**1. Introduction**

The rhythmic beating of the heart is controlled by an intricate and wellorchestrated flux of ions through a process called excitation–contraction coupling (ECC), where the electrical action potential leads to cellular contraction. Among all the ions involved in ECC, calcium (Ca) plays a critical role and serves as the signal for cardiac contraction. Briefly, upon cardiac excitation a small current of Ca enters the cytoplasm through the sarcolemmal L-type Ca channels (LTCC). This triggers a much larger Ca release from the sarcoplasmic reticulum (SR)—the intracellular Ca store—by opening the type 2 ryanodine receptor (RyR2) channel in a process called Ca-induced-Ca release (CICR) [1]. The resultant elevation of cytoplasmic Ca concentration activates the contractile apparatus, thus leading to myocyte contraction. For relaxation to occur, Ca must be extruded from the cytoplasm. Two main

#### **Figure 1.**

*Cardiac excitation-contraction coupling.*

mechanisms are involved in removing cytoplasmic Ca: one is by Ca re-sequestration into the SR to replenish the intracellular store, through the action of the SR Ca ATPase (SERCA). The other is by transporting calcium outside of the cell via the membrane-embedded protein, sodium calcium exchanger (NCX) (**Figure 1**). Other avenues for Ca removal do exist (e.g. sarcolemmal Ca-ATPase and mitochondria Ca uniporter), but only play a minor role in this process [1]. The rhythmic rise and fall of cytoplasmic Ca underlies the systolic and diastolic phases of the cardiac cycle.

#### **2. Dysregulated SR Ca release is linked to cardiac pathologies**

The RyR2 is a large protein with a molecular weight of ~560 kDa that forms homotetrameric channels in the SR membrane (**Figure 2**) [2]. Due to its crucial role in releasing Ca to trigger contraction, it is no surprise that there are a number of auxiliary proteins with likely overlapping/redundant functions acting from both the cytosolic and SR luminal sides to regulate the function of the channel complex. Moreover, the activity of the channel is also subject to regulation by posttranslational modifications, including redox modifications, phosphorylation, and nitrosylation [3]. Unfortunately, both genetic and acquired defects due to mutation or posttranslational modification of the channel complex contribute to its dysfunction [3]. These defects typically make the channel hyperactive or leaky, giving rise to dysregulated Ca release (DCR). DCR is implicated in a spectrum of cardiac dysfunctions [3], and in particular, it directly causes a deadly cardiac arrhythmia syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT) [3, 4].

CPVT is a stress-induced arrhythmia that is triggered by elevated levels of catecholamines [5]. Patients do not exhibit the cardiac remodeling typical of structural heart diseases, which makes the diagnosis particularly challenging. Life-threatening cardiac arrhythmias occur following exercise or emotional stress, which elevates circulating catecholamine levels. CPVT mutations have been identified in the genes encoding the RyR2 channel and its several auxiliary proteins. The remainder of this chapter focuses on the regulation of SR Ca release, the molecular mechanisms of CPVT, and the current and future state of therapies targeted towards CPVT.

*Molecular Mechanism and Current Therapies for Catecholaminergic Polymorphic Ventricular… DOI: http://dx.doi.org/10.5772/intechopen.98767*

**Figure 2.**

*SR Ca release channel RyR2 is regulated by both cytosolic and SR luminal proteins.*

Since dysregulated SR Ca release has been implicated in multiple cardiac disorders, knowledge obtained from CPVT studies will also shed light on the development of therapeutic approaches for these devastating cardiac dysfunctions as a whole.

