**5. Impaired Ca signaling refractoriness and generation of DCR**

Despite the differences in the molecular details on how the various CPVT mutations of the RyR2 channel complex cause the disease, they all seem to make the channel more susceptible to arrhythmogenic diastolic Ca release. Following systolic Ca release, RyR2 becomes functionally suppressed and remains that way for a brief period, known as Ca signaling refractoriness [60]. Refractoriness of the Ca release channel can be measured by myocyte experiments employing a two-pulse protocol to record the process of Ca transient restitution. It's been demonstrated that full recovery of Ca transient takes ~1 s (anywhere from ~0.8 − 1.5 s, depending on species) [61–65]. If Ca signaling refractoriness is impaired, the RyR2 channel is expected to recover earlier from the functionally suppressed state, thereby promoting the generation of DCR, thus causing cellular arrhythmogenesis. Indeed, multiple CPVT mutations have been found to shorten refractoriness of RyR2, including mutations of CASQ2 [65], CaM [66], and RyR2 [67]. Moreover, shortened Ca signaling refractoriness can also occur due to oxidation/hyperphosphorylation of RyR2 as seen in models of acquired heart diseases [63]. Thus, both genetic and acquired defects of RyR2 channel complex seem to converge on shortening Ca signaling refractoriness to cause arrhythmogenic Ca release. Further evidence supporting shortened refractoriness as a unifying mechanism for the generation of DCR comes from a recent study using an engineered therapeutic CaM in an attempt to restore refractoriness and treat CPVT [66], as discussed in a later section on future therapies for CPVT. Taken together, these studies suggest that disease mutations may change the SR Ca dynamics, the modulation of RyR2 by cytosolic or luminal proteins, or conformational changes of the channel protein itself, each of which has been experimentally demonstrated to shorten Ca signaling refractoriness and give rise to arrhythmogenic DCR.

#### **6. Cellular arrhythmogenesis: SR Ca load**

At the single myocyte level, DCR is manifested as different forms: Ca sparks, Ca wavelets, and propagating Ca waves. When large enough, DCR activates electrogenic NCX, resulting in an inward current that causes delayed afterdepolarization (DAD) [68–70]. With a large enough amplitude, DADs may surpass the voltage threshold to open Na channels, thus leading to the generation of an ectopic action potential or triggered activity [71, 72]. Both the amplitude and the rate of DCRs are important in determining if it will trigger an ectopic action potential [73]. Localized DCR events in the form of Ca sparks and wavelets are less likely to trigger ectopic action potentials as compared with propagating Ca waves. Ca waves are more likely to occur when SR Ca load is high, such as following activation of β-adrenergic signaling pathways.

β-adrenergic stimulation results in phosphorylation of key EC-coupling proteins and subsequent generation of a larger and faster Ca transient, underlying its positive inotropy (ability to contract) and lusitropy (ability to relax) effect [1]. One of these proteins, phospholamban (PLN), acts as an inhibitor of SERCA. Its inhibition on SERCA is relieved upon β-adrenergic-dependent phosphorylation, thus contributing to a faster Ca transient decay and also higher SR Ca content. This higher SR Ca load facilitates Ca wave generation, and explains the stress-induced arrhythmias that occur in CPVT. Therefore, SERCA's function to refill the SR with Ca is critical to maintain a certain SR Ca load to stimulate the generation of Ca waves. On the other hand, SERCA directs Ca out of the cytosol while refilling the SR with Ca, which opposes the formation of or "breaks" Ca waves.

