Sirt1-inducible Deacetylation Of P21 Promotes Cardiomyocyte Proliferation And Cardiac Regeneration Post-myocardial Infarction

Background and ObjectionMyocardial infarction(MI)causes the rapid death of cardiomyocytes(CMs)with subsequent cardiac dysfunction.Approaches that could induce cardiac regeneration would help increase the number of CMs for the recovery of cardiac function post-MI.Stimulating endogenous cardiac regeneration may be an effective therapeutic strategy to induce cardiac repair and improve cardiac remodeling Post-MI.Recent studies have demonstrated that the induction of cell cycle re-entry could be a promising way for inducing cardiac regeneration.P21,encoded by cyclin-dependent kinase inhibitor 1A(CDKN1A),directly inhibits cyclin-dependent kinases(CDKs)in the cell proliferation.Previous studies have revealed that p21 inhibits various types of cancer cell proliferation and liver,and skeletal muscle regeneration.P21 also exhibits a regeneration-inhibiting role in mammalian heart.A previous study showed that p21 can be acetylated by Tip60 in HCT116 cells.Acetylation makes p21 more stable and unable to be degraded by ubiquitination followed by HCT116 cell proliferation arrest.Herein,the deacetylation of p21 may lead to p21 degradation,reducing p21 abundance,removing CM cell proliferation arrest,and ultimately promoting CM proliferation.Sirtl is one of the Sirtuins family that catalze the deacetylation of protein lysine residues.Not only is Sirtl crucial for cardiac embryogenesis and development,but it also contributes to the cell proliferation during deacetylating targets in previous studies.Sirtl also play a cardioprotection role in cardiovascular diseases.However,whether Sirtl involved in CM proliferation is still unclear.In this study,we hypothesized that Sirtl could modulate CM proliferation and cardiac regeneration through deacetylating p21.Our study provides a novel strategy for p21 deacetylation which could be used as an effective therapeutic approach following myocardial infarction.Materials and MethodsThe animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee at Southern Medical University,and they followed the NIH Guidelines for the Care and Use of Laboratory Animals.1.To explore whether p21 acetylation/deacetylation can affect CM proliferation.(1)To explore whether p21 is acetylated in CMs.(2)To verify the acetylation sites ofp21 in CMs.(3)To elucidate whether p21 acetylation/deacetylation can promote CM proliferation.2.To explore whether p21 is deacetylated by Sirtl in CMs.(1)To explore whether p21 can interact with Sirtl.(2)In vitro overexpression of Sirtl and detection of p21 acetylation.(3)In vitro depletion of Sirtl and detection of p21 acetylation.3.To elucidate whether Sirtl can promote CM proliferation.(1)Differential expression of Sirtl protein in embryonic day 16.5,post-natal day 1,and post-natal day 28 mouse hearts.(2)In vitro overexpression of Sirtl and detection of CM proliferation.(3)In vitro depletion of Sirtl and detection of CM proliferation.4.To explore whether p21 deacetylation is involved in Sirtl-induced CM proliferation.(1)Isolated CMs transfected with p21-WT,p21-KQ,or p21-KQ+Ad-Sirt 1.(2)To explore whether p21 deacetylation is involved in Sirtl-induced CM proliferation.5.To explore whether Sirtl can promote cardiac regeneration post-MI.(1)To verify the effects in CM proliferation in MI mice overexpressed Sirt1.(2)To verify the effects in CM apoptosis in MI muce overexpressed Sirt1.(3)To verify the effects in cardiac function in MI mice overexpressed Sirt1.Results1.P21 is acetylated in CMs,and the two acetylation sites,namely,the lysine residues positioned at sites 156 and 158.2.The acetylation of p21 inhibits CM proliferation,and the deacetylation of p21 removes p21-induced CM cell proliferation arrest.3.Sirtl interacts with and deacetylates p21.4.Sirtl promotes CM proliferation via deacetylating p21.5.Sirtl promotes cardiac regeneration,improves cardiac function in adult mouse hearts post-MI.ConclusionP21 deacetylation by Sirt1 could attenuate cell proliferatione arrest in CMs and subsequently CM proliferation.In addition,Sirtl triggers cardiac regeneration and improves cardiac function post-MI.