Research Article: RyRCa2+ Leak Limits Cardiac Ca2+ Window Current Overcoming the Tonic Effect of Calmodulin in Mice

Date Published: June 6, 2011

Publisher: Public Library of Science

Author(s): María Fernández-Velasco, Gema Ruiz-Hurtado, Angélica Rueda, Patricia Neco, Martha Mercado-Morales, Carmen Delgado, Carlo Napolitano, Silvia G. Priori, Sylvain Richard, Ana María Gómez, Jean-Pierre Benitah, Marcello Rota. http://doi.org/10.1371/journal.pone.0020863

Abstract: Ca2+ mediates the functional coupling between L-type Ca2+ channel (LTCC) and sarcoplasmic reticulum (SR) Ca2+ release channel (ryanodine receptor, RyR), participating in key pathophysiological processes. This crosstalk manifests as the orthograde Ca2+-induced Ca2+-release (CICR) mechanism triggered by Ca2+ influx, but also as the retrograde Ca2+-dependent inactivation (CDI) of LTCC, which depends on both Ca2+ permeating through the LTCC itself and on SR Ca2+ release through the RyR. This latter effect has been suggested to rely on local rather than global Ca2+ signaling, which might parallel the nanodomain control of CDI carried out through calmodulin (CaM). Analyzing the CICR in catecholaminergic polymorphic ventricular tachycardia (CPVT) mice as a model of RyR-generated Ca2+ leak, we evidence here that increased occurrence of the discrete local SR Ca2+ releases through the RyRs (Ca2+ sparks) causea depolarizing shift in activation and a hyperpolarizing shift inisochronic inactivation of cardiac LTCC current resulting in the reduction of window current. Both increasing fast [Ca2+]i buffer capacity or depleting SR Ca2+ store blunted these changes, which could be reproduced in WT cells by RyRCa2+ leak induced with Ryanodol and CaM inhibition.Our results unveiled a new paradigm for CaM-dependent effect on LTCC gating and further the nanodomain Ca2+ control of LTCC, emphasizing the importance of spatio-temporal relationships between Ca2+ signals and CaM function.

Partial Text: Dynamic modulation of cellular Ca2+ flows from either the extracellular space or the intracellular Ca2+ store into the cytoplasm participates in key pathophysiological processes, which depends on the ability of cells to properly sort ‘global’ and ‘local’ Ca2+ signals [1]. In this respect, the functional coupling of the sarcolemmal L-type Ca2+ channels (LTCC) and the sarcoplasmic reticulum (SR) Ca2+ release channels (ryanodine receptor, RyR), plays an important role in ventricular cardiomyocytes [2], [3]. Depolarizing stimuli open voltage-gated LTCC, leading to Ca2+ entry (ICa) and a subsequent rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i). While such [Ca2+]i elevations are initiated by LTCC, they are also influenced by Ca2+ transporting organelles such as the mitochondria and the SR. Notably, in response to these increases in [Ca2+]i, Ca2+ binds to and activates RyRs thereby amplifying the initial Ca2+ signal through the locally controlled Ca2+-induced Ca2+-release (CICR) process to support the excitation-contraction coupling (ECC) and thus heart function [4]. On the other hand, the opening of LTCCs is tightly controlled to prevent intracellular Ca2+overload. A major intrinsic negative feedback mechanism is the Ca2+-dependent inactivation (CDI) of the widely distributed voltage-gated Ca2+ channels [5], [6], [7], [8], [9], [10]. From the pioneering descriptions [11], CDI manifests as the hallmark time-dependent current decay during prolonged depolarization but also determines the voltage-dependent availability of Ca2+ channel during double-pulse protocols. Early studies of CDI were mainly focused on Ca2+ entry, but SR Ca2+ release also contributes significantly to the CDI [5]. In cardiac myocytes, depletion of SR Ca2+ stores or abolition of SR Ca2+ release causes a reduction of CDI [3], [12], whereas increasing SR Ca2+ loading results in CDI enhancement [13]. CDI depends linearly on the rate and magnitude of SR Ca2+ release from the RyRs [12]. Thus, ∼70% of CDI that occurs during ECC in rat ventricular myocytes arises from SR Ca2+ released, which might reduce Ca2+ influx during action potential up to 50% [14], [15]. Furthermore, it has been shown that SR Ca2+ release dominates CDI initially, then, as [Ca2+]i decreases due to SR Ca2+ reuptake, the SR dependent contribution declines with participation from Ca2+ entry via ICa dominating [16]. Now, SR Ca2+ release from the RyRs results in discrete and localized rises of [Ca2+]i (Ca2+ sparks) triggered by ICa[4]. The large local releases of Ca2+during CICR modulate in turn LTCC [3], [12], suggesting that discrete Ca2+ cross-signaling occurs in the microdomains of LTCC-RyRs [17].

The purpose of our study was to examine whether discrete and local increase of SR Ca2+ release has any effect on ICa with respect to nanodomain control of LTCCs. Using CPVT mice as a model with high spontaneous RyR-generated Ca2+ leak, our results pinpoint that the increase in RyRCa2+ leak caused opposite shifts in activation and in activation voltage curves of the cardiac LTCC. The resulting reduction of window current is prevented by manoeuvres that minimize variations in [Ca2+]i due to SR Ca2+ release. Surprisingly, application of CaM antagonists did not have any effect on the cells from the CPVT mouse but altered the inactivation and activation curves in the WT mouse to make these more like the CPVT mouse, revealing a new paradigm for CaM-dependent effect on LTCC gating. These effects might represent an adaptive mechanism to cytotoxic SR Ca2+ leak and Ca2+ overload.

Experiments were performed on male and female heterozygous RyR2R4497C mice (CPVT) and their WT littermates (F3 to F5 generation), as previously described [20], [22], in accordance to the ethical principles laid down by the French (Ministry of Agriculture) and ECC directive 96/609/EEC and was approved by the Comité Régional d’ Ethique sur l’expérimentation animale of Languedoc-Roussillon on the Use and Care of Animals. All persons who participated in the experiments had the training and authorization to do so (authorization B34-172-16 for animal facility manager).

Source:

http://doi.org/10.1371/journal.pone.0020863