Abstract 797: Neuronal Nitric Oxide Synthase Deficiency Increases Sarcoplasmic Reticulum Calcium Leak and Arrhythmogenesis in Ventricular Myocytes
Background In heart failure, neuronal nitric oxide synthase (NOS1) translocates from the sarcoplasmic reticulum (SR) to the plasmalemma, but whether this is adaptive or maladaptive remains highly controversial. Accumulating biochemical and cellular electrophysiologic data support the hypothesis that NOS1-derived NO may directly regulate the activity of the ryanodine receptor (RyR2) in the SR, thereby affecting excitation-contraction coupling. Accordingly, we tested the prediction that NOS1 deficiency alters RyR activity leading to diastolic Ca2+ leak that has been associated with arrhytmias and contractile dysfunction.
Methods and Results We measured [Ca2+]i transients and sarcomere shortening in ventricular myocytes from wild type (WT), NOS1−/−, NOS3−/− and double NOS1/NOS3−/− deficient mice (n=3–10 mice/ strain). Diastolic [Ca2+]i was significantly elevated and sarcomere shortening was decreased in NOS1 −/− and NOS1/NOS3−/− but not in NOS3−/− myocytes (paced from 2– 8 Hz) compared to WT (n=7–10 cells/ strain p<0.05, ANOVA). Also, total SR [Ca2+] was decreased in NOS1−/− and NOS1/NOS3−/− but not in NOS3−/− cells, compared to WT. The load-leak relationship in each strain was quantified using tetracaine to block RyR2. As predicted, the leak was increased in NOS1−/− and NOS1/NOS3−/− myocytes but not in NOS3−/− cells compared to WT. Moreover, NOS1−/− myocytes exhibited delayed after depolarizations (DADS) after pacing, associated with diastolic leak. Pharmacological inhibition of NOS1 (S-methyl-tiocitrulline, 1 μM) in Wt myocytes recapitulated the effect of NOS1 gene inactivation, producing DADS. Kinetic analysis of [Ca2+]i transients revealed no differences in SERCA and NCX activity and Western blotting revealed no differences of these proteins in NOS1−/− vs. Wt. Also, the stoichiometry of FKBP12 to RyR was not altered in NOS1−/− myocytes.
Conclusions We conclude that NOS1-derived NO regulates the activity of RyR during diastole and its deficiency leads to Ca2+ leak, with important consequences for myocardial contractile reserve and electrophysiological stability. These data support the idea that loss of NOS1 from the SR may have important pathophysiological consequences in states like heart failure or after myocardial infarction.