Lucia Brunello

Decreased RyR2 refractoriness determines myocardial synchronization of aberrant Ca2+ release in a genetic model of arrhythmia

Dysregulated intracellular Ca2+ signaling is implicated in a variety of cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia. Spontaneous diastolic Ca2+ release (DCR) can induce arrhythmogenic plasma membrane depolarizations, although the mechanism responsible for DCR synchronization among adjacent myocytes required for ectopic activity remains unclear. We investigated the synchronization mechanism(s) of DCR underlying untimely action potentials and diastolic contractions (DCs) in a catecholaminergic polymorphic ventricular tachycardia mouse model with a mutation in cardiac calsequestrin. We used a combination of different approaches including single ryanodine receptor channel recording, optical imaging (Ca2+ and membrane potential), and contractile force measurements in ventricular myocytes and intact cardiac muscles. We demonstrate that DCR occurs in a temporally and spatially uniform manner in both myocytes and intact myocardial tissue isolated from cardiac calsequestrin mutation mice. Such synchronized DCR events give rise to triggered electrical activity that results in synchronous DCs in the myocardium. Importantly, we establish that synchronization of DCR is a result of a combination of abbreviated ryanodine receptor channel refractoriness and the preceding synchronous stimulated Ca2+ release/reuptake dynamics. Our study reveals how aberrant DCR events can become synchronized in the intact myocardium, leading to triggered activity and the resultant DCs in the settings of a cardiac rhythm disorder.

Neuronal Na+ channel blockade suppresses arrhythmogenic diastolic Ca2+ release

AIMS Sudden death resulting from cardiac arrhythmias is the most common consequence of cardiac disease. Certain arrhythmias caused by abnormal impulse formation including catecholaminergic polymorphic ventricular tachycardia (CPVT) are associated with delayed afterdepolarizations resulting from diastolic Ca2+ release (DCR) from the sarcoplasmic reticulum (SR). Despite high response of CPVT to agents directly affecting Ca2+ cycling, the incidence of refractory cases is still significant. Surprisingly, these patients often respond to treatment with Na+ channel blockers. However, the relationship between Na+ influx and disturbances in Ca2+ handling immediately preceding arrhythmias in CPVT remains poorly understood and is the object of this study. METHODS AND RESULTS We performed optical Ca2+ and membrane potential imaging in ventricular myocytes and intact cardiac muscles as well as surface ECGs on a CPVT mouse model with a mutation in cardiac calsequestrin. We demonstrate that a subpopulation of Na+ channels (neuronal Na+ channels; nNav) colocalize with ryanodine receptor Ca2+ release channels (RyR2). Disruption of the crosstalk between nNav and RyR2 by nNav blockade with riluzole reduced and also desynchronized DCR in isolated cardiomyocytes and in intact cardiac tissue. Such desynchronization of DCR on cellular and tissue level translated into decreased arrhythmias in CPVT mice. CONCLUSIONS Thus, our study offers the first evidence that nNav contribute to arrhythmogenic DCR, thereby providing a conceptual basis for mechanism-based antiarrhythmic therapy.

Store-dependent deactivation: Cooling the chain-reaction of myocardial calcium signaling

In heart cells, Ca(2+) released from the internal storage unit, the sarcoplasmic reticulum (SR) through ryanodine receptor (RyR2) channels is the predominant determinant of cardiac contractility. Evidence obtained in recent years suggests that SR Ca(2+) release is tightly regulated not only by cytosolic Ca(2+) but also by intra-store Ca(2+) concentration. Specifically, Ca(2+)-induced Ca(2+) release (CICR) that relies on auto-catalytic action of Ca(2+) at the cytosolic side of RyR2s is precisely balanced and counteracted by RyR2 deactivation dependent on a reciprocal decrease of Ca(2+) at the luminal side of RyR2s. Dysregulation of this inherently unstable Ca(2+) signaling is considered to be an underlying cause of triggered arrhythmias, and is associated with genetic and acquired forms of sudden cardiac death. In this article, we present an overview of recent advances in our understanding of the regulatory role luminal Ca(2+) plays in Ca(2+) handling, with a particular emphasis on the role of Ca(2+)release refractoriness in aberrant Ca(2+) release.

Rationally engineered Troponin C modulates in vivo cardiac function and performance in health and disease

Treatment for heart disease, the leading cause of death in the world, has progressed little for several decades. Here we develop a protein engineering approach to directly tune in vivo cardiac contractility by tailoring the ability of the heart to respond to the Ca2+ signal. Promisingly, our smartly formulated Ca2+-sensitizing TnC (L48Q) enhances heart function without any adverse effects that are commonly observed with positive inotropes. In a myocardial infarction (MI) model of heart failure, expression of TnC L48Q before the MI preserves cardiac function and performance. Moreover, expression of TnC L48Q after the MI therapeutically enhances cardiac function and performance, without compromising survival. We demonstrate engineering TnC can specifically and precisely modulate cardiac contractility that when combined with gene therapy can be employed as a therapeutic strategy for heart disease.

