Abstract 252: Neuronal Nitric Oxide Synthase Regulates the Activity of Cardiac Myocyte Ryanodine Receptors
The regulation of cardiac contractility by neuronal nitric oxide synthase (NOS1) is unclear and the mechanism(s) is unknown. It is known that NOS1 localizes to the sarcoplasmic reticulum (SR) and that exogenous nitric oxide is able to modulate the SR Ca2+ release channel (ryanodine receptor, RyR2). Thus, the purpose of this study is to examine if NOS1 regulates RyR2 activity. Ventricular myocytes were isolated from NOS1 knockout (NOS1−/ −) and wildtype (WT) mice. SR Ca2+ leak measurements, measured as the tetracaine (RyR inhibitor)-induced shift in diastolic [Ca2+] (measured with Fluo-4), showed that NOS1−/ − myocytes had a decrease in SR Ca2+ leak compared to WT (0.017±0.004 vs 0.032±0.004 Fratio, P<0.05). Since NOS1 knockout may lead to compensatory adaptations, we measured SR Ca2+ leak in WT myocytes with acute and specific NOS1 inhibition (S-methyl-L-thiocitrulline). NOS1 inhibition in WT myocytes also lead to a decrease in SR Ca2+ leak (0.015±0.006 Fratio, P<0.05 vs WT). Since SR Ca2+ leak is dependent upon SR Ca2+ load, we measured load via caffeine-induced Ca2+ transients and found a significant decrease in NOS1−/ − vs WT myocytes (0.59±0.10 vs 0.92±0.09 F/Fo, P<0.05). However, when the SR Ca2+ leak vs SR Ca2+ load was plotted, we observed that NOS1−/ − myocytes had a rightward shift, indicative of decreased RyR2 activity. Ca2+ spark frequency, another measure of RyR2 activity, was measured with Fluo-4 and confocal microscopy. Ca2+ spark frequency was also reduced in NOS1−/ − vs WT myocytes (1.4±0.1 vs 2.1±0.2 sparks/100 μm/s, P<0.05). RyR2 channels isolated from NOS1−/ − and WT hearts were incorporated into lipid bilayers. The open probability of NOS1−/ − RyR2 channels was reduced compared to WT RyR2 channels (0.09±0.04 vs 0.42±0.09, P<0.05). These data demonstrate that NOS1 knockout or inhibition leads to decreased RyR2 activity. Most studies have shown that NOS1−/ − myocytes have decreased contractility. Thus, this reduction in RyR2 activity may be responsible, in part, for the observed decreased contractility in NOS1−/ − myocytes.