### **3. Modulation of RyR2 Ca release**

RyR2s form physically separated/isolated clusters to act as functionally independent Ca release units [6]. Within dyads, the structural element formed by the close apposition of T-tubules and junctional SR, Ca influx via LTCC in the T-tubule triggers more Ca release from RyR2 clusters in the junctional SR to initiate contraction (**Figure 1**). Ca release from individually activated RyR2 clusters are known as Ca sparks and can be experimentally observed during diastole [7]. The systolic Ca transient is the summation of tens of thousands of Ca sparks due to the synchronized Ca release of RyR2 clusters following sarcolemmal depolarization. The positive-feedback nature of CICR suggests that SR Ca release should terminate upon depletion of SR Ca store. However, only a fraction of the SR Ca store is released during EC coupling [8, 9]. This begs the question of how the Ca release process is terminated. Cytosolic Ca-dependent inactivation of RyR2 has been proposed as a mechanism for the termination of Ca release [10], but lacks widespread support. In contrast, evidence from different research groups collectively points to a mechanism that works to inhibit Ca release from the SR luminal side.

The first piece of evidence comes from an *in vitro* study conducted with single RyR2 channels reconstituted into lipid bilayers. This study demonstrated that the opening of RyR2 is significantly reduced at lowered luminal Ca [11]. The channel's luminal accessory proteins, calsequestrin 2 (CASQ2), junctin, and triadin, are required for this luminal Ca-dependent inhibition of the channel [12]. More

convincing evidence comes from a subsequent cellular study that manipulated the SR Ca buffering capacity by introducing exogenous Ca chelators into the SR to result in a slower Ca depletion [13]. Enhanced SR Ca buffering drastically increased the amplitude of Ca release (both Ca sparks and global Ca transients) and slowed its termination, hence supporting the role of SR luminal Ca in controlling RyR2 Ca release. Mathematical modelling studies provided further support that luminal Ca-dependent deactivation of RyR2 is involved in termination of Ca release [14].

While the role of luminal Ca-dependent deactivation of RyR2 has been established, it is unclear what the specific molecular mechanism is. There is evidence supporting either direct activation of RyR2 or through its luminal accessory proteins, i.e. CASQ2, junctin, and triadin, which form the SR Ca release unit with RyR2. Studies performed in human embryonic kidney cells (HEK293) overexpressing recombinant RyR2 support a direct activation of RyR2 by luminal Ca. Despite a lack of several Ca handling proteins, HEK293 cells with exogenously expressed RyR2 mutants of CPVT exhibit dysregulated Ca release that is sensitive to SR Ca load in a process called store-overload-induced Ca release (SOICR) [15]. The authors found that CPVT mutations of RyR2 reduce the threshold for SOICR, which is expected to increase the propensity of dysregulated Ca release, hence contributing to cellular arrhythmogenesis. Further, a more recent study from the same group proposed the amino acid E4872 as the luminal Ca sensor for the direct activation of RyR2 [16]. A point mutation of E4872A completely abolishes luminal Ca activation of RyR2 in single channel studies, and markedly reduces dysregulated Ca release in HEK293 and HL-1 cardiac cells. Moreover, mice harboring a heterozygous mutation of E4872Q are resistant to SOICR and protected from ventricular arrhythmias *in vivo*. E4872 is localized in the S6 helix bundle-crossing region, the putative cation binding pocket of RyR2. Despite recent breakthroughs in resolving the structure of RyR2, the composition of this putative cation binding pocket remains undetermined [17], and it is thought that other key amino acids in this region such as E4878 may also play a critical role in luminal Ca activation of RyR2.

