Luigi Venetucci

Increasing Ryanodine Receptor Open Probability Alone Does Not Produce Arrhythmogenic Calcium Waves Threshold Sarcoplasmic Reticulum Calcium Content Is Required

Diastolic waves of Ca2+ release have been shown to activate delayed afterdepolarizations as well as some cardiac arrhythmias. The aim of this study was to investigate whether increasing ryanodine receptor open probability alone or in the presence of β-adrenergic stimulation produces diastolic Ca release from the sarcoplasmic reticulum (SR). When voltage-clamped rat ventricular myocytes were exposed to caffeine (0.5 to 1.0 mmol), diastolic Ca2+ release was seen to accompany the first few stimuli but was never observed in the steady state. We attribute the initial phase of diastolic Ca2+ release to a decrease in the threshold SR Ca2+ content required to activate Ca2+ waves and its subsequent disappearance to a decrease of SR content below this threshold. Application of isoproterenol (1 &mgr;mol/L) increased the amplitude of the systolic Ca2+ transient and also the SR Ca2+ content but did not usually produce diastolic Ca2+ release. Subsequent addition of caffeine, however, resulted in diastolic Ca2+ release. We estimated the time course of recovery of SR Ca2+ content following recovery from emptying with a high (10 mmol/L) concentration of caffeine. Diastolic Ca2+ release recommenced only when SR content had increased back to its final level. We conclude that increasing ryanodine receptor open probability alone does not produce arrhythmogenic diastolic Ca2+ release because of the accompanying decrease of SR Ca2+ content. β-Adrenergic stimulation increases SR content and thereby allows the increased ryanodine receptor open probability to produce diastolic Ca2+ release. The implications of these results for arrhythmias associated with abnormal ryanodine receptors are discussed.

Increasing ryanodine receptor open probability alone does not produce arrhythmogenic Ca waves: threshold SR Ca content is required

Diastolic waves of Ca2+ release have been shown to activate delayed afterdepolarizations as well as some cardiac arrhythmias. The aim of this study was to investigate whether increasing ryanodine receptor open probability alone or in the presence of β-adrenergic stimulation produces diastolic Ca release from the sarcoplasmic reticulum (SR). When voltage-clamped rat ventricular myocytes were exposed to caffeine (0.5 to 1.0 mmol), diastolic Ca2+ release was seen to accompany the first few stimuli but was never observed in the steady state. We attribute the initial phase of diastolic Ca2+ release to a decrease in the threshold SR Ca2+ content required to activate Ca2+ waves and its subsequent disappearance to a decrease of SR content below this threshold. Application of isoproterenol (1 &mgr;mol/L) increased the amplitude of the systolic Ca2+ transient and also the SR Ca2+ content but did not usually produce diastolic Ca2+ release. Subsequent addition of caffeine, however, resulted in diastolic Ca2+ release. We estimated the time course of recovery of SR Ca2+ content following recovery from emptying with a high (10 mmol/L) concentration of caffeine. Diastolic Ca2+ release recommenced only when SR content had increased back to its final level. We conclude that increasing ryanodine receptor open probability alone does not produce arrhythmogenic diastolic Ca2+ release because of the accompanying decrease of SR Ca2+ content. β-Adrenergic stimulation increases SR content and thereby allows the increased ryanodine receptor open probability to produce diastolic Ca2+ release. The implications of these results for arrhythmias associated with abnormal ryanodine receptors are discussed.

Neuronal Nitric Oxide Synthase Signaling in the Heart Is Regulated by the Sarcolemmal Calcium Pump 4b

Background— Neuronal nitric oxide synthase (nNOS) has recently been shown to be a major regulator of cardiac contractility. In a cellular system, we have previously shown that nNOS is regulated by the isoform 4b of plasma membrane calcium/calmodulin-dependent ATPase (PMCA4b) through direct interaction mediated by a PDZ domain (PSD 95, Drosophilia Discs large protein and Zona occludens-1) on nNOS and a cognate ligand on PMCA4b. It remains unknown, however, whether this interaction has physiological relevance in the heart in vivo. Methods and Results— We generated 2 strains of transgenic mice overexpressing either human PMCA4b or PMCA ct120 in the heart. PMCA ct120 is a highly active mutant form of the pump that does not interact with or modulate nNOS function. Calcium was extruded normally from PMCA4b-overexpressing cardiomyocytes, but in vivo, overexpression of PMCA4b reduced the &bgr;-adrenergic contractile response. This attenuated response was not observed in ct120 transgenic mice. Treatment with a specific nNOS inhibitor (N&ohgr;-propyl-l-arginine) reduced the &bgr;-adrenergic response in wild-type and ct120 transgenic mice to levels comparable to those of PMCA4b transgenic animals. No differences in lusitropic response were observed in either transgenic strain compared with wild-type littermates. Conclusions— These data demonstrate the physiological relevance of the interaction between PMCA4b and nNOS and suggests its signaling role in the heart.

