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

J Mol Cell Cardiol. 2013 Aug:61:164-71. doi: 10.1016/j.yjmcc.2013.04.013. Epub 2013 Apr 20.

Abstract

In the heart, Na/K-ATPase regulates intracellular Na(+) and Ca(2+) (via NCX), thereby preventing Na(+) and Ca(2+) overload and arrhythmias. Here, we test the hypothesis that nitric oxide (NO) regulates cardiac intracellular Na(+) and Ca(2+) 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 μM), 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 Ca(2+)-chelation (EGTA 10mM) or NOS inhibition l-NAME (1mM). Exogenously added NO (spermine-NONO-ate) stimulated Na/K-ATPase (EC50=3.8 μM; n=6/grp), via decrease in Km, 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 (2 μM 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 Ca(2+) overload and arrhythmias. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".

Keywords: 2,3-butanedione monoxime; ARVM; Arrhythmia; BDM; Bis; EGTA; FXYD-1; GC; N(G)-nitro-l-arginine methyl ester; NO; Nitric oxide; PKC; PLB; PLM; Phospholemman; Protein kinase C; Sodium pump; VASP; VF; adult rat ventricular myocytes; bisindolylmaleimide; ethylene glycol tetraacetic acid; guanylate cyclase; l-NAME; nitric oxide; phospholamban; phospholemman; protein kinase C; vasodilatory protein; ventricular fibrillation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Action Potentials
  • Animals
  • Calcium / metabolism*
  • Calcium-Binding Proteins / metabolism
  • Cytoplasm / metabolism
  • Electric Stimulation
  • Heart Ventricles / cytology
  • In Vitro Techniques
  • Male
  • Membrane Proteins / metabolism*
  • Mice
  • Myocytes, Cardiac / physiology
  • NG-Nitroarginine Methyl Ester / pharmacology
  • Nitric Oxide / physiology*
  • Nitric Oxide Synthase / antagonists & inhibitors
  • Nitric Oxide Synthase / metabolism
  • Patch-Clamp Techniques
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Protein Kinase C-epsilon / metabolism*
  • Protein Processing, Post-Translational
  • Rats
  • Sodium / metabolism*
  • Sodium-Potassium-Exchanging ATPase / metabolism*

Substances

  • Calcium-Binding Proteins
  • Membrane Proteins
  • Phosphoproteins
  • phospholamban
  • phospholemman
  • Nitric Oxide
  • Sodium
  • Nitric Oxide Synthase
  • Protein Kinase C-epsilon
  • Sodium-Potassium-Exchanging ATPase
  • Calcium
  • NG-Nitroarginine Methyl Ester