Abstract 1116: 4-Hydroxy-2-nonenal Induces Calcium Overload via NADPH Oxidase-derived Reactive Oxygen Species in Isolated Rat Cardiac Myocytes
Background: Oxidative stress has been implicated in the pathogenesis of heart failure. Recently, we and other investigators have reported that the amount of 4-hydroxy-2-nonenal (HNE), which is a major lipid peroxidation product and a cytotoxic aldehyde, is increased in the failing myocardium. However, the effect of HNE as a mediator of oxidative stress in cardiomyocytes has not been assessed. This study was therefore designed to determine whether HNE has a pro-oxidant effect in cardiac myocytes and whether HNE increases intracellular Ca2+ concentration ([Ca2+]i).
Methods and Results: HNE (10 to 100 μmol/L) induced the production of reactive oxygen species (ROS) within isolated rat ventricular myocytes in a dose-dependent manner, as assessed by 2′, 7′-dichlorofluorescin diacetate fluorescence. Diphenyleneiodonium (DPI) (100 μmol/L), which is an NADPH oxidase inhibitor, significantly inhibited the generation of ROS (P<0.0001, 10 μmol/L HNE versus HNE +DPI; P<0.0001, 100 μmol/L HNE versus HNE +DPI; n=8 experiments). A high level of HNE (400 μmol/L), which is available in pathological conditions, increased [Ca2+]i as assessed by fura-2 ratio in a time-dependent manner. After 20 min of HNE exposure, [Ca2+]i was significantly increased compared with that in the control medium (P<0.0001, HNE: 198±16% of the baseline versus control: 102±7% of the baseline, n=15) and hypercontracture was induced in 67% of HNE-treated cells. Catalase (100 U/mL), an antioxidative enzyme, significantly attenuated the increase in [Ca2+]i induced by HNE (P<0.0005, HNE versus HNE+catalase: 106±3% of the baseline) and completely inhibited HNE-induced hypercontracture. Furthermore, carvedilol (0.1 μmol/L), a beta-blocker with potent antioxidant activity, significantly attenuated the increase in [Ca2+]i induced by HNE (P=0.002, HNE versus HNE+calvedilol: 105±10% of the baseline) and completely inhibited HNE-induced hypercontracture, but propranolol (0.2 μmol/L) did neither (P=NS, HNE versus HNE+propranolol: 187±38% of the baseline).
Conclusion: HNE causes intracellular Ca2+ overload via NADPH oxidase-derived ROS formation in cardiomyocytes. HNE plays a crucial role as a mediator of oxidative stress in cardiomyocytes.