
Garry
Daniel J
DJ
D000818:Animals; D002460:Cell Line; D002465:Cell Movement; D049109:Cell Proliferation; D004179:Cobra Cardiotoxin Proteins; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D017930:Genes, Reporter; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018101:Mice, Inbred mdx; D024510:Muscle Development; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000196:embryology / Q000378:metabolism* / Q000503:physiopathology; D009135:Muscular Diseases / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D018006:Myogenic Regulatory Factors / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D012038:Regeneration; D020133:Reverse Transcriptase Polymerase Chain Reaction; D032496:Satellite Cells, Skeletal Muscle / Q000378:metabolism*; D053671:Syndecan-4 / Q000378:metabolism; D013997:Time Factors; D015533:Transcriptional Activation; D015854:Up-Regulation
D004179:Cobra Cardiotoxin Proteins; D004268:DNA-Binding Proteins; D018006:Myogenic Regulatory Factors; D009687:Nuclear Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C508306:Sdc4 protein, mouse; D053671:Syndecan-4; C118478:Xin protein, mouse