OBJECTIVEType 1 diabetes leads to impairments in growth, function, and regenerative capacity of skeletal muscle; however, the underlying mechanisms have not been clearly defined.RESEARCH DESIGN AND METHODSWith the use of Ins2WT/C96Y mice (model of adolescent-onset type 1 diabetes), muscle regeneration was characterized in terms of muscle mass, myofiber size (cross-sectional area), and protein expression. Blood plasma was analyzed for glucose, nonesterified fatty acids, insulin, and plasminogen activator inhibitor-1 (PAI-1). PAI-039, an effective inhibitor of PAI-1, was orally administered to determine if PAI-1 was attenuating muscle regeneration in Ins2WT/C96Y mice.RESULTSIns2WT/C96Y mice exposed to 1 or 8 weeks of untreated type 1 diabetes before chemically induced muscle injury display significant impairments in their regenerative capacity as demonstrated by decreased muscle mass, myofiber cross-sectional area, myogenin, and Myh3 expression. PAI-1, a physiologic inhibitor of the fibrinolytic system and primary contributor to other diabetes complications, was more than twofold increased within 2 weeks of diabetes onset and remained elevated throughout the experimental period. Consistent with increased circulating PAI-1, regenerating muscles of diabetic mice exhibited excessive collagen levels at 5 and 10 days postinjury with concomitant decreases in active urokinase plasminogen activator and matrix metalloproteinase-9. Pharmacologic inhibition of PAI-1 with orally administered PAI-039 rescued the early regenerative impairments in noninsulin-treated Ins2WT/C96Y mice.CONCLUSIONSTaken together, these data illustrate that the pharmacologic inhibition of elevated PAI-1 restores the early impairments in skeletal muscle repair observed in type 1 diabetes and suggests that early interventional studies targeting PAI-1 may be warranted to ensure optimal growth and repair in adolescent diabetic skeletal muscle.
Xin is a striated muscle-specific actin-binding protein whose mRNA expression has been observed in damaged skeletal muscle. Here we demonstrate increased Xin protein expression early postinjury (≤ 12 h) and localization primarily to the periphery of damaged myofibers. At 1 day postinjury, Xin is colocalized with MyoD, confirming expression in activated satellite cells (SCs). By 5 days postinjury, Xin is evident in newly regenerated myofibers, with a return to preinjury levels by 14 days of regeneration. To determine whether the increased Xin expression is functionally relevant, tibialis anterior muscles of wild-type mice were infected with Xin-short hairpin RNA (shRNA) adenovirus, whereas the contralateral tibialis anterior received control adenovirus (Control). Four days postinfection, muscles were harvested or injured with cardiotoxin and collected at 3, 5, or 14 days thereafter. When compared with Control, Xin-shRNA infection attenuated muscle regeneration as demonstrated by Myh3 expression and fiber areas. Given the colocalization of Xin and MyoD, we isolated single myofibers from infected muscles to investigate the effect of silencing Xin on SC function. Relative to Control, SC activation, but not proliferation, was significantly impaired in Xin-shRNA-infected muscles. To determine whether Xin affects the G0-G1 transition, cell cycle reentry was assessed on infected C2C12 myoblasts using a methylcellulose assay. No difference in reentry was noted between groups, suggesting that Xin contributes to SC activation by means other than affecting G0-G1 transition. Together these data demonstrate a critical role for Xin in SC activation and reduction in Xin expression results in attenuated skeletal muscle repair.
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