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

Based on these seemingly contradictory effects of SERCA activity on Ca wave generation, an interesting question arises: what will be the consequences of upregulating SERCA activity in the setting of CPVT? Both beneficial and deleterious effects have been reported from studies conducted by different groups. When overexpressing a skeletal isoform of SERCA1a in the CPVT model of CASQ2 KO, the resultant CPVT-SERCAox mice developed severe Ca-dependent cardiomyopathy [74]. These mice suffered from early mortality and contractile dysfunction. Myocytes isolated from the hypertrophied hearts of these animals also displayed enhanced levels of DCR. While these results clearly demonstrate a detrimental effect, the severe cardiomyopathy phenotype due to chronic SERCA overexpression masks the effect of the genetic manipulation on arrhythmias. A follow-up study from the same group conditionally overexpressed SERCA2a in the same CASQ2 KO mice and found that both atrial and ventricular arrhythmias were exacerbated due to acute upregulation of SERCA activity [75]. In contrast, in another study employing a different CPVT model of RyR2 knock in mouse (R4496C+/<sup>−</sup>), upregulation of SERCA activity by knocking out its inhibitor PLN suppressed arrhythmias *in vivo*. In cellular experiments, Ca waves were also suppressed, due to propagating Ca waves being converted into non-arrhythmogenic mini waves and Ca sparks [76]. Interestingly, a different study showed that although enhancing SERCA activity by PLB ablation alleviated arrhythmias, it exacerbated myocardial infarction and cardiac damage in a RyR2 model featuring elevated DCR due to a mutation (S2814D) mimicking constitutive CaMKII-mediated phosphorylation of RyR2 [77].

#### **7. Synchronization of DCR in myocardium**

It is well established how DCR triggers ectopic action potentials at the cellular level. However, the heart contains billions of cardiomyocytes, and how arrhythmogenesis at the level of isolated myocytes translates into arrhythmias at the level of a multi-cellular tissue preparation or even the whole heart remains unknown. Within the myocardium, individual myocytes are electrically coupled to their neighboring cells, hence Ca-dependent depolarizing currents generated in any random, isolated cells should be easily absorbed by neighboring cells that act as a current sink (the source-sink mismatch theory) [78]. Therefore, cellular depolarization, if happening randomly in individual cells, cannot generate sufficient current to trigger tissuelevel depolarization.

Computational simulation studies suggest a very large number of cells—nearly 7x105 —have to depolarize simultaneously to overcome source-sink mismatch and trigger depolarization to generate an ectopic beat in normal myocardium. This number is reduced by modeling disease conditions such as fibrosis or heart failure related electrical remodeling, but the number of requisite cells still remains quite large [78]. Therefore, it's been proposed that DCR happens in a synchronous way in multiple cells of the CPVT hearts to cause a tissue-wide ectopic beat. Experimental evidence has been provided in support of the synchronization of DCR in a CPVT model carrying the CASQ2 R33Q mutation [65]. This study quantified the degree of DCR synchronization by measuring the latency, or the time interval to the first DCR, following systolic Ca release. It was found that DCR occurs in a highly synchronized way in both myocytes and cardiac muscle tissue obtained from the R33Q CPVT model. Importantly, two factors are important for the synchronization of DCR: 1) shortened Ca signaling refractoriness that increases the propensity of release sites to fire synchronously by facilitating CICR, and 2) the presence of a preceding systolic action potential acting as a synchronizing event that temporally aligns the release sites and primes them for recovery from refractoriness.

### **8. The cellular origin of CPVT: Purkinje cells or ventricular myocytes?**

Purkinje fibers are a specialized network of electrically excitable cells found in the conduction system of the heart. They radiate throughout the ventricular muscle to ensure a rapid propagation of electrical impulse and a coordinated ventricular contraction. Compared with the myocardium, the Purkinje system has a smaller source-sink mismatch [78]. Based on this and other structural features [79], Purkinje cells have been proposed as the cellular origin of many arrhythmias including CPVT. Experimental evidence obtained from the CPVT model of RyR2 R4496C+/− mouse supports this hypothesis [80]. Optical mapping of R4496C+/<sup>−</sup> hearts demonstrates that ventricular tachycardia (VT) may originate from the His-Purkinje system in both ventricles. Cellular studies also found that Purkinje cells had a significantly higher rate of DCR and triggered activity compared to ventricular myocytes [81, 82].

However, a recent study attempting to establish the causal link between Purkinje cells and CPVT did not provide such evidence [83]. In this study, CASQ2 was conditionally knocked out in the cardiac conduction system, but not the myocardium, using a conduction system-specific Cre recombinase. Ablation of CASQ2 in the Purkinje fibers failed to produce a CPVT phenotype. Considering CASQ2 ablation is an established molecular cause of CPVT as demonstrated by a global CASQ2 KO model [20], this result argues against Purkinje cells as the origin of CPVT, at least not on their own. On the other hand, in support of myocytes as the origin of CPVT, cells isolated from the myocardium of CPVT mouse models have been shown to exhibit DCR, DAD, and ectopic action potentials in multiple studies [20, 66, 67, 80]. Human iPSC-derived cardiomyocytes generated from biopsies of human CPVT patients also displayed DCR, DAD, and ectopic action potentials characteristic of the abovementioned CPVT cells [84, 85]. Drug studies based on isolated myocytes also serve as good indicators of drug efficacy in both mouse models and humans [22, 86, 87]. More evidence regarding the cellular origin of CPVT are discussed elsewhere [22].

