RyR(R4496C) Mutant Mice Model Reveals a New Paradigm on Local Ca 2+ Control of I CaL

Abstract

The Ca2+-dependent inactivation (CDI) of the widely distributed L-type Ca2+ channels is a major intrinsic feedback mechanism for Ca2+ homeostasis. CDI manifests by the time-dependent current decay during prolonged depolarization but also the voltage-dependent availability of Ca2+ channel during double-pulse protocols. Whereas the first one has received most of the attention cumulating with the identification of calmodulin (CaM) as an important Ca2+ sensor that mediates CDI, the latter has not received extensive investigation. Here we evidence that Ca2+ sparks control L-type Ca2+ current (ICaL) availability in cardiomyocytes and consequently the steady-state window current. Comparing RyRR4496C catecholaminergic polymorphic ventricular tachycardia (CPVT) mutant mice to wild type (WT) littermates, we show that whereas peak density of ICaL and global [Ca2+]i transient are not modified, RyR2R4496C myocytes demonstrated a significantly lower ICaL density at −30 and −20 mV. This decrease is the consequence of a depolarizing shift in steady-state activation and a hyperpolarizing shift of steady-state inactivation that result in a decreased window current. These differences are eliminated in presence of fast the Ca2+ buffer BAPTA and by depleting the sarcoplasmic reticulum (SR) with thapsigargin, pointing out to a local control by the observed increase in Ca2+ spark occurrence at low voltages in CPVT mice. Somewhat surprisingly, CaM antagonists did not have any effect on CPVT cells but altered the inactivation and activation curves in the WT cells to make these similar to the CPVT. Our data echo the retrograde coupling of RyR and L-type Ca2+ channel observed in skeletal muscle, representing a novel mechanism to counteract excessive SR Ca2+ leak and to preserve CPVT mice from arrhythmic trigger activity such as early afterdepolarization due to enhancement of ICaL window current.