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

Abstract

In heart cells, Ca²⁺ 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 Ca²⁺ release is tightly regulated not only by cytosolic Ca²⁺ but also by intra-store Ca²⁺ concentration. Specifically, Ca²⁺-induced Ca²⁺ release (CICR) that relies on auto-catalytic action of Ca²⁺ at the cytosolic side of RyR2s is precisely balanced and counteracted by RyR2 deactivation dependent on a reciprocal decrease of Ca²⁺ at the luminal side of RyR2s. Dysregulation of this inherently unstable Ca²⁺ 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 Ca²⁺ plays in Ca²⁺ handling, with a particular emphasis on the role of Ca²⁺ release refractoriness in aberrant Ca²⁺ release. This article is part of a Special Issue entitled “Calcium Signaling in Heart”.

1. Introduction

During each heartbeat, coordinated contraction and subsequent relaxation of the billions of cardiomyocytes in the mammalian heart is attained through a synchronized effort of two high fidelity signaling systems: The first is mediated by the action potential (AP) and the other by intracellular Ca²⁺ transient. In the former case, opening of voltage-dependent Na⁺ channels generates a depolarizing inward Na⁺ current that initiates the AP [1]. In the latter, Ca²⁺ influx through voltage-sensitive L-type Ca²⁺ channels in response to electrical depolarization activates Ca²⁺-sensitive ryanodine receptors channels (RyR2) on the surface of the sarcoplasmic reticulum (SR), leading to a self-regenerating process known as Ca²⁺-induced Ca²⁺ release (CICR), which initiates contraction [2]. Although the intracellular Ca²⁺ storage site, the SR, takes up only 3.5% of the total myocyte volume, it contains sufficient Ca²⁺ not only for the generation of systolic contraction but also for a sizable physiological SR Ca²⁺ reserve [3]. This in part is due to the presence of the SR Ca²⁺-adenosine triphosphatase (SERCA) pump on the SR membrane that raises the free intra-SR (luminal) Ca²⁺ concentration ([Ca²⁺]) close to 1 mM [3], as well as to the low affinity, high capacity luminal Ca²⁺ buffering protein calsequestrin (CASQ2) that virtually doubles the SR Ca²⁺ storage capacity [4].

It is important to note that both membrane depolarization and CICR, because of their positive feedback mechanisms, are intrinsically prone to instabilities and spontaneous activation. For this reason, these high fidelity, self-regenerating signaling processes require effective means for signal termination and containment. For instance, the termination of the AP and the resultant restoration of the resting membrane potential is achieved by the inactivation of Na⁺ and Ca²⁺ channels as well as by the repolarizing currents carried by multitude of K⁺ channels that include the delayed rectifier current with its multiple components [5]. Resting membrane potential between APs, on the other hand, is stabilized by the inward rectifier K⁺ current. Analogously, termination of SR Ca²⁺ release and restoration of the diastolic Ca²⁺ level within the cytosol of the cardiomyocyte is predominantly attained through inactivation/deactivation of RyR2s as well as by SERCA-dependent resequestration of Ca²⁺ into the SR, and to a lesser extent by extrusion of Ca²⁺ by the sarcolemmal Na⁺-Ca²⁺ exchanger (NCX) [6]. These processes responsible for signal termination ensure that the membrane potential and CICR remain functionally silent or refractory during the diastolic period preventing thereby inappropriately timed APs or contractions.

The concepts of refractoriness and repolarization reserve have been key to understanding the pathophysiology and treatment of rhythm disorders associated with abnormal membrane excitability [7,8]. Despite, growing evidence indicating that regulation of RyR2 by luminal Ca²⁺ is critical for controlling physiologic SR Ca²⁺ release (for recent reviews see references [4,9–11]) key questions as to the molecular components involved in luminal Ca²⁺ regulation of SR Ca²⁺ release and its refractoriness in the development of cardiac disease remains to be resolved. In this review, we will highlight recent advances that have furthered our understanding of the mechanisms underlying the control of SR Ca²⁺ release, termination and refractoriness by the interaction of luminal Ca²⁺ with RyR2 complex. We will also discuss some of the important unresolved issues regarding these processes in normal physiologic and cardiac disease conditions.