#### **9. Therapies for CPVT**

Symptoms of CPVT vary from palpitations, syncope, or even cardiac arrest. Although a rare disease, the mortality rate of CPVT can reach as high as ~50% in untreated individuals before the age of 40 [23]. In this section, we will first discuss traditional therapies that are currently available to CPVT patients. Next, we will focus on novel therapeutic approaches, based on recent advances in understanding the molecular mechanisms of this life-threatening arrhythmia syndrome.

#### **9.1 Current therapies for CPVT**

#### *9.1.1 Beta-blockers*

Beta-blockers are the first-line drug therapy to treat CPVT. As discussed above, in CPVT, the β-adrenergic-dependent increase in SR Ca load is important in triggering DCR and subsequent cellular arrhythmogenesis. Thus, blocking the β-adrenergic signaling pathway is expected to decrease DCR and suppress arrhythmias. The most effective beta-blocker at the time of writing is nadolol [88, 89], but it remains unknown why it is more effective than other beta-blockers. Unfortunately, betablockers only offer limited protection even with the maximal tolerated dose. It has been reported that more than 30% patients still suffer from arrhythmic events after receiving beta-blockers [90].

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

Carvedilol, a beta-blocker that is highly effective in preventing VT in heart failure, has been shown to suppress CPVT through a dual inhibitory action on both β-adrenergic signaling and RyR2 channel activity [91]. Experimentally, an analog of carvedilol with minimal beta-blocking activity still prevented VT in a CPVT mouse model and exhibited improved efficacy when combined with a selective beta-blocker [91]. Nevertheless, further studies are required to assess its effectiveness in CPVT patients. However, this provides a new potential pharmacological approach where a combination of RyR2 channel inhibition and beta-blockade could provide a more effective therapeutic approach than current options based solely on beta-blockers.

#### *9.1.2 Na channel blockers and flecainide*

Na channel blockers may serve as anti-arrhythmic drugs due to the critical role of the Nav 1.5 channel in the depolarization phase of action potential. Flecainide, an FDA approved drug to treat arrhythmias, was originally thought to work by blocking the Na channel. Recent studies have found that flecainide prevents CPVT both in mouse models and human patients through a dual inhibition mechanism: inhibiting Na channels as well as RyR2 [86]. Studies show that flecainide appears to be a promising therapy for CPVT patients not responding well to beta-blockers. However, the working mechanism of flecainide was controversial.

A study conducted on the CPVT model of RyR2 R4496C+/<sup>−</sup> showed that while flecainide was effective in preventing arrhythmias, it did not reduce DCR in the cellular experiments. Instead, it increased the threshold for triggered activity, thus pointing to the other possibility: that the drug works by solely acting as a Na channel blocker [92]. Several follow-up studies attempted to reconcile this discrepancy. Evidence has been provided that flecainide is effective in reducing DCR in cells harboring the RyR2 R4496C+/<sup>−</sup> mutation, but this effect could be masked by experimental conditions such as Ca overload [93]. On the other hand, more convincing evidence comes from a recent study that employed a synthesized analog of flecainide with reduced inhibition on RyR2 activity but unaltered inhibition on Na channel [94]. This analog failed to reduce DCR at cellular level and arrhythmia burden *in vivo,* indicating that flecainide acts through inhibition of RyR2 activity. In support of this, flecainide reduced DCR in permeabilized CPVT cells lacking membrane-residing Na channels, and intact cells pretreated with Na channel blocker. Similar to flecainide, another approved drug propafenone also seems to work through dual inhibition of Na channel and RyR2 [95]. Further studies are required to fully understand its working mechanism.

