Abstract 17163: A Recombinant Atrial Natriuretic Peptide Prevents Aberrant Ca2+ Leakage Through Ryanodine Receptor by Suppression of Mitochondrial Reactive Oxygen Species Production in Failing Cardiomyocytes

Introduction: Catecholamine induces intracellular reactive oxygen species (ROS), enhances diastolic Ca2+ leak through the ryanodine receptor in failing myocytes. The aim of the present study is to clarify the mechanism by which an exogenous atrial natriuretic peptide (ANP) exerts cardioprotective effect in failing cardiomyocytes. Methods: Myocytes was isolated from the LV of canine heart failure (HF) model by 4 weeks rapid ventricular pacing. First, mitochondrial oxidized DNA damage was evaluated by double immunohistochemical (IHC) staining using anti-VDAC antibody for mitochondria and anti 8-hydroxy-2′-deoxyguanosine (8-OHdG) antibody for oxidized DNA. Then, we investigated the effect of ANP on intracellular ROS production by DCFH-DA, diastolic Ca2+ sparks by confocal microscopy using Fluo 4-AM, and survival rate of myocytes after 48h. Results: The double IHC study revealed that isoproterenol (ISO) markedly increased oxidized DNA in mitochondria in failing myocytes (not normal myocytes), and the ISO-induced DNA damage was markedly inhibited by the co-presence of ANP (10 nM). Intracellular ROS production was not increased in normal myocytes (relative fluorescence intensity of DCFH-DA; 100%), whereas the ROS production was already increased even at baseline in failing myocytes (180%). Moreover, the ROS was much increased in the presence of ISO (250%). The Ca2+ spark frequency in failing myocytes (CaSF: 313%) was significantly increased as compared with CaSF in normal myocytes (100%). The CaSF was much increased by addition of ISO (625%), in parallel with an increase in the ROS. Interestingly, in failing myocytes, ANP (10 nM) significantly suppressed the ISO-induced ROS production (170%) and CaSF (237%), respectively. Eventually, the survival rate (%) in failing myocytes was significantly lower in the presence of ISO than in its absence, while it was significantly increased by co-incubation of ISO plus ANP (Survival rate; Baseline 59%, ANP 63%, ISO 33%, ISO plus ANP 55%). Conclusions: ANP has a potent antioxidant action against ISO-induced ROS, which may correct aberrant diastolic Ca2+ sparks, eventually contribute to improvement of myocytes survival in HF. The mechanism may be attributed to the suppression of ISO-induced ROS in mitochondria.