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±2μM), PKCε 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 10mM) or NOS inhibition l-NAME (1mM). Exogenously added NO (spermine-NONO-ate) stimulated Na/K-ATPase (EC50=3.8μM; n=6/grp), via decrease in K_m, in PLM^WT but not PLM^KO or PLM^3SA myocytes (where phospholemman cannot be phosphorylated) as measured by whole-cell perforated-patch clamp. Field-stimulation with l-NAME or PKC-inhibitor (2μM Bis) resulted in elevated intracellular Na⁺ (22±1.5 and 24±2 respectively, vs. 14±0.6mM 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 PLM^3SA mouse hearts but not PLM^WT and PLM^KO. 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".