On the other hand, independent labs have provided evidence supporting the participation of luminal proteins in regulating RyR2 Ca release. Lipid bilayer single-channel studies found that CASQ2 serves as a sensor to inhibit opening of RyR2 at low luminal [Ca], which notably required the presence of junctin and triadin, thus suggesting these proteins form a regulatory complex to control luminal Ca-dependent deactivation of RyR2 [12]. Additionally, increasing or decreasing the expression of CASQ2 in rat cardiac myocyte not only changes the SR Ca storage capacity, consistent with CASQ2's Ca buffer function, but also affects SR Ca release [18]. In particular, decreased expression of CASQ2 leads to dysregulated arrhythmogenic Ca release, supporting CASQ2 function as an inhibitor of RyR2 [18]. Interestingly, the expression of a competitive peptide in myocytes to disrupt the interaction between CASQ2 and triadin impairs the ability of CASQ2 to stabilize the Ca release channel [19], thus echoing conclusions from earlier bilayer studies that CASQ2 interacts with other luminal proteins to regulate RyR2 activity. The development of a genetic model of CASQ2 KO mouse provided additional evidence supporting CASQ2's regulation of SR Ca release [20]. CASQ2 KO mice phenocopied human CPVT by exhibiting catecholamine-induced tachyarrhythmias in vivo. Myocytes isolated from these mice are characterized with β agonist-induced dysregulated Ca release, a hallmark of cellular arrhythmogenesis. However, besides resulting in CPVT, the ablation of major SR Ca buffer protein CASQ2 does not seem to result in more severe cardiac dysfunctions, suggesting the existence of other Ca buffer proteins with similar function at the SR luminal side. Surprisingly, deletion of CASQ2 and another Ca binding protein histidine-rich calcium binding protein (HRC) in a double knockout (DKO) mouse model alleviates arrhythmias as

*Molecular Mechanism and Current Therapies for Catecholaminergic Polymorphic Ventricular… DOI: http://dx.doi.org/10.5772/intechopen.98767*

compared with the CASQ2 KO mouse [21]. HRC binds to RyR2 through the same CASQ2-binding domain on triadin, and results from this DKO mouse study suggest that rather than having redundant roles, CASQ2 and HRC play opposing roles to regulate RyR2 Ca release. Taken together, these studies not only support the notion that luminal accessory proteins of RyR2 participate in controlling SR Ca release, but also highlight the intricate nature of such regulation.

#### **4. Molecular mechanisms of CPVT**

CPVT mutations have been identified in 6 genes encoding 4 different proteins of the Ca release channel complex: *RYR2*, *CASQ2*, *TRDN*, *CALM1*, *CALM2*, and *CALM3* [22]. Among them, the 3 genes of calmodulin (*CALM1*, *CALM2*, and *CALM3*) encode the same protein. Of note, these mutations account for up to 60–75% of CPVT cases, with the genetic cause of the remaining clinical cases unknown [23, 24]. It is likely that more disease mutations will be discovered in other proteins of the Ca release channel complex. In this section, we will summarize the proposed molecular mechanisms for different genetic forms of CPVT.

#### **4.1 CPVT linked to RyR2 mutations**

Among the genetically confirmed cases of CPVT, over 90% are due to mutations of RyR2 [23]. CPVT linked to RyR2 mutations is autosomal dominant. Up to date, more than 200 gain-of-function mutations in RyR2 have been discovered. RyR2 loss-of-function mutations have also been detected but are less frequent and are associated with arrhythmias distinct from CPVT [25]. Additionally, the loss-offunction mutation appears to cause arrhythmias through an early afterdepolarization (EAD)-based mechanism [26] which is much less studied compared to the classic DAD-based mechanism. Never the less, EADs have been observed in CPVT patient specific induced pluripotent stem cells (iPSC)-derived cardiomyocytes [27] in addition to myocytes isolated from a mouse model harboring a loss-of-function CPVT mutation of RyR2 [26]. The focus of the rest of the chapter will be on the arrhythmias evoked by DADs, rather than EADs.

Three main theories of how gain-of-function RyR2 mutations lead to CPVT have been proposed by different groups. The first comes from the observation that CPVT mutants of RyR2 expressed in HEK293 cells decreased the threshold to induce SOICR [15]. Based on these results, it is proposed that CPVT mutations make RyR2 more sensitive to luminal Ca, thus susceptible to dysregulated arrhythmogenic Ca release. The second theory proposes that CPVT mutations reduce the binding of RyR2 to FKBP12.6, a cytosolic protein thought to stabilize the channel, thus increasing RyR2 activity and giving rise to arrhythmogenic diastolic calcium release [28]. However, this theory has been challenged by several labs [29–31], a conserved binding between CPVT mutant RyR2 and FKBP has been reported [32]. The majority of RyR2 mutations are found at three "hot spots" which are located in the N-terminal domain (amino acids 1–600), central domain (amino acids ~2100–2500) and C terminal domain (amino acids ~3900–end) of the protein [33, 34]. Structural studies show that many of them are found at the domain-domain interfaces, thus giving rise to the third theory that mutations impair the inter-domain interactions of RyR2 to cause CPVT. Specifically, the interaction between N-terminal and central domains of RyR2 is responsible for the so-called domain "zipping" and is thought to stabilize the channel; the third theory posits that CPVT mutants impair this interaction (causing domain unzipping) and causes channel dysfunction. This model of domain zipping-unzipping has been supported by experimental evidence [35–37].

