HDAC6 Reduction Promotes Aortic Valve Calcification Via ER Stress-mediated Osteogenic Pathways

Background:The pathophysiological process of Aortic valve(AoV)calcification includes endoplasmic reticulum(ER)stress activation,osteoblastic differentiation,apoptosis and inflammation of valvular interstitial cells(VICs).Histone deacetylase-6(HDAC6)is a member of the HDAC Ⅱb subfamily and inhibiting HDAC6 in breast cancer cells triggers ER stress.We first found that HDAC6 expression was reduced in aortic valve tissue in patients with calcific aortic valve disease.Studies have demonstrated that ER stress is involved in the progression of AoV calcification.Therefore,this study aims to elucidate the role and mechanism of HDAC6 in AoV calcification.Methods and results:Human VICs(hVICs)were cultured in osteogenic media to establish a model of AoV calcification in vitro.We incubated hVICs with tubacin(10 μM),a specific inhibitor of HDAC6 for 48h,Tubacin treatment markedly increased the expressions of Runx2 in hVICs.The result was further confirmed by HDAC6 silencing,that knockdown of HDAC6 significantly induced Runx2 expression in hVICs.Then we found HDAC6 inhibition by tubacin significantly triggered the expressions of ER stress markers including p-PERK,p-IREla,p-eif2α,and ATF4 in hVICs.Moreover,knockdown of HDAC6 by siRNA in hVICs showed the same results as Tubacin treatment,that HDAC6 silencing markedly induced ER stress in hVICs.Tauroursodeoxycholic acid(TUDCA)is a chemical chaperone that has been well demonstrated to be a classical inhibitor against ER stress both in vitro and in vivo.TUDCA(100 μM)pre-incubation significantly relieved ER stress activation as well as Runx2 expression after tubacin treatment or HDAC6 silencing.ATF4 is a key member of ER stress signaling pathway,knockdown of ATF4 significantly suppressed Runx2 expression which are induced by tubacin treatment as well as HDAC6 silencing in hVICs.Finally,hVICs were transfected with lentiviral HDAC6 shRNA or scramble shRNA.The transfected cells were incubated with or without TUDCA and cultured in osteogenic media for 14 days.Media were changed every 3 days.Alizarin red staining showed that knockdown of HDAC6 promoted calcified nodules formation,while inhibition of ER stress by TUDCA suppressed the effect.Conclusion:HDAC6 reduction promotes AoV calcification via ER stress-mediated osteogenic pathways.These data provide novel evidence that HDAC6 participates in AoV calcification development and suggest that HDAC6 may be a novel target for AoV calcification prevention and treatment.