Endothelial nitric oxide synthase decreases β-adrenergic responsiveness via inhibition of the L-type Ca2+ current

Signaling via endothelial nitric oxide synthase (NOS3) limits the heart's response to beta-adrenergic (beta-AR) stimulation, which may be protective against arrhythmias. However, mechanistic data are limited. Therefore, we performed simultaneous measurements of action potential (AP, using patch clamp), Ca2+ transients (fluo 4), and myocyte shortening ( edge detection). L-type Ca2+ current (I-Ca) was directly measured by the whole cell ruptured patchclamp technique. Myocytes were isolated from wild-type (WT) and NOS3 knockout ( NOS3(-/-)) mice. NOS3(-/-) myocytes exhibited a larger incidence of beta-AR ( isoproterenol, 1 mu M)-induced early afterdepolarizations (EADs) and spontaneous activity ( defined as aftercontractions). We also examined ICa, a major trigger for EADs. NOS3(-/-) myocytes had a significantly larger beta-AR-stimulated increase in ICa compared with WT myocytes. In addition, NOS3(-/-) myocytes had a larger response to beta-AR stimulation compared with WT myocytes in Ca2+ transient amplitude, shortening amplitude, and AP duration (APD). We observed similar effects with specific NOS3 inhibition [L-N-5-(1-iminoethyl)-ornithine(L-NIO),10 mu M] in WT myocytes as with NOS3 knockout. Specifically, L-NIO further increased isoproterenol-stimulated EADs and aftercontractions. L-NIO also further increased the isoproterenol-stimulated ICa, Ca2+ transient amplitude, shortening amplitude, and APD ( all P < 0.05 vs isoproterenol alone). L-NIO had no effect in NOS3(-/-) myocytes. These results indicate that NOS3 signaling inhibits the beta-AR response by reducing ICa and protects against arrhythmias. This mechanism may play an important role in heart failure, where arrhythmias are increased and NOS3 expression is decreased.