2. Luminal Ca²⁺ governs its own release

In cardiomyocytes during contractions, where most of the Ca²⁺ required for contractile activation is supplied by the SR, luminal [Ca²⁺] itself is an important determinant of contractility. However, as suggested by the initial studies, intra-SR Ca²⁺ is not merely acting as a passive reservoir of available Ca²⁺, but plays anactive role in controlling the Ca²⁺ release process [4,12–15]. More precisely, high luminal [Ca²⁺] (load) facilitates Ca²⁺ release from the SR, while a sharp decrease of release is observed at reduced SR [Ca²⁺] load [16,17]. Two principal mechanisms have been proposed by which this regulation could be accomplished. The first is the modulation of RyR2 activity via RyR2 luminal Ca²⁺ sensing sites. The other, termed feed-through, posits that leak of Ca²⁺ from the SR (via RyR2) then alters cytosolic [Ca²⁺] in the vicinity of the “leaky” RyR2 and thereby affects the open probability of that very same and neighboring RyR2s via cytosolic Ca²⁺ sensing sites [4]. Delineating the contributions of these mechanisms in isolated myocytes is complicated by the difficulty of controlling [Ca²⁺] on both sides of the RyR2 channel and by the possibility that these mechanisms may operate in tandem.

Compelling evidence for the regulation of RyR2 gating by luminal Ca²⁺ was obtained in planar lipid bilayers, which allowed not only a direct control over the luminal environment surrounding RyR2s but also prevented the possibility of the feed-through effects [18,19]. It was possible in these studies to isolate the true luminal Ca²⁺ effects and minimize the possibility of feed-through mechanism(s) by either setting a cytosolic-to-luminal electrochemical Ca²⁺ gradient [18] or by verifying the luminal localization of the gating effect by trypsin digestion of the luminal aspects of RyR2 [20]. These studies demonstrated that RyR2 open probability changes as a function of luminal Ca²⁺ with a K_d value of about 1 mM, which corresponds to the SR [Ca²⁺] in a cardiomyocyte at rest [18,21]. Taken together, these studies suggest that in addition to CICR, luminal effects contribute to the regulatory role of SR Ca²⁺ load on Ca²⁺ transient/release by potentiating this effect at elevated SR Ca²⁺ loads. Importantly, these studies also raised the possibility that reduced luminal [Ca²⁺] could serve as a negative regulator of RyR2 by inhibiting Ca²⁺ release at decreased SR Ca²⁺ content [18].

3. Luminal Ca²⁺ alters the sensitivity of cytosolic Ca²⁺ activation site

A number of studies using isolated cardiomyocytes and reconstituted RyR2s demonstrated that luminal Ca²⁺ alters the sensitivity of RyR2 to cytosolic Ca²⁺. Single channel studies demonstrated that rather than uniformly scaling RyR2 activity at different cytosolic [Ca²⁺], increases in luminal Ca²⁺ shift the cytosolic Ca²⁺ sensitivity to lower [Ca²⁺] [18,22,23]. As already mentioned, such investigation of the interplay between the cystosolic and luminal Ca²⁺ on RyR2 function is challenging in the setting of isolated myocytes. To better understand the mechanisms of SR Ca²⁺ release regulation by Ca²⁺ on both sides of the SR membrane, Stevens et al. [24] investigated the effects of a wide range (1–100 µM) of cytosolic [Ca²⁺] on SR Ca²⁺ release in permeabilized cardiomyocytes by monitoring luminal [Ca²⁺]with Fluo-5N, a low-affinity Ca²⁺ indicator. At any given cytosolic [Ca²⁺], including levels as high as 50 µM, luminal Ca²⁺ evidenced periodic oscillations. Since feed-through effects of luminal Ca²⁺ on SR Ca²⁺ release should beminimal at such high cytosolic [Ca²⁺], these intra-SR Ca²⁺ oscillations were attributed to RyR2 alternating between low and high cytosolic Ca²⁺-sensitivity states as determined by the filling status of the SR (low and high Ca²⁺ load, respectively). A similar conclusion regarding dynamic allosteric regulation of RyR2 functional activity by cytosolic and luminal Ca²⁺ was also reached by MacQuaide et al. [25] in their analysis of the effects of tetracaine on spontaneous Ca²⁺ release in cardiomyocytes. Of note, a recent study by Tencerová et al. [26] demonstrated that luminal Ca²⁺ influences RyR2 gating by allosterically modulating affinity of the adenosine-5′-triphosphate (ATP) binding site and thereby RyR2 activation by ATP and low levels of cytosolic [Ca²⁺]s (<500 nM). Taken together, these results are consistent with the view that RyR2 is an allosteric protein whose activity is regulated not only by luminal Ca²⁺ but also by numerous ligands, both endogenous (Mg²⁺, ATP, calmodulin) and exogenous (caffeine, ryanodine, tetracaine) that modulate each others' effects on RyR2 activity [27].

