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.
Xin, an actin binding protein, has been implicated in the regulation of satellite cell function and is hypothesized to be essential for skeletal muscle regeneration following injury. To investigate the role of Xin during skeletal muscle regeneration, we inhibited Xin expression using a Xin shRNA adenoviral (XinAd) injection into the right tibialis anterior (TA) of 6 week old male mice, while the left TA served as a control receiving an injection of the virus lacking Xin shRNA. Injury was induced in both TAs 4 days later via cardiotoxin injection. At 5 days post‐injury, muscle fiber area was significantly smaller (Control: 1007 ± 62 μm2 ; Xin Ad: 785± 73 μm2) and the number of centrally located nuclei per muscle fiber was significantly increased (134 ± 9%) in XinAd regenerating muscle compared to control. These deficits in fiber area (Control: 3136±278 μm2;Xin Ad: 2273 ±167 μm2) and centrally located nuclei (137 ± 9% of control) in the XinAd muscles persisted to 14 days regeneration suggesting that the regenerating muscle injected with XinAd is less mature at 5 and 14 days of regeneration. A significant increase in apoptosis (TUNEL) at 5 and 14 days of regeneration (126±6%; 141±12% of control) was observed in the XinAd TA compared to control. These data support the hypothesis that Xin is important for muscle regeneration and future work will investigate the roles of Xin on satellite cell activation and maturation.
Xin, an actin binding protein, has been implicated in the regulation of satellite cells during skeletal muscle regeneration. It is believed to exert effects on the cytoskeleton through interactions with other structural proteins like filamin c, f‐actin and desmin. In order to investigate the role of Xin in muscle regeneration following injury, we injected Xin shRNA adenovirus (XinAd) into the right tibialis anterior (R‐TA) of male C57B6 mice while the left tibialis anterior (L‐TA) served as a control. Muscle injury in both legs was induced 4 days later using a cardiotoxin injection. As expected, XinAd reduced endogenous Xin expression by 67.8 ± 7.4% at 0 days. Surprisingly, at 3 days Xin Ad injected muscle displayed 29.3 ± 8.1% increase in filamin c, 12.6 ± 5.6% increase in f‐actin and a 41.2 ± 12.9% decrease in desmin expression compared to the L‐TA. Histochemically, the treatment and control tissues illustrated similar signs of degradation and regeneration at all time points. Therefore, while silencing Xin modifies actin‐associated protein expression patterns, it appears that other factors may compensate for its disappearance in terms of whole muscle regeneration.
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