Abstract 15464: Mitochondrial Function of Adventitial Fibroblasts in Pulmonary Hypertension is Regulated Through Sirt3 Deacetylase Activity

Abstract

Introduction: Pulmonary hypertension (PH) is a heterogeneous and life-threatening cardiopulmonary disorder in which mitochondrial dysfunction drive pathogenesis. Although metabolic changes in PH draw attention of extensive investigation, the underlying mechanisms remain unclear. Hypothesis: Decreased Sirt3 mitochondrial deacetylase activity leads to increased mitochondrial enzyme acetylation level and mitochondrial dysfunction in pulmonary artery adventitial fibroblast cells isolated from IPAH patients (PH-Fibs). Methods: 1) qPCR and western blot to examine Sirt3 gene and protein levels in PH-Fibs and CO-Fibs, 2) western blotting of isolated mitochondrial protein acetylation level to examine the Sirt3 deacetylase activity, 3) immunostaining of mitochondrial SOD2 (direct target of Sirt3) acetylation level in lung tissues from PAH patients to examine Sirt3 deacetylase activity in vivo, 4) respiratory analysis and fluorescent lifetime imaging microscopy (FLIM) to analyze the glycolytic and mitochondrial function in cells, 5) overexpressing Sirt3 and supplement NAD + to investigate the therapeutic potential of regulating mitochondrial enzyme acetylation level and mitochondrial function in PH-Fibs. Results: PH-Fibs have decreased Sirt3 mRNA and protein levels compared to CO-Fibs. NAD + , a Sirt3 co-factor, is also decreased in PH-Fibs. Increased mitochondrial protein pan-acetylation levels and mitochondrial specific Sirt3 target (MnSOD2) were identified in cultured PH-Fibs, as well as lung vasculatures of IPAH patients, indicating the decreased mitochondrial deacetylase activity of Sirt3 in vitro and in vivo. We demonstrated increased glycolytic capacity and impaired mitochondrial function in PH-Fibs compared to CO-Fibs. Finally, by overexpressing Sirt3 level and at the same time increase NAD + availability, we proved that Sirt3 and NAD + can serve as therapeutic methods to synergistically improve mitochondrial function by regulating mitochondrial enzyme acetylation level in PH-Fibs. Conclusions: This study demonstrated that the mitochondrial deacetylase activity of Sirt3 as a critical mechanism regulating mitochondrial protein acetylation and mitochondrial function of fibroblast cells in IPAH patients.