Nitric Oxide Can Mediate Beta-Adrenergic- and CaMKII-Dependent Spontaneous Ca2+ Waves in Cardiac Myocytes, Independent of PKA Activation

Abstract

Increased diastolic SR Ca leak can initiate spontaneous Ca waves (SCaWs). SCaWs activate inward Na/Ca exchanger current causing an arrhythmogenic delayed afterdepolarization. Here we examine SCaWs in ventricular myocytes isolated from rabbit hearts. Myocytes did not exhibit SCaWs at baseline conditions, but 43% did when exposed to isoproterenol (ISO). This ISO-induced increase in activity was reversed by inhibition of CaMKII by KN93, but not with PKA inhibition by H89. At similar [Ca]SRT (121 μM) myocytes treated with ISO plus KN93 had significantly fewer SCaWs versus those treated with ISO or ISO plus H89 (0.2±0.28 vs. 1.1±0.28 & 1.29±0.39 SCaWs cell−1, respectively). We attribute this increase in activity to the previously characterized CaMKII-dependent increase in RyR-dependent leak. We also find that SR Ca leak is increased by the nitric oxide (NO) donor, SNAP; and this NO-dependent effect is also completely reversed by KN-93. We also show the increase in leak to be dependent on nitric oxide synthase 1 (NOS1) activity. At comparable SR Ca load (132 μM) ISO treated myocytes have significantly higher leak vs. control (8.4 vs. 3.8 μM). The ISO-induced leak (at constant SR Ca load) was attenuated by the NOS1 inhibitor, SMLT, but not the NOS3 inhibitor, L-NIO (3.5 vs. 6.8 μM). Moreover, ISO causes an upward trend in myocyte [NO] (sensed by the NO-dependent dye, DAF-2 A), and the NOS inhibitor, L-NAME significantly attenuated the development of SCaWs. Together this data suggests a novel pathway in which β1-adrenergic receptor activation stimulates NO production via NOS1, which in turn activates CaMKII to increase RyR gating, SR Ca leak, SCaWs and delayed afterdepolarizations.