Abstract 39: Organic Nitrates Improve Calcium Handling and Myofilament Sensitivity Impaired by Nitroso-Redox Disequilibrium

Abstract

Oxidative stress is a key factor in the dysfunctional calcium (Ca 2+ ) handling and contractile performance in heart disease. Organic nitrates are used for the treatment of cardiovascular afflictions, however endogenous nitric oxide (NO) has a broad spectrum of actions and the mechanisms by which it regulates Ca 2+ cycling and contractility is poorly understood. Neuronal NO synthase (nNOS) deficiency induces a nitroso-redox disequilibrium characterized by oxidative stress and altered contractility and calcium handling. We hypothesize that treatment of nNOS −/− mice with organic nitrates such as nitroglycerin (TNG) or isosorbide dinitrate (ISDN) reduces cytosolic calcium levels and restores myofilament responsiveness to calcium with no changes in contractility. Cardiomyocytes (CMs) were isolated from nNOS −/− (N=7) and wild type (WT) mice (N=3). Cells were loaded with fura-2 and then electrically evoked intracellular Ca 2+ and sarcomere length were simultaneously measured in an IonOptix system. It has been shown that L-type Ca 2+ current is exacerbated in nNOS −/− cardiomyocytes. In consequence, at 1 Hz pacing rate, Ca 2+ peak and transient amplitude were increased in nNOS −/− (Peak Ca 2+ : 571 ± 38 nM) compared to WT (401 ± 32 nM; p = 0.012) cardiomyocytes, which was reduced toward normal by 10 nmol/L TNG (453 ± 55 nM; p = 0.11) as well as 10 nmol/L ISDN (394 ± 65; p = 0.035). Phospholamban phosphorylation has been shown to be reduced in nNOS −/− CMs, possibly due to the enhanced activity of protein phosphatases induced by oxidative stress. Thus, Ca 2+ decay and sarcomere relaxation were slower in nNOS −/− compared to WT CMs ( p < 0.0001). Treatment with TNG ( p < 0.0001 vs. NOS1 −/− ) or ISDN ( p < 0.0001 vs. NOS1 −/− ) accelerated Ca 2+ decay and relaxation. Ca 2+ sensitivity was impaired in nNOS −/− ( p < 0.021 vs. WT). Although organic nitrates either reduce or do not affect myofilament sensitivity in normal myocytes, TNG and ISDN increased their responsiveness to Ca 2+ in nNOS −/− . In conclusion, restoration of NO availability and subsequent attenuation of the nitroso-redox imbalance, improved excitation-contraction coupling in nNOS −/− CMs, decreased intracellular Ca 2+ which is counteracted by the improved myofilament sensitivity resulting in no net change in contractility.