The Mechanism Of Peptidase Inhibitory Protein PI16 Inhibiting Angiotensin II-induced Cardiac Hypertrophy And Cardiac Fibrosis By Down-regulating HDAC1

Aims: Cardiac hypertrophy and fibrosis are the primary causes of heart failure due to non-ischemia heart disease.To date,no specific therapy exists for cardiac fibrosis due to the largely unknown mechanisms of disease and lack of applicable therapeutic targets.In this study,we aimed to explore the role and associated mechanism of peptidase inhibitor 16(PI16)in cardiac hypertrophy and fibrosis induced by angiotensin II or pressure overload.Methods: After generation of PI16 transgenic(Tg)mice,eight to twelve-week-old male PI16-Tg mice and their wild-type(WT)littermates(control)were subjected to Ang II or stroke-physiological saline solution(control)treatment for 4 weeks.Echocardiography and hematoxylin-eosin staining were used for analysis of heart size assessment.Sirius red and Masson's trichrome staining were used to analyze fibrosis level.Markers of hypertrophy and fibrosis were determined by western blotting and q PCR.Newborn rat ventricular myocytes(NRVMs)and cardiac fibroblasts(NRCFs)were isolated and cultured for in vitro experiments.After infected with recombinant adenovirous Ad-PI16 and treated with angiotensin II,protein levels,including HDACs,p53,Ac H3 and Ac H4,were detected by western blotting.NRCFs were co-infected with recombinant adenovirous Ad-PI16 and Ad-HDAC1 for over expression of HDAC1,cell proliferation was determined with RTCA.Protein levels were assessed by western blotting.The mechanism of p53 down regulation of PI16 was explored by Chromatin Immunoprecipitation(Ch IP).Immunofluorescent staining was used to test the changes of HDAC1 and Ac H3 in vivo.The role of PI16 in cardiac hypertrophy and fibrosis induced by pressure overload was determined in another model with transverse aortic coarctation(TAC).Results: Echocardiography showed that PI16 transgenic(Tg)mice have smaller left ventricle mass than wild-type mice.Histological analysis data showed that PI16 Tg mice demonstrated smaller cardiomyocyte size and less collagen deposition than wildtype mice.The protein levels of PI16 showed that PI16 is highly and specifically expressed by cardiac fibroblasts(CFs),not cardiomyocytes.PI16 overexpression in cardiomyocytes with an adenovirus PI16 vector did not affect cell size or hypertrophic protein expression.In CFs,overexpressed PI16 significantly inhibited CF proliferation and the levels of fibrosis-associated proteins.Further analysis of epigenetic changes of CF revealed that overexpressed PI16 decreases the nuclear level of histone deacetylase1(HDAC1)after angiotensin II treatment,resulting in increased histone 3 acetylation in K18 and K27 lysine.However,overexpression of HDAC1 by an adenovirus vector in CFs reversed these changes.The effects of PI16 on HDAC1 and histone 3 were confirmed in PI16 Tg mice using immunostaining.Conclusions: Our data suggest that PI16 is a HDAC1 regulator specifically in CFs,and PI16 overexpression prevents cardiac hypertrophy and fibrosis by inhibiting stressinduced CF activation.