Conversely, Jiang et al. [28] using recombinant channels expressed in HEK cells reported that luminal Ca²⁺ acts without influencing cytosolic Ca²⁺ sensitivity of RyR2. Furthermore, these investigators reported that the application of caffeine, considered a sensitizer of cytosolic activation sites [27], actuated RyR2 Ca²⁺ release by sensitizing the luminal sites without affecting cytosolic sensitivity. However, these results were challenged by the work of Porta et al. [29] which reaffirmed previous observations that caffeine indeed acts by sensitizing the RyR2 cytosolic activation sites [27]. Thus, most available evidence suggests that luminal Ca²⁺ allosterically influences the sensitivity of RyR2 to cytosolic Ca²⁺. As discussed below, the mode of action by which RyR2 is regulated by luminal Ca²⁺ is relevant to understanding the mechanistic control of SR Ca²⁺ release by intra-store Ca²⁺ in normal physiology and disease.

4. Luminal Ca²⁺ and SR Ca²⁺ release termination

Although Ca²⁺ release from the SR should be a self-limiting process because of the restricted size of the Ca²⁺ store, only a fraction of available Ca²⁺ is released during a regular Ca²⁺ transient, thus leaving a substantial Ca²⁺ reserve in the SR [30,31]. Growing evidence, both theoretical and experimental, points to the importance of luminal Ca²⁺ for release termination. Computer simulations by Sobie et al. [15] demonstrated the feasibility of local depletion of SR Ca²⁺ as a signal for the closure of RyR2. At the same time, Terentyev et al. [12,13] used low affinity Ca²⁺ buffers directed to the SR to demonstrate that changes in luminal [Ca²⁺] play a critical role in Ca²⁺ release termination and refractoriness, whereby a decline in the SR Ca²⁺ store resulted in functional deactivation of RyR2s. Subsequent introduction of luminal Ca²⁺ measurement techniques (with SR-entrapped low-affinity Ca²⁺ indicators) made possible direct detection of Ca²⁺ depletion during both unitary Ca²⁺ release events (Ca²⁺ sparks) as well as during cell-wide Ca²⁺ transients [32,33]. Through paired measurements of cytosolic and luminal [Ca²⁺], it has been demonstrated that Ca²⁺ spark termination occurs at a certain steady-state threshold of intra-SR [Ca²⁺] [34–36]. These findings thus offered further support for the role of luminal [Ca²⁺] in release termination. However, it is important to note that this seemingly premature termination of SR Ca²⁺ release in cardiac myocytes that does not completely deplete the SR of Ca²⁺ can also have other explanations. For example, activation of only a fraction of release sites, a scenario where all the activated sites become fully depleted while idle sites maintain their corresponding SR Ca²⁺ content, could also account for the partial release of the total SR [Ca²⁺]. Consistent with this notion, Hake et al. [37] using mathematical modeling to compare the true extent of local junctional SR depletion as reported by Ca²⁺ blinks concluded that blinks may underestimate the extent of local SR depletion. Critical to the role of SR depletion in termination of local Ca²⁺ release is the concept of Ca²⁺ mobility within the SR. Indeed, high Ca²⁺ mobility would tend to prevent severe depletion by hastening Ca²⁺ redistribution from adjacent SR elements. On the other hand, low Ca²⁺ mobility would facilitate SR emptying by restricting the aforementioned redistribution of Ca²⁺ within the SR network. However, studies of the intra-SR diffusion yielded conflicting results with nearly an order of magnitude difference in the apparent diffusion coefficients (8 µm²/s [38] vs. 60 µm²/s [39,40]). Resolving the precise role of local SR Ca²⁺ depletion in termination of Ca²⁺ release will require future investigation into Ca²⁺ diffusion through the SR network as well as further advances in the techniques for measurement of luminal Ca²⁺ in cardiac myocytes.

5. Refractoriness of SR Ca²⁺ release

Following the discharge of Ca²⁺ from the SR, the release mechanism enters a refractory period when it is unresponsive to stimulation by cytosolic Ca²⁺ [41,42]. There is a growing consensus that this refractoriness of Ca²⁺ signaling involves a change of [Ca²⁺] on the luminal side of the SR [4,12,14,24,42,43]. For example, experimental interventions that either accelerate or slow SR refilling corresponding accelerate or slow Ca²⁺ release restitution, respectively, independent of changes in cytosolic Ca²⁺ [12,13,43]. Importantly, the functional recovery of release lags significantly behind refilling of the SR Ca²⁺ store, an observation made on the level of focal as well as global SR Ca²⁺ release using both direct and indirect methods for assessing intra-SR Ca²⁺ recovery [14,43–46].

The specific components of Ca²⁺ signaling restitution following SR Ca²⁺ release were recently defined by Belevych et al. [45] by concomitant assessment of L-type Ca²⁺ current along with cytosolic and luminal Ca²⁺ recorded from myocytes isolated from normal and post-infarction canine hearts. As illustrated in Fig. 1, SR Ca²⁺ release is accompanied by a transient decrease in luminal Ca²⁺ content. The total time from this initial release to a spontaneous Ca²⁺ release event is composed of a period of refilling of the SR Ca²⁺ stores and a subsequent latency period during which the intra-SR Ca²⁺ levels remain constant. Two-pulse experiments demonstrated that the latency period is comprised of a refractory phase and an “idle” phase. As suggested by theoretical analysis performed by Maltsev et al. [47], the idle period can be attributed to a delay required for synchronization of stochastic release sites that have recovered from refractoriness through a process resembling a phase transition from unitary events to global Ca²⁺ release. Of note, the refractoriness and not the idle period are significantly shortened in post-infarction myocytes thereby accounting for increased vulnerability of these myocytes to diastolic spontaneous Ca²⁺ waves. The distinction between a refilling time and a latency period has also been described on the level of Ca²⁺ spark. Using both isolated cardiomyocytes as well as computational simulations, Ramay et al. [48] separated the recovery of Ca²⁺ spark amplitude from recovery of spark triggering probability for recurring events triggered by ryanodine. These investigators concluded that the former depended only on local SR refilling whereas the latter depended on both refilling and on recovery from RyR2 refractoriness. Accordingly, mathematical theory developed by Rovetti et al. [49] proposed that luminal Ca²⁺-dependent refractoriness of release units is one of the important variables required for stability of CICR that governs the appearance of arrhythmogenic phenomena such Ca²⁺ waves and alternans. Thus, in contrast to Ca²⁺ release termination for which the role of functional effects of luminal Ca²⁺ on RyR2 gating is yet to be distinguished from SR Ca²⁺ depletion, release refractoriness does have a distinct component due to the effects upon RyR2 gating (i.e. deactivation).

Fig. 1.

Prior to membrane depolarization, the SR Ca²⁺ interacts with CASQ2 functionally detaching it from the RyR2 complex rendering RyR2 primed for activation (primed cytosolic RyR2 activation site highlighted in red) by cytosolic Ca²⁺ (1). During RyR2 activation and the resulting systolic Ca²⁺ release, SR Ca²⁺ content is partially reduced thereby facilitating CASQ2 interaction with the RyR2 complex [via triadin (TRD) and/or junctin (JNT) (2). This interaction results in an allosteric inhibition of cytosolic activation site of the RyR2. The time between systolic SR Ca²⁺ release and spontaneous diastolic Ca²⁺ release is composed by refilling of SR Ca²⁺ store and latency during which the Ca²⁺ within the SR remains constant (2 and 3). The refractory phase reflects the time required for recovery of RyR2 from luminal Ca²⁺ deactivation and can be determined by a 2-pulse protocol (insets 1 and 2). At the completion of the refractory phase, refilling of the SR Ca²⁺ stores leads to dissociation of CASQ2 rendering RyR2 again functionally primed (primed cytosolic RyR2 activation site highlighted in red, 3) underlying thereby an idle phase during which stochastic activation of the recovered SR Ca²⁺ release sites triggers spontaneous diastolic Ca²⁺ waves (SCW). Phosphorylation (P) and redox modification (R) of RyR2 along with mutations in CASQ2 or the SR Ca²⁺ release complex (M) promote shortened recovery of RyR2 from luminal Ca²⁺ deactivation reducing thereby the refractory period and the time to spontaneous diastolic Ca²⁺ waves.

6. The intermediary luminal Ca²⁺ sensor

Consistent with the presence of a RyR2 Ca²⁺-mediated modulation site on the luminal side of the SR, it is possible that luminal Ca²⁺ can directly regulate SR Ca²⁺ release by binding directly to the luminal side of RyR2 or via intermediate luminal auxiliary proteins such as triadin (TRD), junctin (JNT) and CASQ2. The notion of direct luminal regulation was further advanced in experiments using recombinant RyR2 variants (expressed in HEK cells devoid of RyR2 luminal auxiliary proteins) with altered responsiveness to luminal Ca²⁺ [28,50,51]. Based on these experiments it was proposed that direct activation of RyR2 by luminal Ca²⁺ independent of accessory proteins and effects of cytosolic Ca²⁺ causes the release of Ca²⁺ from the SR [28,50]. However, a body of evidence has accumulated, suggesting that intermediate regulatory proteins play a role in modulation of SR Ca²⁺ release by luminal Ca²⁺ [13,22,52–57]. Moreover as noted above, most available evidence indicates that luminal Ca²⁺ acts by allosterically affecting RyR2's responsiveness to cytosolic Ca²⁺, and hence CICR, rather than by directly activating the RyR2 [24–27].

Studies from several laboratories have demonstrated that CASQ2 is capable of conferring luminal Ca²⁺ sensitivity to RyR2s reconstituted in planar lipid bilayers [22,52–54]. The effects of CASQ2 are dependent on the presence of luminal TRD and JNT and are influenced by cytosolic factors such as ATP. In RyR2s activated by cytosolic Ca²⁺ and ATP association with CASQ2 has been reported to decrease RyR2 activity at low luminal Ca²⁺ (<1 mM) and facilitate the channel response to high luminal Ca²⁺ [52,54]. The effects of CASQ2 were mediated by TRD (and/or JNT) as they relied on the presence of luminal TRD and JNT [52]. In the absence of activating ATP, CASQ2 conferred luminal Ca²⁺ dependency by increasing RyR2 activity at high (>1 mM) but not low luminal Ca²⁺ (at which open probability was close to zero) [22]. In both settings (i.e. in the presence or absence of ATP) the ability of CASQ2 and luminal Ca²⁺ to modulate RyR2 function was compromised by arrhythmogenic mutations linked to Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) [22,35]. Future RyR2 reconstitution studies will have to address the precise contributions of direct Ca²⁺ binding and intermediate luminal Ca²⁺ sensor(s) on the RyR2 as well as the role of intracellular affecters (including ATP, Mg²⁺, calmodulin) in modulation of RyR2 complex gating by luminal Ca²⁺.

The role of CASQ2 in Ca²⁺ handling has been further demonstrated in studies utilizing myocytes deficient in CASQ2 [13,55,56] and those expressing inhibitory peptides targeting CASQ2-TRD interaction [57]. These studies revealed that the absence of CASQ2 or specifically targeting CASQ2 interaction with the RyR2 complex leads to dysregulated SR Ca²⁺ release. Such alterations in the function of the RyR2 complex manifest an increased frequency of diastolic Ca²⁺ sparks and Ca²⁺ waves. Taken together these results provided further evidence that CASQ2 can act as a luminal Ca²⁺ sensor that cross-talks (possibly through TRD) with RyR2, thereby regulating SR Ca²⁺ release and its termination and/or refractoriness by stabilizing the deactivation of RyR2.

Based on the combination of studies conducted using single RyR2 and myocyte reviewed above it was proposed that RyR2 modulation by CASQ2 and luminal Ca²⁺ involves the following steps [24]: At low luminal [Ca²⁺], CASQ2 interacts with the RyR2 complex rendering the cytosolic activation site on RyR2 allosterically inhibited (Fig. 1). Refilling the SR Ca²⁺ stores by SERCA during the recovery (restitution) phase once again increases luminal Ca²⁺, causing CASQ2 to dissociate from the RyR2s rendering these SR Ca²⁺ release channels once more sensitized for yet another activation by cytosolic Ca²⁺. There is also evidence that has implicated other proteins such as TRD [56,58], JNT [59] and histidine-rich Ca²⁺ binding protein [60] in regulation of RyR2. The potential role of these proteins in mediating the effects of luminal Ca²⁺ on RyR2 function, as well as that of Ca²⁺-dependent CASQ2 polymerization-depolymerization as part of the luminal signaling cascade [35] requires further investigation.

7. The luminal Ca²⁺ threshold level for spontaneous SR Ca²⁺ release revisited

Over forty years ago Fabiato & Fabiato [2] reported that in addition to CICR, mechanically skinned cardiac cells can exhibit another form of SR Ca²⁺ release that occurs secondary to increased SR Ca²⁺ load, independent of a cytosolic Ca²⁺ trigger. These unprovoked, aberrant SR Ca²⁺ releases, also visualized as propagating Ca²⁺ waves, have been examined in many subsequent studies. A common observation that emerged from these investigations was that whenever luminal Ca²⁺ reached a “threshold” level, this resulted in spontaneous Ca²⁺ release [61,62] Moreover, this apparent intra-SR threshold has been reported to be reduced in cardiac disease as a consequence of either genetic mutations or acquired modifications in the RyR2 channel complex thus accounting for the increased arrhythmogenic propensity in these cell types [28,61,63,64]. These results have sometimes been taken as an indication that spontaneous Ca²⁺ release is directly activated by luminal Ca²⁺ independent of canonical CICR, a process referred to as store-overload-induced Ca²⁺ release (SOICR) [28,50,51]. This view however may not be sufficiently justified.

As recent studies have demonstrated with the aid of direct luminal Ca²⁺ monitoring, spontaneous Ca²⁺ release does not occur once the intra-SR Ca²⁺ returns to its baseline level, but a distinct time delay is present before another spontaneous Ca²⁺ release event may occur (Fig. 1) [45,48,65]. Importantly, this latency to spontaneous Ca²⁺ release is shortened in disease despite lowered diastolic intra-SR [Ca²⁺] [45,65]. Thus direct monitoring of [Ca²⁺] SR failed to confirm existence of a luminal [Ca²⁺] threshold as a sufficient factor for activation of diastolic Ca²⁺ release. The concept of threshold SR Ca²⁺ content while being useful for characterization of SR Ca²⁺ release at steady-state, it may be less applicable to beat-to-beat Ca²⁺ cycling dynamics as occurs in the beating heart and in arrhythmias. The aforementioned behavior further challenges the notion of direct activation of Ca²⁺ release by luminal Ca²⁺, and as alluded to earlier instead points to recovery from refractoriness as the underlying mechanism responsible for the timing of spontaneous Ca²⁺ release. A correlation between incidents of diastolic release and SR refilling rate consistent with a role for store-dependent refractoriness has been also demonstrated in intact beating hearts [46].

Considering the intrinsic instability of CICR, which requires mechanisms able to exquisitely control it, it is difficult to envision a physiologically useful role for direct luminal Ca²⁺ activation of RyR2. Rather than operating as a stimulator of Ca²⁺ release, luminal modulation appears to constrain RyR2 activity when intra-SR Ca²⁺ level is low such as following SR Ca²⁺ release. As growing evidence suggest [12–14,43,45], following Ca²⁺ release the decrease of luminal Ca²⁺ provides a signal for deactivation of RyR2s thereby contributing to diastolic refractoriness of Ca²⁺ release. Thus CICR that relies on auto-catalytic action of Ca²⁺ at the cytosolic side of the SR is precisely balanced and counteracted by a deactivation mechanism dependent on a reciprocal decrease of Ca²⁺ inside the SR. Importantly, the impairment of this regulatory mechanism accounts for the spontaneous Ca²⁺ release observed in various pathologies as discussed below.

8. Genetic and acquired arrhythmias associated with altered luminal Ca²⁺ control of SR Ca²⁺ release

The importance of RyR2 modulation by luminal Ca²⁺ in healthy heart is highlighted by recent findings indicating that genetic defects in RyR2 and its auxiliary luminal proteins including CASQ2 and TRD are associated with inherited arrhythmias such CPVT [66–68]. CPVT has been attributed to “leaky” RyR2s resulting in spontaneous Ca²⁺ release during diastole [51,69,68].Whereas the link between spontaneous Ca²⁺ release and the consequent membrane depolarization (delayed after depolarization, DAD) is well established and involves NCX stimulated by elevated cytosolic Ca²⁺ [62,70–72], the mechanism responsible for spontaneous Ca²⁺ release in CPVT has been an area of intense investigation. Based on studies using recombinant RyR2, Chen and colleagues suggested that spontaneous release associated with CPVT is due to direct activation of SR Ca²⁺ release by luminal Ca²⁺ [28,51]. However, CASQ2 and other auxiliary luminal proteins were absent from the experimental preparations used by these investigators making their interpretation of the role of luminal Ca²⁺ in a physiological milieu difficult. Furthermore, as already stated the concept of direct activation of RyR2 by luminal Ca²⁺ is not supported in cardiomyocytes by direct experimental measurements of spontaneous Ca²⁺ release either on the level of cytosolic or luminal Ca²⁺ dynamics [45,65]. Conversely, evidence provided by other laboratories suggests that rather than acting by direct luminal Ca²⁺ activation, CPVT-associated genetic mutations of CASQ2 destabilize the deactivation of RyR2 [44,68,73,74]. This is accomplished by an alteration of luminal Ca²⁺ control of RyR2 and thereby shortening Ca²⁺ release refractoriness resulting in premature spontaneous Ca²⁺ release (Fig. 1).

Growing evidence suggests that similar alterations in RyR2 function as those ascribed to CPVT-associated mutations accompany some of the much more common acquired forms of cardiac diseases including both ischemic and non-ischemic cardiomyopathy. In the case of these acquired diseases, post-translational modification of RyR2s by either phosphorylation (via Protein Kinase A and/or Calmodulin-dependent Protein Kinase II) and/or by reactive oxygen and nitrogen species have been implicated as molecular mechanisms of altered Ca²⁺ release function [45,64,65,73,75]. Moreover, research into alterations of RyR2 complex in these acquired forms of cardiac disease provided new mechanistic insight into the role of altered Ca²⁺ signaling and its refractoriness in arrhythmogenesis. Specifically, RyR2 phosphorylation and oxidation evidenced in post-infarction cardiomyocytes resulted in shortened Ca²⁺ signaling refractoriness that contributed to the increased rate of spontaneous Ca²⁺ waves observed in these cells [45]. Thus it appears that genetic arrhythmogenic mutations within the components of the RyR2 complex along with posttranslational modifications of the Ca²⁺ release channel associated with acquired cardiac disease act through a common mechanism, which affects the RyR2 luminal to cytosolic Ca²⁺ regulatory axis (Fig. 1).

Taken together these studies highlight the importance of RyR2 function and its regulation by its auxiliary luminal proteins and luminal Ca²⁺ in the maintenance of dynamic stability of SR Ca²⁺ release. The loss of such regulatory mechanism results in shortened Ca²⁺ release refractoriness that in turn underlies arrhythmogenic spontaneous Ca²⁺ release and DADs. Refractoriness has been extensively investigated in relation to electrical repolarization and its reserve [8,76]. By analogy it appears Ca²⁺ release refractoriness and CICR stability reserve hold similar promise for understanding and treatment of arrhythmias. Future studies will need to elucidate the role of Ca²⁺ release refractoriness as well as other mechanism(s) responsible for synchronization of Ca²⁺ waves and the ensuing membrane potential depolarization between individual cardiac cells.

Glossary

AP

Action potential

ATP

Adenosine-5′-triphosphate

[Ca²⁺]

Ca²⁺ concentration

CICR

Ca²⁺-induced Ca²⁺ release

CASQ2

Calsequestrin

JNT

Junctin

NCX

Na⁺–Ca²⁺ exchanger

RyR2

Ryanodine receptors channels

SR

Sarcoplasmic reticulum

SERCA

SR Ca²⁺-adenosine triphosphatase

SOICR

Store-overload-induced Ca²⁺ release

TRD

Triadin