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

TY - JOUR

T1 - Nitric oxide regulates cardiac intracellular Na+ and Ca2+ by modulating Na/K ATPase via PKC epsilon and phospholemman-dependent mechanism

AU - Pavlovic, Davor

AU - Hall, Andrew R.

AU - Kennington, Erika J.

AU - Aughton, Karen

AU - Boguslavskyi, Andrii

AU - Fuller, William

AU - Despa, Sanda

AU - Bers, Donald M.

AU - Shattock, Michael J.

PY - 2013/8

Y1 - 2013/8

N2 - 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.

AB - 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.

KW - Nitric oxide

KW - Protein kinase C

KW - Phospholemman

KW - FXYD-1

KW - Sodium pump

KW - Arrhythmia

KW - PROTEIN-KINASE-C

KW - VENTRICULAR MYOCYTES

KW - K-ATPASE

KW - PHOSPHOLAMBAN PHOSPHORYLATION

KW - NA,K-ATPASE ACTIVITY

KW - PUMP FUNCTION

KW - IN-VITRO

KW - STIMULATION

KW - ACTIVATION

KW - SYNTHASE

U2 - 10.1016/j.yjmcc.2013.04.013

DO - 10.1016/j.yjmcc.2013.04.013

M3 - Article

SN - 0022-2828

VL - 61

SP - 164

EP - 171

JO - Journal of Molecular and Cellular Cardiology

JF - Journal of Molecular and Cellular Cardiology

ER -