Unacylated Ghrelin Promotes Skeletal Muscle Regeneration Following Hindlimb Ischemia via SOD‐2–Mediated miR‐221/222 Expression

<jats:sec xml:lang="en"> <jats:title>Background</jats:title> <jats:p xml:lang="en"> Surgical treatment of peripheral artery disease, even if successful, does not prevent reoccurrence. Under these conditions, increased oxidative stress is a crucial determinant of tissue damage. Given its reported antioxidant effects, we investigated the potential of unacylated‐ghrelin (Un <jats:styled-content style="fixed-case">AG</jats:styled-content> ) to reduce ischemia‐induced tissue damage in a mouse model of <jats:styled-content style="fixed-case">peripheral artery disease</jats:styled-content> . </jats:p> </jats:sec> <jats:sec xml:lang="en"> <jats:title>Methods and Results</jats:title> <jats:p xml:lang="en"> We show that Un <jats:styled-content style="fixed-case">AG</jats:styled-content> but not acylated ghrelin ( <jats:styled-content style="fixed-case">AG</jats:styled-content> ) induces skeletal muscle regeneration in response to ischemia via canonical p38/mitogen‐actived protein kinase signaling Un <jats:styled-content style="fixed-case">AG</jats:styled-content> protected against reactive oxygen species–induced cell injuries by inducing the expression of superoxide dismutase‐2 ( <jats:styled-content style="fixed-case">SOD</jats:styled-content> ‐2) in satellite cells. This led to a reduced number of infiltrating <jats:styled-content style="fixed-case">CD</jats:styled-content> 68 <jats:sup>+</jats:sup> cells and was followed by induction of the myogenic process and a reduction in functional impairment. Moreover, we found that miR‐221/222, previously linked to muscle regeneration processes, was up‐regulated and negatively correlated with p57 <jats:sup>Kip2</jats:sup> expression in Un <jats:styled-content style="fixed-case">AG</jats:styled-content> ‐treated mice. Un <jats:styled-content style="fixed-case">AG</jats:styled-content> , unlike <jats:styled-content style="fixed-case">AG</jats:styled-content> , promoted cell‐cycle entry in satellite cells of mice lacking the genes for ghrelin and its receptor ( <jats:styled-content style="fixed-case">GHSR</jats:styled-content> 1a). Un <jats:styled-content style="fixed-case">AG</jats:styled-content> ‐induced p38/mitogen‐actived protein kinase phosphorylation, leading to activation of the myogenic process, was prevented in <jats:styled-content style="fixed-case">SOD</jats:styled-content> ‐2–depleted <jats:styled-content style="fixed-case">SC</jats:styled-content> s. By si <jats:styled-content style="fixed-case">RNA</jats:styled-content> technology, we also demonstrated that <jats:styled-content style="fixed-case">SOD</jats:styled-content> ‐2 is the antioxidant enzyme involved in the control of miR‐221/222–driven posttranscriptional p57 <jats:sup>Kip2</jats:sup> regulation. Loss‐of‐function experiments targeting miR‐221/222 and local pre–miR‐221/222 injection in vivo confirmed a role for miR‐221/222 in driving skeletal muscle regeneration after ischemia. </jats:p> </jats:sec> <jats:sec xml:lang="en"> <jats:title>Conclusions</jats:title> <jats:p xml:lang="en"> These results indicate that Un <jats:styled-content style="fixed-case">AG</jats:styled-content> ‐induced skeletal muscle regeneration after ischemia depends on <jats:styled-content style="fixed-case">SOD</jats:styled-content> ‐2–induced miR‐221/222 expression and highlight its clinical potential for the treatment of reactive oxygen species–mediated skeletal muscle damage. </jats:p> </jats:sec>