Neuronal Na+ Channels Are Integral Components of Pro-Arrhythmic Na+/Ca2+ Signaling Nanodomain That Promotes Cardiac Arrhythmias During β-Adrenergic Stimulation

Summary Although triggered arrhythmias including catecholaminergic polymorphic ventricular tachycardia (CPVT) are often caused by increased levels of circulating catecholamines, the mechanistic link between β-adrenergic receptor (AR) stimulation and the subcellular/molecular arrhythmogenic trigger(s) is unclear. Here, we systematically investigated the subcellular and molecular consequences of β-AR stimulation in the promotion of catecholamine-induced cardiac arrhythmias. Using mouse models of cardiac calsequestrin-associated CPVT, we demonstrate that a subpopulation of Na+ channels, mainly the neuronal Na+ channels (nNav), colocalize with ryanodine receptor 2 (RyR2) and Na+/Ca2+ exchanger (NCX) and are a part of the β-AR-mediated arrhythmogenic process. Specifically, augmented Na+ entry via nNav in the settings of genetic defects within the RyR2 complex and enhanced sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA)-mediated SR Ca2+ refill is both an essential and a necessary factor for arrhythmogenesis. Furthermore, we show that augmentation of Na+ entry involves β-AR–mediated activation of CAMKII, subsequently leading to nNav augmentation. Importantly, selective pharmacological inhibition as well as silencing of Nav1.6 inhibit myocyte arrhythmic potential and prevent arrhythmias in vivo. Taken together, these data suggest that the arrhythmogenic alteration in Na+/Ca2+ handling evidenced ruing β-AR stimulation results, at least in part, from enhanced Na+ influx through nNav. Therefore, selective inhibition of these channels and of Nav1.6 in particular can serve as a potential antiarrhythmic therapy.

Ablation of HRC alleviates cardiac arrhythmia and improves abnormal Ca handling in CASQ2 knockout mice prone to CPVT

AIMS Cardiac calsequestrin (CASQ2) and histidine-rich Ca-binding protein (HRC) are sarcoplasmic reticulum (SR) Ca-binding proteins that regulate SR Ca release in mammalian heart. Deletion of either CASQ2 or HRC results in relatively mild phenotypes characterized by preserved cardiac structure and function, although CASQ2 knockout (KO), or Cnull, shows increased arrhythmia burden under conditions of catecholaminergic stress. We hypothesized that given the apparent overlap of functions of CASQ2 and HRC, simultaneous ablation of both would deteriorate the cardiac phenotype compared with the single knockouts. METHODS AND RESULTS In contrast to this expectation, double knockout (DKO) mice lacking both CASQ2 and HRC exhibited normal cardiac ejection fraction and ultrastructure. Moreover, the predisposition to catecholamine-dependent arrhythmia that characterizes the Cnull phenotype was alleviated in the DKO mice. At the myocyte level, DKO mice displayed Ca transients of normal amplitude; additionally, the frequency of spontaneous Ca waves and sparks in the presence of isoproterenol were decreased markedly compared with Cnull. Furthermore, restitution of SR Ca release was slowed in DKO myocytes compared with Cnull cells. CONCLUSION Our results suggest that rather than being functionally redundant, CASQ2 and HRC modulate cardiac ryanodine receptor-mediated (RyR2) Ca release in an opposing manner. In particular, while CASQ2 stabilizes RyR2 rendering it refractory in the diastolic phase, HRC enhances RyR2 activity facilitating RyR2 recovery from refractoriness.

Dissociation of Calcium Transients and Force Development following a Change in Stimulation Frequency in Isolated Rabbit Myocardium.

As the heart transitions from one exercise intensity to another, changes in cardiac output occur, which are modulated by alterations in force development and calcium handling. Although the steady-state force-calcium relationship at various heart rates is well investigated, regulation of these processes during transitions in heart rate is poorly understood. In isolated right ventricular muscle preparations from the rabbit, we investigated the beat-to-beat alterations in force and calcium during the transition from one stimulation frequency to another, using contractile assessments and confocal microscopy. We show that a change in steady-state conditions occurs in multiple phases: a rapid phase, which is characterized by a fast change in force production mirrored by a change in calcium transient amplitude, and a slow phase, which follows the rapid phase and occurs as the muscle proceeds to stabilize at the new frequency. This second/late phase is characterized by a quantitative dissociation between the calcium transient amplitude and developed force. Twitch timing kinetics, such as time to peak tension and 50% relaxation rate, reached steady-state well before force development and calcium transient amplitude. The dynamic relationship between force and calcium upon a switch in stimulation frequency unveils the dynamic involvement of myofilament-based properties in frequency-dependent activation.

Synchronization of Intracellular Ca2+ Release in Multicellular Cardiac Preparations

In myocardial tissue, Ca2+ release from the sarcoplasmic reticulum (SR) that occurs via the ryanodine receptor (RyR2) channel complex. Ca2+ release through RyR2 can be either stimulated by an action potential (AP) or spontaneous. The latter is often associated with triggered afterdepolarizations, which in turn may lead to sustained arrhythmias. It is believed that some synchronization mechanism exists for afterdepolarizations and APs in neighboring myocytes, possibly a similarly timed recovery of RyR2 from refractoriness, which enables RyR2s to reach the threshold for spontaneous Ca2+ release simultaneously. To investigate this synchronization mechanism in absence of genetic factors that predispose arrhythmia, we examined the generation of triggered activity in multicellular cardiac preparations. In myocardial trabeculae from the rat, we demonstrated that in the presence of both isoproterenol and caffeine, neighboring myocytes within the cardiac trabeculae were able to synchronize their diastolic spontaneous SR Ca2+ release. Using confocal Ca2+ imaging, we could visualize Ca2+ waves in the multicellular preparation, while these waves were not always present in every myocyte within the trabeculae, we observed that, over time, the Ca2+ waves can synchronize in multiple myocytes. This synchronized activity was sufficiently strong that it could trigger a synchronized, propagated contraction in the whole trabecula encompassing even previously quiescent myocytes. The detection of Ca2+ dynamics in individual myocytes in their in situ setting at the multicellular level exposed a synchronization mechanism that could induce local triggered activity in the heart in the absence of global Ca2+ dysregulation.