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

Davor Pavlovic, Andrew R Hall, Erika J Kennington, Karen Aughton, Andrii Boguslavskyi, William Fuller, Sanda Despa, Donald M Bers, Michael J Shattock

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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".

Original languageEnglish
Pages (from-to)164-171
Number of pages8
JournalJournal of Molecular and Cellular Cardiology
Volume61
DOIs
Publication statusPublished - Aug 2013

Keywords

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

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