#### **4.2 CPVT linked to CASQ2 mutations**

The second most common cause of CPVT is mutation of CASQ2, an SR luminal Ca binding protein thought to regulate deactivation of RyR2. CPVT linked to CASQ2 was considered as an autosomal recessive disease until the recent discovery of autosomal dominant disease-causing mutations [38]. CASQ2 is a low-affinity, high-capacity Ca binding protein. It does not contain Ca binding structural domains such as an EF-hand motif, a helix–loop–helix structural domain, found in "typical" Ca binding proteins (troponin C, calmodulin) [39]. Instead, it has multiple (~60–70) negatively charged amino acids, which facilitates electrostatic interactions between the protein and ~ 40–50 Ca ions [40]. CASQ2 monomers change their structure upon Ca binding, and form protein polymers in a Ca-dependent process. Structural studies show that monomeric CASQ2 contains three highly similar tandem domains, resembling that of bacterial thioredoxin. However, much less is known about the structure of the polymers. Based on *in vitro* biophysical studies by Park et al. [41], the following model of CASQ2 polymerization is proposed: CASQ2s exist as monomers at low luminal [Ca]; as [Ca] increases, CASQ2s form dimers, tetramers, and polymers in a [Ca]-dependent process. Thus, CASQ2s polymerize to bind additional Ca at high luminal Ca, but depolymerize to release Ca at low luminal Ca. Considering the Ca and protein concentrations in SR, CASQ2s likely exist as a mixture of monomers, dimers, and polymers of varying sizes [42]. As described above, monomeric CASQ2 is thought to be anchored to RyR2 via junctin and triadin to deactivate Ca release at low luminal Ca. The intriguing question remains whether this Ca-dependent change in the polymerization states of CASQ2 happens on a beat-to-beat basis in response to SR Ca load to regulate RyR2 Ca release. It has been shown that the polymerization state of CASQ2 changes upon depletion of luminal Ca in fibroblast by fluorescent approaches [43]. Additional evidence comes from studies conducted with skeletal muscle fibers demonstrating luminal-Ca dependent changes in polymerization of CASQ1, the skeletal counterpart of CASQ2 [44]. However, whether Ca-dependent changes in CASQ2 polymerization happens in beating cardiomyocytes at a time scale comparable with the cardiac cycle awaits further investigation.

At least 2 molecular mechanisms are proposed to explain how autosomal recessive CPVT mutations of CASQ2 cause the disease based on its two primary functions, buffering Ca and modulating RyR2 opening [4]. These mutations (nonsense or missense) lead to loss or reduced expression of CASQ2. Subsequently, reduced Ca buffering allows the free Ca to rise faster near the Ca release sites, thereby triggering dysregulated Ca release. Besides reduced Ca buffering power, some missense mutations of CASQ2 appear to work through another mechanism: by impairing RyR2 regulation from the luminal side. It's been shown that the mutation of R33Q leads to abnormal interaction between CASQ2 and the Ca release channel complex [45], and another mutation of D307H reduces the binding between CASQ2 and triadin [46]. These results support the notion that a regulatory complex involving several proteins (CASQ2, triadin, junctin, and potentially others) senses luminal Ca to regulate Ca release, and disruption of interactions between them leads to dysregulation of the channel and disease. Compared with the autosomal recessive mutations, less is known about the autosomal dominant mutations that were more recently identified. Two mutations (K180R and S173I) have been found to interfere the polymerization of CASQ2 [47], likely causing CPVT by reducing the Ca buffering capacity.

