Recombinant Atrial Natriuretic Peptide Prevents Aberrant Ca²⁺ Leakage through the Ryanodine Receptor by Suppressing Mitochondrial Reactive Oxygen Species Production Induced by Isoproterenol in Failing Cardiomyocytes

Catecholamines induce intracellular reactive oxygen species (ROS), thus enhancing diastolic Ca2+ leakage through the ryanodine receptor during heart failure (HF). However, little is known regarding the effect of atrial natriuretic peptide (ANP) on ROS generation and Ca2+ handling in failing cardiomyocytes. The aim of the present study was to clarify the mechanism by which an exogenous ANP exerts cardioprotective effects during HF. Cardiomyocytes were isolated from the left ventricles of a canine tachycardia-induced HF model and sham-operated vehicle controls. The degree of mitochondrial oxidized DNA was evaluated by double immunohistochemical (IHC) staining using an anti-VDAC antibody for the mitochondria and an anti-8-hydroxy-2′-deoxyguanosine antibody for oxidized DNA. The effect of ANP on ROS was investigated using 2,7-dichlorofluorescin diacetate, diastolic Ca2+ sparks assessed by confocal microscopy using Fluo 4-AM, and the survival rate of myocytes after 48 h. The double IHC study revealed that isoproterenol (ISO) markedly increased oxidized DNA in the mitochondria in HF and that the ISO-induced DNA damage was markedly inhibited by the co-presence of ANP. ROS production and Ca2+ spark frequency (CaSF) were increased in HF compared to normal controls, and were further increased in the presence of ISO. Notably, ANP significantly suppressed both ISO-induced ROS and CaSF without changing sarcoplasmic reticulum Ca2+ content in HF (p<0.01, respectively). The survival rate after 48 h in HF was significantly decreased in the presence of ISO compared with baseline (p<0.01), whereas it was significantly improved by the co-presence of ANP (p<0.01). Together, our results suggest that ANP strongly suppresses ISO-induced mitochondrial ROS generation, which might correct aberrant diastolic Ca2+ sparks, eventually contributing to the improvement of cardiomyocyte survival in HF.儿茶酚胺可诱导细胞内活性氧簇（reactive oxygen species, ROS）生成，进而在心力衰竭（heart failure, HF）过程中通过雷诺丁受体增强舒张期钙离子漏出。然而，目前对于心房利钠肽（atrial natriuretic peptide, ANP）对衰竭心肌细胞中ROS生成及钙离子处理的影响尚不清楚。本研究旨在阐明外源性ANP在HF过程中发挥心脏保护作用的机制。本研究从犬心动过速诱导的HF模型及假手术载体对照组的左心室中分离心肌细胞。采用双重免疫组化（immunohistochemical, IHC）染色法评估线粒体氧化DNA的水平：以抗电压依赖性阴离子通道（VDAC）抗体标记线粒体，以抗8-羟基-2'-脱氧鸟苷抗体标记氧化DNA。通过2,7-二氯荧光素二乙酸酯探究ANP对ROS的影响，采用Fluo 4-AM结合共聚焦显微镜评估舒张期钙火花，并检测48小时后心肌细胞的存活率。双重IHC结果显示，异丙肾上腺素（isoproterenol, ISO）可显著增加HF组线粒体中的氧化DNA水平，而ANP的共同孵育可显著抑制ISO诱导的DNA损伤。与正常对照组相比，HF组的ROS生成量及钙火花频率（CaSF）均升高，且在ISO存在时进一步升高。值得注意的是，ANP可在不改变HF组肌浆网钙含量的前提下，显著抑制ISO诱导的ROS生成及CaSF（两组均p<0.01）。与基线水平相比，ISO存在时HF组48小时细胞存活率显著降低（p<0.01），而ANP的共同孵育可显著提升该存活率（p<0.01）。综上，本研究结果表明ANP可强力抑制ISO诱导的线粒体ROS生成，这或许能够纠正异常的舒张期钙火花，最终改善HF状态下心肌细胞的存活率。