What role does modulation of the ryanodine receptor play in cardiac inotropy and arrhythmogenesis

In this article we review the role of the Ryanodine Receptor (RyR) in cardiac inotropy and arrhythmogenesis. Most of the calcium that activates cardiac contraction comes from the sarcoplasmic reticulum (SR) from where it is released through the RyR. The amplitude of the systolic Ca transient depends steeply on the SR Ca content and it is therefore important that SR content be regulated. This regulation occurs via changes of SR Ca content affecting systolic Ca and thence sarcolemmal Ca fluxes. In the steady state, the cardiac myocyte must be in Ca flux balance on each beat and this has implications for understanding even simple inotropic manoeuvres. The main part of the review considers the effects of modulating the RyR on systolic Ca. Potentiation of RyR opening produces an increase of the amplitude of the Ca transient but this effect disappears within a few beats because the increased sarcolemmal efflux of Ca decreases SR Ca content. We conclude that it is therefore unlikely that potentiation of the RyR by phosphorylation plays a dominant role in the actions of positive inotropic agents such as beta-adrenergic stimulation. Some cardiac arrhythmias result from release of Ca from the SR in the form of waves. This is best known to occur when the SR is overloaded with calcium. Mutations in the RyR also produce cardiac arrhythmias attributed to Ca waves due to leaky RyRs and a similar leak has been suggested to contribute to arrhythmias in heart failure. We show that, due to compensatory changes of SR Ca content, simply making the RyR leaky does not produce Ca waves in the steady state and that SR Ca content is critical in determining whether Ca waves occur.

In the RyR2R4496C Mouse Model of CPVT, β-Adrenergic Stimulation Induces Ca Waves by Increasing SR Ca Content and Not by Decreasing the Threshold for Ca Waves

Rationale: Mutations of the ryanodine receptor (RyR) cause catecholaminergic polymorphic ventricular tachycardia (CPVT). These mutations predispose to the generation of Ca waves and delayed afterdepolarizations during adrenergic stimulation. Ca waves occur when either sarcoplasmic reticulum (SR) Ca content is elevated above a threshold or the threshold is decreased. Which of these occurs in cardiac myocytes expressing CPVT mutations is unknown. Objective: We tested whether the threshold SR Ca content is different between control and CPVT and how it relates to SR Ca content during &bgr;-adrenergic stimulation. Methods and Results: Ventricular myocytes from the RyR2 R4496C+/− mouse model of CPVT and wild-type (WT) controls were voltage-clamped; diastolic SR Ca content was measured and compared with the Ca wave threshold. The results showed the following. (1) In 1 mmol/L [Ca2+]o, &bgr;-adrenergic stimulation with isoproterenol (1&mgr;mol/L) caused Ca waves only in R4496C. (2) SR Ca content and Ca wave threshold in R4496C were lower than those in WT. (3) &bgr;-Adrenergic stimulation increased SR Ca content by a similar amount in both R4496C and WT. (4) &bgr;-Adrenergic stimulation increased the threshold for Ca waves. (5) During &bgr;-adrenergic stimulation in R4496C, but not WT, the increase of SR Ca was sufficient to reach threshold and produce Ca waves. Conclusions: In the R4496C CPVT model, the RyR is leaky, and this lowers both SR Ca content and the threshold for waves. &bgr;-Adrenergic stimulation produces Ca waves by increasing SR Ca content and not by lowering threshold.

Calcium flux balance in the heart

This article reviews the consequences of the need for the cardiac cell to be in calcium flux balance in the steady state. We first discuss how this steady state condition affects the control of resting [Ca(2+)]i. The next section considers how sarcoplasmic reticulum (SR) Ca content is controlled by a feedback mechanism whereby changes of SR Ca affect the amplitude of the Ca transient and this, in turn, controls sarcolemmal Ca fluxes. Subsequent sections review the effects of altering the activity of individual Ca handling proteins. Increasing the activity of the SR Ca-ATPase (SERCA) increases both the amplitude and rate constant of decay of the systolic Ca transient. The Ca flux balance condition requires that this must be achieved with no change of Ca efflux placing constraints on the magnitude of change of amplitude and decay rate. We analyze the quantitative dependence of Ca transient amplitude and SR content on SERCA activity. Increasing the open probability of the RyR during systole is predicted to have no steady state effect on the amplitude of the systolic Ca transient. We discuss the effects of changing the amplitude of the L-type Ca current in the context of both triggering Ca release from the SR and loading the cell with calcium. These manoeuvres are considered in the context of the effects of β-adrenergic stimulation. Finally, we review calcium flux balance in the presence of Ca waves.

