Nitric oxide regulates cardiac intracellular Na+ and Ca2+ by modulating Na/K ATPase via PKCε and phospholemman-dependent mechanism

In the heart, Na/K-ATPase regulates intracellular Na+ and Ca2+ (via NCX), thereby preventing Na+ and Ca2+ overload and arrhythmias. Here, we test the hypothesis that nitric oxide (NO) regulates cardiac intracellular Na+ and Ca2+ and investigate mechanisms and physiological consequences involved. Effects of both exogenous NO (via NO-donors) and endogenously synthesized NO (via field-stimulation of ventricular myocytes) were assessed in this study. Field stimulation of rat ventricular myocytes significantly increased endogenous NO (18 +/- 2 mu M), PKC epsilon activation (82 +/- 12%), phospholemman phosphorylation (at Ser-63 and Ser-68) and Na/K-ATPase activity (measured by DAF-FM dye, western-blotting and biochemical assay, respectively; p < 0.05, n = 6) and all were abolished by Ca2+-chelation (EGTA 10 mM) or NOS inhibition L-NAME (1 mM). Exogenously added NO (spermine-NONO-ate) stimulated Na/K-ATPase (EC50 = 3.8 mu M; n = 6/grp), via decrease in K-m, in PLMWT but not PLMKO or PLM3SA myocytes (where phospholemman cannot be phosphorylated) as measured by whole-cell perforated-patch clamp. Field-stimulation with L-NAME or PKC-inhibitor (2 mu M Bis) resulted in elevated intracellular Na+ (22 +/- 1.5 and 24 +/- 2 respectively, vs. 14 +/- 0.6 mM in controls) in SBFI-AM-loaded rat myocytes. Arrhythmia incidence was significantly increased in rat hearts paced in the presence of L-NAME (and this was reversed by L-arginine), as well as in PLM3SA mouse hearts but not PLMWT and PLMKO. We provide physiological and biochemical evidence for a novel regulatory pathway whereby NO activates Na/K-ATPase via phospholemman phosphorylation and thereby limits Na+ and Ca2+ overload and arrhythmias. This article is part of a Special Issue entitled Na+ Regulation in Cardiac Myocytes. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved.