Chain-reaction Ca²⁺ signaling in the heart

Mutations in Ca²⁺-handling proteins in the heart have been linked to exercise-induced sudden cardiac death. The best characterized of these have been mutations in the cardiac Ca²⁺ release channel known as the ryanodine receptor type 2 (RyR2). RyR2 mutations cause “leaky” channels, resulting in diastolic Ca²⁺ leak from the sarcoplasmic reticulum (SR) that can trigger fatal cardiac arrhythmias during stress. In this issue of the JCI, Song et al. show that mutations in the SR Ca²⁺-binding protein calsequestrin 2 (CASQ2) in mice result not only in reduced CASQ2 expression but also in a surprising, compensatory elevation in expression of both the Ca²⁺-binding protein calreticulin and RyR2, culminating in premature Ca²⁺ release from cardiac myocytes and stress-induced arrhythmia (see the related article beginning on page 1814). In the context of these findings and other recent reports studying CASQ2 mutations, we discuss how CASQ2 influences the properties of Ca²⁺-dependent regulation of RyR2 and how this contributes to cardiac arrhythmogenesis.

In the normal heart, the cycling of intracellular Ca²⁺ in cardiomyocytes is critical to the heart’s mechanical contraction and relaxation. On a beat-to-beat basis, Ca²⁺ entry through voltage-gated Ca²⁺ channels in the sarcolemma (including the transverse tubules) locally activates cardiac ryanodine receptor type 2 (RyR2) Ca²⁺-release channels found in large clusters in the junctional sarcoplasmic reticulum (jSR) across a very narrow (15 nm) junctional gap (Figure 1A). The release of Ca²⁺ from functional clusters of RyR2s depends on this Ca²⁺ influx and works by the mechanism of Ca²⁺-induced Ca²⁺ release (CICR). The release of Ca²⁺ from the RyR2 clusters is visualized as Ca²⁺ sparks. The Ca²⁺ sparks amplify the initial Ca²⁺ influx trigger signal and combine to produce an elevation of cell-wide myoplasmic [Ca²⁺] called the Ca²⁺ transient. This increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) leads to activation of the contractile proteins and hence to generation of the heartbeat (1). Ca²⁺ release to the cytosol is accompanied by a reciprocal decline in the [Ca²⁺] within the SR ([Ca²⁺]_SR) for both the [Ca²⁺]_i transient (2) and for Ca²⁺ sparks (3). This reduction in [Ca²⁺]_SR contributes to deactivation or closure of RyR2s, resulting in Ca²⁺ release termination and induction of a refractory state that prevents Ca²⁺ release during the diastole (4–6). Relaxation occurs following Ca²⁺ reuptake into the SR through the phospholamban-regulated (PLN-regulated) sarcoplasmic or endoplasmic reticulum Ca²⁺ ATPase 2 (SERCA2a). Ordered Ca²⁺ cycling is essential to normal rhythmic activity of the heart, and disturbances in Ca²⁺ handling have previously been shown to underlie diverse Ca²⁺-dependent cardiac arrhythmias (7–9). In this issue of the JCI, catecholamine-induced polymorphic ventricular tachycardia (CPVT) caused by mutations in calsequestrin 2 (CASQ2) is the subject of the study by Song et al. (10); Ca²⁺ signaling plays a central role in the dysfunction.

Figure 1

Intracellular Ca²⁺ handling in cardiomyocytes. (A) Calcium transients begin with the initial influx of Ca²⁺ via L-type Ca²⁺ channels followed by Ca²⁺ release from the SR via RyR2s, which culminates in contraction. During relaxation, Ca²⁺ reuptake occurs via the PLN-regulated Ca²⁺ pump SERCA2a. The major Ca²⁺ buffering protein in the SR is CASQ2. High [Ca²⁺]_SR converts monomeric CASQ2 (bound to the RyR2-triadin-junctin complex) to the polymeric CASQ2 form that buffers Ca²⁺ and remains close to the complex in the jSR. Calstabin2 and monomeric CASQ2 bind to the complex and stabilize RyR2 activity. (B) Altered Ca²⁺ handling in CASQ2-deficient myocytes. As Song et al. report (10), in CASQ2-deficient mouse myocytes, RyR2 expression is significantly upregulated and calreticulin abundance is slightly increased. There is a decrease in Ca²⁺ in the SR. Despite altered Ca²⁺ handling in these animals under resting conditions, these compensatory changes in protein expression appear to help maintain relatively normal heart function. However, under catecholamine- or exercise-induced stress, RyR2 instability increases, leading to an increased risk of cardiac arrhythmia. nSR, nonjunctional SR.

It is appreciated that RyR2 activity underlies SR Ca²⁺ release and generation of the cytosolic Ca²⁺ transient that is required for muscle contraction. However, just how RyR2 is activated, how sensitive it is to [Ca²⁺] in both cytosolic and lumenal compartments, and how RyR2-mediated Ca²⁺ release from the SR is regulated depends in complex ways on five interacting proteins: the SR transmembrane proteins, RyR2, junctin, and triadin; CASQ2, located within the SR lumen; and FKBP12.6 (also known as calstabin2), which is tightly bound to the large cytosolic regulatory domain of RyR2 (11–13) (Figure 1A).

CASQ2 is the major Ca²⁺-binding and -buffering protein that resides entirely within the SR and binds the Ca²⁺ that is released during Ca²⁺ sparks and during the [Ca²⁺]_i transient (5, 11, 14). It normally exists in monomeric and polymeric forms, with the polymers dynamically self assembling when [Ca²⁺]_SR is in submillimolar range (15, 16). Full CASQ2 polymerization is thought to occur at high mM [Ca²⁺]_SR. Polymeric CASQ2 has a high Ca²⁺-binding capacity (16) and is located close (~5 nm) to the clustered RyR2s (17) that are organized in a paracrystalline array in the jSR membrane. Monomeric CASQ2 forms a quaternary complex with RyR2 and the intrinsic membrane proteins triadin and junctin, and this conformation decreases the likelihood or probability that the RyR2 channel will be triggered to open by the low diastolic [Ca²⁺]_i. The low open probability (P_o) of RyR2 channels under physiological conditions prevents the RyR2s from opening when they are not triggered by Ca²⁺ influx across the sarcolemmal and transverse tubule membranes, and thus provides a margin of safety. The interactions among CASQ2 and other members of this complex are weakened by elevated [Ca²⁺]_SR. Consequently, at elevated [Ca²⁺]_SR, the P_o of RyR2s increases and results in increased Ca²⁺ release from the SR in cardiomyocytes. Thus, CASQ2 appears to play at least two different roles in cardiac myocytes: as a Ca²⁺ storage reservoir in the SR and as an active modulator of the Ca²⁺ release process. As a Ca²⁺ storage molecule, CASQ2 is thought to supply the bulk of Ca²⁺ required for contractile activation. As a modulator of Ca²⁺ release, CASQ2 controls RyR2 P_o (via protein-protein interactions involving triadin and junctin) in a manner that depends on the amount of Ca²⁺ within the SR lumen. Given the importance of these SR proteins for Ca²⁺ handling, it is not surprising that genetic alterations of these proteins lead to cardiac disease.

Disturbances in the regulation of intracellular Ca²⁺ in the heart were linked explicitly to electrical abnormalities and arrhythmogenesis over 30 years ago. These arrhythmias developed during Ca²⁺ overload, a state of the cardiac myocyte in which intracellular Ca²⁺ levels are elevated (7–9, 18). During Ca²⁺ overload, increased Ca²⁺ instability within the myocyte was observed, and during diastole, the rate of appearance of elementary Ca²⁺ release events, Ca²⁺ sparks, changed from rare to frequent. Additionally, Ca²⁺ sparks could trigger a chain reaction in the form of propagating waves of CICR within the myocytes (1, 19, 20) whereas under control conditions, Ca²⁺ waves are not seen at all. Increases in lumenal [Ca²⁺] sensitized RyR2s to activation by cytosolic Ca²⁺, contributing to generation of Ca²⁺ waves above a certain [Ca²⁺]_SR threshold (21, 22). Such cellular arrhythmogenesis during Ca²⁺ overload underlies changes in both automaticity and electrical conduction that contribute to arrhythmogenesis (23–25).

CPVT is a type of Ca²⁺-dependent triggered arrhythmia that was initially identified as resulting from mutations in RyR2 (26) and more recently from mutations in CASQ2 (27). CPVT occurs in the absence of structural heart disease and is characterized by episodes of syncope, seizures, or sudden death, usually elicited during physical activity or stress. Seven autosomal recessive mutations in CASQ2 are linked to CPVT (28–31) in addition to the more than 60 arrhythmogenic mutations in RyR2 (32, 33). The fact that mutations in the SR Ca²⁺ release channel (RyR2) and in the SR Ca²⁺-binding protein CASQ2 both result in the same phenotype (exercise-induced sudden cardiac death or CPVT) suggests a common mechanism linked to aberrant regulation of SR Ca²⁺ release. The study by Song et al. reported in this issue of the JCI (10) examines the consequences of mutating (or deleting) CASQ2 in mice and how the changes in expression of the CASQ2 protein and other proteins involved in Ca²⁺ signaling lead to cardiac arrhythmias, specifically CPVT. These mouse models, as well as a CASQ2 knockout mouse described by Knollmann et al. in a recent issue of the JCI (34), emulate the exercise-induced CPVT that has been linked to mutations in the CASQ2 gene in humans (30, 31).

Song et al. (10) constructed two lines of mutant mice: first, homozygous CASQ^307/307 mice, which possessed a D307H missense mutation in their CASQ2 gene, a mutation that has previously been identified in a number of Bedouin families in Israel with recessive CPVT (31). The second animal line, homozygous CASQ^ΔE9/ΔE9 mice, possessed a truncation mutation causing loss of CASQ2 exon 9. Complete absence of CASQ2 (homozygous 62delA and 532+1 G/A) was previously shown to be associated with CPVT in patients with otherwise functionally and structurally normal hearts (30). The finding that these mutations did not cause structural heart disease in humans was puzzling given the presumed importance of CASQ2 to Ca²⁺ handling and the expectation that absence of this protein would be incompatible with life. Both the study by Song et al. (10) and the recent work with CASQ2 knockout mice (34) help to resolve this enigma. The results show that there are a multitude of compensatory mechanisms that develop when CASQ2 is mutated or knocked out. Song et al. (10) demonstrate that CASQ2-deficient mice show a compensatory (albeit small) increase in expression of the lumenal Ca²⁺-binding protein calreticulin. Additionally, RyR2 expression was dramatically (6-fold) enhanced in a possible attempt by the cells to compensate for the reduced SR Ca²⁺ content (Figure 1B). The logic behind such compensatory changes may be that, along with increased SR volume demonstrated by Knollmann et al. (34), these changes should help the SR to maintain its Ca²⁺ storage function. However, these compensatory mechanisms clearly proved inadequate to fully restore normal Ca²⁺ handling. Indeed the SR Ca²⁺ content remained significantly reduced in CASQ2-deficient myocytes. Moreover, under conditions of stress, the mice developed malignant arrhythmias characteristic of CPVT. Given the similar manifestations of arrhythmia caused by CASQ2 mutations and arrhythmia associated with Ca²⁺ overload, one may expect that these two disease states have the same underlying mechanism. However, in reality the situation is more complex, as detailed below.

A critical factor in this story is determination of Ca²⁺ concentration within the SR itself (6), which is assessed by measuring the average Ca²⁺ efflux or “leak,” the Ca²⁺ reaccumulation by SERCA2a, and levels of the Ca²⁺-buffer CASQ2. One paradox that has emerged from the current work of Song et al. (10) and another recent study (34) is that CASQ2-deficient myocytes have low [Ca²⁺]_SR yet possess an increased probability of Ca²⁺ release — a phenomenon known as the overload paradox. Although the paradox is not fully resolved in these studies, key questions are raised and specific future experiments suggested. The overload paradox is found in other arrhythmic diseases including heart failure (35–37) and CPVT due to RyR2 mutations (38). A common feature in these arrhythmic diseases, including those associated with CASQ2 mutations, is increased SR Ca²⁺ leak. This term refers to the loss of Ca²⁺ from the SR by any means. Ca²⁺ sparks represent a clear and visible loss of Ca²⁺ from the SR; indeed, whenever any RyR2 opens, there is a loss of SR Ca²⁺. In addition, whenever either RyR2s or inositol-1,4,5-trisphosphate receptors open, Ca²⁺ leaks out of all of the Ca²⁺ storage organelles (SR, ER, and nuclear envelope) because they are interconnected (39). Mutations in both RyR2 and CASQ2 lead to CPVT and result in a leaky SR. The question is thus raised, How does SR leak tie in with arrhythmogenesis? This will be discussed here in the context of the role played by CASQ2 under normal and disease conditions.

Under control conditions, SR Ca²⁺ leak is low and is thought to occur almost exclusively through RyR2 Ca²⁺ release channels. Clustered RyR2s (20–300 in number) in the jSR produce Ca²⁺ sparks when activated. In contrast, single or very small clusters of RyR2s (“rogue” RyR2s) may produce SR Ca²⁺ release that may not be readily visible as Ca²⁺ sparks (40). The low SR Ca²⁺ leak in ventricular myocytes is due to the low P_o of the RyR2 Ca²⁺ release channels under diastolic conditions and the low sensitivity of RyR2s to [Ca²⁺]_i. Importantly, however, RyR2 sensitivity to [Ca²⁺]_i can be modulated by many factors, including [Ca²⁺]_SR (41, 42), phosphorylation of critical proteins such as RyR2 itself, and possibly important proteins such as CASQ2, junctin, triadin, and calstabin2 (11, 13, 43).

Although Ca²⁺ sparks do not normally trigger other Ca²⁺ sparks, during Ca²⁺ overload, a Ca²⁺ spark chain reaction can occur, and this reaction appears as propagated waves of elevated [Ca²⁺]_i (19). Such reactions may also arise when RyR2 sensitivity to [Ca²⁺]_i is increased (e.g., by increased [Ca²⁺]_SR, mutations in RyR2 (38, 44), or mutations of CASQ2). As the RyR2 P_o increases, so does the SR Ca²⁺ leak rate. Under steady state conditions, the increased leak tends to deplete the SR of Ca²⁺ whether the leak is due to Ca²⁺ sparks or to rogue RyR2 openings. While the probability of arrhythmogenic Ca²⁺ waves increases with RyR2 P_o, SR Ca²⁺ content tends to decrease (Figure 2). Thus, there is a biphasic relationship between the probability of an arrhythmogenic wave and P_o. The common thread in cellular arrhythmogenesis is a disturbance in Ca²⁺-signaling stability, and this instability underlies multicellular conductance abnormalities (24, 38). This work raises the question, How do CASQ2 mutations lead to arrhythmogenesis?

Figure 2

Ca²⁺-dependent arrhythmogenesis. (A) Relationship between [Ca²⁺]_SR and diastolic [Ca²⁺]_i. As [Ca²⁺]_i increases, so does [Ca²⁺]_SR. (B) As [Ca²⁺]_SR increases, so does SR Ca²⁺ leak. Any additional features that increase RyR2 openings (P_o) will also increase Ca²⁺ leak. (C) As leak increases, there is an increasing loss of Ca²⁺ in the SR. (D) Probability of generating a cellular arrhythmia (i.e., a wave) (P_wave). [Ca²⁺]_SR is the primary factor in Ca²⁺ overload arrhythmogenesis because it affects P_o. However, as the leak increases, there is loss of Ca²⁺ from within the SR. Thus, increasing P_o has a biphasic effect on P_wave. The relationship is biphasic because at low P_o, [Ca²⁺]_SR remains sufficiently high to produce substantial Ca²⁺ efflux and sustain the propagation of Ca²⁺ waves. When P_o is very high, the Ca²⁺ leak outpaces SERCA2a; there is a net loss of [Ca²⁺]_SR, and P_wave decreases. Increased SERCA2a activity (red curve) shifts the curve (48). The physiological range occurs at very low RyR2 P_o (about 10^–4 s^–1).

Catecholamines activate protein kinase A (PKA), which phosphorylates RyR2 and also PLN (Figure 2B). When phosphorylated, PLN no longer inhibits SERCA2a and [Ca²⁺]_SR increases. This phosphorylation-dependent modulation of [Ca²⁺]_SR is an important physiological modulation of cardiac Ca²⁺ signaling by catecholamines and is not arrhythmogenic. However, when CASQ2 is mutated, the relationship between [Ca²⁺]_SR and RyR2 behavior may be different, and this difference underlies CPVT. As discussed above, CASQ2 affects RyR2 sensitivity to [Ca²⁺]_i by binding to the homologous SR transmembrane proteins triadin and junctin (11, 12). At low [Ca²⁺]_SR, CASQ2 is tightly bound to triadin and junctin (Figure 1), and in this four-protein complex conformation, RyR2 is inhibited. As [Ca²⁺]_SR increases, the CASQ2-RyR2 complex is weakened and RyR2 becomes more sensitive to [Ca²⁺]_i. In the absence of normal CASQ2, which is capable of inhibiting RyR2 in a Ca²⁺-dependent manner, the RyR2 complex will be more sensitive to [Ca²⁺]_i, increased Ca²⁺ leak from the SR would be expected to be seen, and there will be a propensity for Ca²⁺ waves and arrhythmogenesis at lower [Ca²⁺]_SR (28). The compensatory increase in the Ca²⁺-binding protein calreticulin is small, and calreticulin does not seem to interact with the RyR2-triadin-junctin complex. The effects of the known mutations in increasing the sensitivity of RyR2 to [Ca²⁺]_i combine with the actions of PKA on RyR2 and on PLN to produce the CICR chain reaction and the CPVT phenotype (28, 45). The resolution of the overload paradox in CPVT may simply be that in CPVT, the Ca²⁺ overload phenomenon occurs at much lower [Ca²⁺]_SR.

The compensatory increase in RyR2 abundance may further exacerbate the problem if the RyR2 proteins are added to the already large jSR clusters (at this point, however, we do not know the fate of the overexpressed RyR2 proteins). More RyR2 proteins at the jSR may increase the overall likelihood that one is activated to produce the Ca²⁺ chain reaction. Additionally, calstabin2 expression, which remained unchanged compared with wild type, may become insufficient for stabilizing RyR2 activity due to a significantly reduced calstabin2/RyR2 ratio.

Using an animal model to investigate an important disease provides significant insight into molecular pathophysiology but also highlights the tools we use and the complexity of the diseases we study. CPVT is recapitulated in mouse models with the CASQ2 D307H mutation as shown in the Song et al. study (10). There are, however, many questions raised by this investigation that motivate future work. Previous studies did show expression of the mutant CASQ2 protein, but they demonstrated that the D307H mutation impairs monomeric CASQ2 binding to triadin or/and disrupts formation of polymeric CASQ2 (45–47). This loss of CASQ2 function in animals with the D307H mutation may accelerate CASQ2 protein degradation, and the disparities in CASQ2 abundance may depend on differences in the genetic background of the mice used in the various studies. This raises the question of how Ca²⁺ signaling remains quasinormal in the D307H mice when there is virtually no CASQ2 to buffer the SR Ca²⁺. As Song et al. suggest (10), the overexpression of calreticulin (which is normally only present in cardiomyocytes at low levels) may substitute for CASQ2 loss. Finally, does calreticulin interact with the RyR2-junctin-triadin complex to modulate signaling? The cellular investigation of these questions remains to be carried out. A possibly even more perplexing question is raised by the elevation of RyR2 expression: How does this feature of the CPVT phenotype contribute to the disease? Finally, it is interesting to note that the two functional knockouts in the Song et al. study exhibit a different set of compensatory mechanisms compared with the mutant CASQ2 mouse described by Knollmann et al. (34). Whereas the principal changes reported in the Song et al. study are increased RyR2 and calreticulin levels, Knollmann et al. (34) observed expansion of SR volume and downregulation of triadin and junctin. In future studies, it will be interesting to determine how these different adaptive changes support the same disease phenotype. In summary then, either mutations in the RyR2 channel that render it leaky due to decreased binding of calstabin2 as previously shown (44) or those in CASQ2 that impair inhibition of the RyR2 channel from the luminal SR side can result in a diastolic SR Ca²⁺ leak that triggers fatal cardiac arrhythmias.

We thank the National Heart, Lung, and Blood Institute for continued support of S. Györke and W.J. Lederer and the National Institute of Arthritis and Musculoskeletal and Skin Diseases and the Muscle Training Program, University of Maryland School of Medicine for support to B.M. Hagen.

Address correspondence to: W.J. Lederer, Institute of Molecular Cardiology, Medical Biotechnology Center, University of Maryland Biotechnology Institute, 725 West Lombard Street, Baltimore, Maryland, USA. Phone: (410) 706-8181; Fax: (410) 510-1545; E-mail: lederer@umbi.umd.edu.

Nonstandard abbreviations used: [Ca²⁺]_i, cytosolic Ca²⁺ concentration; [Ca²⁺]_SR, SR Ca²⁺ concentration; CASQ2, calsequestrin 2; CICR, Ca²⁺-induced Ca²⁺ release; CPVT, catecholamine-induced polymorphic ventricular tachycardia; jSR, junctional SR; PLN, phospholamban; P_o, open probability; RyR2, ryanodine receptor type 2; SERCA2a, sarcoplasmic or endoplasmic reticulum Ca²⁺ ATPase 2; SR, sarcoplasmic reticulum.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.117:1758–1762 (2007). doi:10.1172/JCI32496.

See the related article at Calsequestrin 2 (CASQ2) mutations increase expression of calreticulin and ryanodine receptors, causing catecholaminergic polymorphic ventricular tachycardia.

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