Class I And Ii Histone Deacetylases Orchestrate Muscle Regeneration Program Following Hind-Limb Ischemia

INTRODUCTION: Although a regulatory and functional cross-talk between class I and II HDACs have been hypothesized, the role of these molecules in the context of muscle regeneration following acute hind-limb ischemia has never been addressed and represents the object of this research. METHODS & RESULTS: mice (C57BLJ10) were made ischemic by surgical excision of femoral artery and treated either with the selective class IIa inhibitor MC1568 (40 mg/Kg/day; n=12), the selective class I inhibitor MS275 (5 mg/Kg/day; n=12) or an equivalent amount of solvent (DMSO; n=12). Untreated ischemic (n=12) or sham operated animals (n=12) were used as further controls. Histological analyses revealed, from 3 to 7 days after damage, the prevalent localization of class IIa HDAC4 and 5 outside the nucleus. Following damage, dystrophin and nNOS proteins were down-regulated and decreased phosphatase PP2A activity was observed. Between 14 and 21 days after ischemia dystrophin and nNOS expression was reconstituted and paralleled by HDAC4 and 5 relocalization to the nucleus. Remarkably, the class IIa inhibitor MC1568 delayed muscle regeneration and immature fibers could be still detected 21 days after damage compared to ischemic untreated animals, while the class I selective inhibitor MS275 totally prevented postischemia muscle regeneration determining muscle atrophy, inflammation and fibrosis suggesting that members of different HDAC classes could be involved at different postischemia regeneration stages. Chromatin immunoprecipitation (ChIp) experiments demonstrated that A) during muscle regeneration class IIa HDACs bind and repress the promoter regions of proliferation-associated genes such as c-fos, telomerase, cyclin A1 and B1; B) class IIa HDACs bind class I HDAC2 promoter repressing its expression in late regeneration phases (>14 days post-ischemia). Accordingly, in MC1568-treated ischemic mice HDAC2 protein levels were increased as a consequence of class IIa inactivation. CONCLUSION: These findings indicate a) opposite to class I nuclear localization of class IIa HDACs is important during late muscle regeneration after hind-limb ischemia; b) this molecular switch controls gene expression providing a functional link between different classes of HDACs.