Rad GTPase deletion increases L-type calcium channel current leading to increased cardiac contraction

J Am Heart Assoc. 2013 Dec 12;2(6):e000459. doi: 10.1161/JAHA.113.000459.

Abstract

Background: The small GTPase Rad is a negative regulator of voltage-dependent L-type calcium channel current (ICaL); however, the effects of Rad ablation on cardiomyocyte function are unknown. The objective of this study is to test the hypothesis that Rad-depletion causes positive inotropic effects without inducing cardiac hypertrophy.

Methods and results: Ventricular myocytes from adult Rad(-/-) mice were isolated and evaluated by patch-clamp recordings for I(Ca,L) and action potentials, Ca(2+) transients, and sarcomere shortening. Maximum I(CaL) is elevated in Rad(-/-) (maximal conductance 0.35 ± 0.04 picoSiemens/picoFarad (pS/pF) wild-type; 0.61 ± 0.14 pS/pF Rad(-/-)), decay kinetics are faster, and I(Ca,L) activates at lower voltages (activation midpoint -7.2 ± 0.6 wild-type; -11.7 ± 0.9 Rad(-/-)) mimicking effects of β-adrenergic receptor stimulation. Diastolic and twitch calcium are elevated in Rad(-/-) (F340/380: 1.03 diastolic and 0.35 twitch for wild-type; 1.47 diastolic and 0.736 twitch for Rad(-/-)) and sarcomere shortening is enhanced (4.31% wild-type; 14.13% Rad(-/-)) at lower pacing frequencies. Consequentially, frequency-dependence of Ca(2+) transients is less in Rad(-/-), and the frequency dependence of relaxation is also blunted. In isolated working hearts, similar results were obtained; chiefly, +dP/dt was elevated at baseline and developed pressure was relatively nonresponsive to acute β-adrenergic receptor stimulation. In single cells, at subphysiological frequencies, nonstimulated calmodulin-dependent protein kinase-sensitive calcium release is observed. Remarkably, Rad(-/-) hearts did not show hypertrophic growth despite elevated levels of diastolic calcium.

Conclusions: This study demonstrates that the depletion of Rad GTPase is equivalent to sympathomimetic β-adrenergic receptor, without stimulating cardiac hypertrophy. Thus, targeting Rad GTPase is a novel potential therapeutic target for Ca(2+)-homeostasis-driven positive inotropic support of the heart.

Keywords: L‐type calcium current; RGK; Rad; inotropy; β‐adrenergic stimulation.

Publication types

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

MeSH terms

  • Action Potentials
  • Adrenergic beta-Agonists / pharmacology
  • Animals
  • Calcium Channels, L-Type / metabolism*
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism
  • Cardiac Pacing, Artificial
  • Cardiotonic Agents / pharmacology
  • Excitation Contraction Coupling* / drug effects
  • Gene Deletion*
  • Genotype
  • Heart Rate
  • Kinetics
  • Mice
  • Mice, 129 Strain
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Myocardial Contraction* / drug effects
  • Myocytes, Cardiac / drug effects
  • Myocytes, Cardiac / enzymology*
  • Phenotype
  • Sarcomeres / metabolism
  • Up-Regulation
  • Ventricular Pressure
  • ras Proteins / deficiency*
  • ras Proteins / genetics

Substances

  • Adrenergic beta-Agonists
  • Calcium Channels, L-Type
  • Cardiotonic Agents
  • Rrad protein, mouse
  • Calcium-Calmodulin-Dependent Protein Kinases
  • ras Proteins