#### **4.3 CPVT linked to triadin mutations**

CPVT mutations have also been identified in triadin, a trans-SR membrane protein that helps anchor CASQ2 to the RyR2 channel complex. Triadin has a short *Molecular Mechanism and Current Therapies for Catecholaminergic Polymorphic Ventricular… DOI: http://dx.doi.org/10.5772/intechopen.98767*

N-terminal region located on the cytosolic side of SR, a single membrane spanning domain, and a highly charged C-terminal region that comprises most of the protein and resides in the SR luminal side. The C-terminal tail of the protein contains KEKE motifs formed by stretches of alternating residues with opposite charges. A single KEKE motif consisting of 15 residues (210–224) has been suggested as the CASQ2 binding region [48]. The binding between triadin and CASQ2 is Ca-dependent and they dissociate at high [Ca] (10 mM). In contrast, triadin's binding to RyR2 is Ca-independent [48]. Due to its role of anchoring CASQ2 to Ca release sites, triadin is thought to facilitate SR Ca release by allowing CASQ2 to buffer Ca near the Ca release channel.

It is also proposed that triadin may play a direct role in regulating RyR2 channel activity. Both overexpression and knockout mouse models of triadin have been created to decipher its function. The overexpression model displayed hypertrophy and altered Ca handling, accompanied by compensatory changes in the expression of several proteins of the RyR2 channel complex, thus masking the functional role of triadin [49]. Similarly, loss of triadin in the KO model also caused compensatory changes [50]. Drastic reduction in the interface of junctional SR and T-tubules occurred due to structural remodeling, thus impaired the coupling between RyR2 and LTCC. As a result, inactivation of LTCC is reduced, which increased Ca current, prolonged action potential, and subsequently increased cellular and SR Ca. Due to Ca overload, myocytes from triadin KO model displayed elevated levels of arrhythmogenic Ca release, especially when stimulated with catecholamines [50]. While both mouse models support the notion that triadin plays an important role in myocyte Ca handling, the massive compensatory changes in these chronic models makes an explanation of the data challenging. Nevertheless, acute overexpression of triadin in cultured myocytes increased RyR2 opening, dysregulated Ca release, and membrane depolarization, mimicking the cellular phenotype of CPVT [51]. Relatively few CPVT mutations of triadin have been reported as of yet. In a 2012 study, three autosomal recessive mutations of triadin were discovered, with two of them (one deletion, one nonsense) resulting in loss of the protein. The third one, a missense mutation of T59R, results in a protein that is more susceptible to degradation [52]. Thus, loss or decreased expression of triadin appear to cause CPVT. Another two autosomal recessive triadin mutations were identified in a 2015 study [53], although the underlying disease-causing mechanisms await further investigation.

#### **4.4 CPVT linked to calmodulin mutations**

Calmodulin (CaM) is an EF-hand Ca binding protein that binds RyR2 from the cytosolic side to regulate Ca release. CaM has a dumbbell-shaped structure, with its two globular domains connected by a flexible central helix. Each of the two domains contain two EF-hand Ca binding sites. The N-domain of the protein has a lower Ca binding affinity than the C-domain [54, 55]. Upon Ca binding, the hydrophobic pockets in both domains become exposed, thereby allowing CaM to bind its several intracellular targets, including RyR2, LTCC and Na channel (Nav 1.5) [56]. Mutations of CaM have been linked to different types of arrhythmias, such as CPVT, long QT syndrome, and idiopathic ventricular fibrillation, likely due to its impaired regulation of various target proteins [22]. Following systolic Ca release and the ensuing increasing in Ca on the cytosolic side of RyR2, CaM binds to the channel and inhibits its opening during the diastole phase of the cardiac cycle [57, 58]. CPVT mutations of CaM appear to have impaired ability to inhibit the channel and promoted the generation of DCR in the form of Ca waves and Ca sparks in a cellular study [59]. They also exhibited higher binding affinity to RyR2 than WT CaM, thereby contributing to the autosomal dominant mode of action [59].