Na/Ca Exchange

Abstract:  The major effect of Na/Ca exchange (NCX) on the systolic Ca transient is secondary to its effect on the Ca content of the sarcoplasmic reticulum (SR). SR Ca content is controlled by a mechanism in which an increase of SR Ca produces an increase in the amplitude of the systolic Ca transient. This, in turn, increases Ca efflux on NCX as well as decreasing entry on the L‐type current resulting in a decrease of both cell and SR Ca content. This control mechanism also changes the response to other maneuvers that affect excitation–contraction coupling. For example, potentiating the opening of the SR Ca release channel (ryanodine receptor, RyR) with caffeine produces an immediate increase in the amplitude of the systolic Ca transient. However, this increases efflux of Ca from the cell on NCX and then decreases SR Ca content until a new steady state is reached. Changing the activity of NCX (by decreasing external Na) changes the level of SR Ca reached by this mechanism. If the cell and SR are overloaded with Ca then Ca waves appear during diastole. These waves activate the electrogenic NCX and thereby produce arrhythmogenic‐delayed afterdepolarizations. A major challenge is how to remove this arrhythmogenic Ca release without compromising the normal systolic release. We have found that application of tetracaine to decrease RyR opening can abolish diastolic release while simultaneously potentiating the systolic release.

Life, sudden death, and intracellular calcium

See related articles, pages 283–291 and 292–298 Since the original studies of Ringer,1 calcium (Ca2+) has become almost synonymous with contraction in the heart. Two papers in the current issue of Circulation Research focus on other roles of this cation.1 Wu and Bers2 show that Ca2+ in the nuclear envelope is in a store that is functionally interconnected with the sarcoplasmic reticulum (SR), a result that may have implications for the role of calcium in controlling gene transcription.2 The other area and the main focus of this editorial is the role of Ca2+ ions in arrhythmogenesis. Liu et al3 present important data concerning the occurrence of arrhythmias in a mouse expressing a mutant SR Ca2+ release channel (ryanodine receptor [RyR]). To discuss this result, we will first briefly summarize current concepts in the area. It is now known that most of the calcium that activates contraction comes from an intracellular store (the SR) and is released through the RyR. Release occurs through the process of calcium-induced calcium release. This depends on the fact that the probability of the RyR being open is increased by an increase of cytoplasmic Ca2+ concentration ([Ca2+]i). The entry of a small amount of Ca2+ into the cell through the L-type Ca2+ current thereby triggers much more release from the SR. It has been appreciated for a long time that …

Biphasic decay of the Ca transient results from increased sarcoplasmic reticulum Ca leak

Ca leak from the sarcoplasmic reticulum through the ryanodine receptor (RyR) reduces the amplitude of the Ca transient and slows its rate of decay. In the presence of β‐adrenergic stimulation, RyR‐mediated Ca leak produces a biphasic decay of the Ca transient with a fast early phase and a slow late phase. Two forms of Ca leak have been studied, Ca‐sensitising (induced by caffeine) and non‐sensitising (induced by ryanodine) and both induce biphasic decay of the Ca transient. Only Ca‐sensitising leak can be reversed by traditional RyR inhibitors such as tetracaine. Ca leak can also induce Ca waves. At low levels of leak, waves occur. As leak is increased, first biphasic decay and then slowed monophasic decay is seen. The level of leak has major effects on the shape of the Ca transient.

Disulfide-activated protein kinase G Iα regulates cardiac diastolic relaxation and fine-tunes the Frank-Starling response

The Frank–Starling mechanism allows the amount of blood entering the heart from the veins to be precisely matched with the amount pumped out to the arterial circulation. As the heart fills with blood during diastole, the myocardium is stretched and oxidants are produced. Here we show that protein kinase G Iα (PKGIα) is oxidant-activated during stretch and this form of the kinase selectively phosphorylates cardiac phospholamban Ser16—a site important for diastolic relaxation. We find that hearts of Cys42Ser PKGIα knock-in (KI) mice, which are resistant to PKGIα oxidation, have diastolic dysfunction and a diminished ability to couple ventricular filling with cardiac output on a beat-to-beat basis. Intracellular calcium dynamics of ventricular myocytes isolated from KI hearts are altered in a manner consistent with impaired relaxation and contractile function. We conclude that oxidation of PKGIα during myocardial stretch is crucial for diastolic relaxation and fine-tunes the Frank–Starling response.