부산대학교 도서관

Introduction: Sildenafil, a phosphodiesterase-5 inhibitor (PDE5i), is used to treat heart and circulatory failure (HF) in single ventricle congenital heart disease (SV). We previously demonstrated that human SVHF myocardium is characterized by increased PDE5 activity and impaired mitochondrial energetics, which are improved with PDE5i. Using an in vitro model in combination with SV myocardial tissue, we test the hypothesis that mitochondrial fatty acid β-oxidation (FAO) is improved with PDE5i therapy via activation of sirtuin-3 (Sirt3) and resulting protein deacetylation.Methods: Primary cardiomyocytes (neonatal rat ventricular myocytes, NRVMs) were treated with 2% serum from SVHF patients or non-failing (NF) controls +/-PDE5i (72 hours). To mechanistically interrogate whether PDE5i acts via Sirt3, we utilized siRNAs against Sirt3 +/-PDE5i. FAO was directly assessed via the Seahorse Bioanalyzer (Agilent). Mitochondrial protein levels and acetylation profiles were quantified via Western blot. Sirt3 activity was measured in isolated mitochondria from SVHF myocardium using a fluorescent-based enzymatic assay. Statistical analysis was performed using a Welch ANOVA with post-hoc Dunnett’s multiple comparisons test (FAO) or a paired T test (Sirt3 activity).Results: NRVMs treated with SVHF serum have impaired mitochondrial FAO and while PDE5i therapy results in improved FAO, siRNA knockdown of Sirt3 abolishes the PDE5i protective effect (A). Mitochondria isolated from SVHF myocardium are hyperacetylated, and ex vivo PDE5i treatment enhances Sirt3 deacetylase activity (B-C), and deacetylates mitochondrial proteins.Conclusions: SVHF serum treatment of cardiomyocytes is a novel in vitro platform for mechanistic investigations of SV-related pathophysiology. These data suggest PDE5i directly augments mitochondrial FAO via Sirt3-mediated protein deacetylation and provides important insight into the myocardial specific mechanisms of PDE5i.