#### *9.1.3 Other treatment options*

Ca channel blockers (LTCC blockers) such as verapamil, have been tested in cellular and animal studies, as well as clinical studies, to examine their efficacy in treating CPVT. Consistently, these studies found Ca channel blockers only confer limited benefits in both cellular preparations and patients already on beta-blockers [96–98]. However, it has been shown to be beneficial for some patients when used in combination with other pharmacological approaches including beta-blockers [98].

Left cardiac sympathetic denervation serves as an alternative treatment. It works by preventing the release of catecholamines from the sympathetic nerve endings. The procedure appears to be effective in reducing major arrhythmic events in clinical studies [99, 100], and thus has been recommended for patients who don't respond to more conventional pharmacological treatments such as beta-blocker therapy. In one case employing an extrapleural approach, the lower part of the

stellate ganglion was ablated together with the second and third thoracic ganglia [99]; another case used the thoracoscopic, the transaxillary, and the supraclavicular approaches as the main surgical approaches [100].

Implantable cardiac defibrillators (ICD) have been utilized in patients who still experience symptoms despite drug therapy and/or sympathetic denervation. A recent study systematically analyzed the efficacy of ICDs using existing clinical data containing 505 CPVT patients implanted with ICDs [101]. It was found that although effective for ventricular fibrillation, ICDs were not protective for VT. Another study of 136 CPVT patients also suggests ICD implant did not confer survival benefits [102]. Considering the potential complications and psychological burden of implantation, especially for pediatric patients, ICDs are not an optimal treatment for CPVT patients.

#### **9.2 Potential future therapies for CPVT**

The molecular mechanisms underlying CPVT have been intensively studied in the past several decades. Several novel therapeutic strategies have been proposed and tested in animal models and even pre-clinical studies. In this section, we will discuss these novel approaches, with a focus on gene therapy.

#### *9.2.1 Gene therapy*

With advances in the adeno-associated virus (AAV) vector-based gene transfer technology in the past a few decades, using gene therapy to treat CPVT is starting to become technically feasible. Several proof-of-principle studies have been conducted to test the efficacy of different therapeutic strategies. Considering several CPVT mutations, especially the ones identified in CASQ2, cause loss or reduced expression of the associated protein, it would seem that the most straightforward therapeutic approach is to deliver a normal gene encoding the protein. Indeed, AAV9-mediated gene transfer of a WT CASQ2 to both CASQ2 KO and R33Q mouse models restored the normal expression of CASQ2, improved abnormal electrophysiological properties of cells, and reduced arrhythmia burden *in vivo* [103, 104]. However, this gene replacement approach is limited by the size of the AAV vector, thus hindering the delivery of a normal gene for the large RyR2 protein, which accounts for the majority of the CPVT mutations. To solve this problem, AAV9 was instead used to deliver siRNA to silence mutant mRNA of RyR2 in an allele-specific way [105]. This RNA silencing approach increased the ratio of WT-RNA vs. mutant RNA, proving to be effective at normalizing cardiac electrophysiology, alleviating abnormal ultrastructural remodeling, and inhibiting *in vivo* VT when tested in the RyR2 R4496C+/<sup>−</sup> mice. Alternatively, another study attempted *in vivo* genome editing using the CRISPR/Cas9 system delivered by AAV and also obtained promising results in a different CPVT model of RyR2 (R176Q+/<sup>−</sup>) [106].

While theses gene therapy strategies seem effective, one of the prerequisites for applying this technology is knowing the genetic cause of CPVT. However, the genetic cause of ~30–40% of clinical cases of CPVT remains undetermined. Several groups have developed novel approaches to tackle this problem. It has been found that the Ca binding properties of CaM—in particular, the kinetics of Ca dissociation from CaM—affects RyR2 refractoriness [66]. Based on this, a therapeutic CaM (TCaM) that specifically targets RyR2 and prolongs its refractoriness by slowing Ca dissociation from CaM was engineered. TCaM reduced DCR in CPVT cells and alleviated arrhythmias *in vivo* when delivered to a CPVT model of CASQ2 R33Q mice [66]. Instead of targeting the specific disease-causing gene, TCaM targets the

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

impaired RyR2 refractoriness, and thus it could potentially serve as a therapeutic avenue for distinct forms of CPVT. Another study chose to target CaMKII, an adrenergically activated kinase that is implicated in arrhythmogenesis and pathological remodeling in multiple cardiac disorders, including CPVT. Pharmacological inhibitors of CaMKII are limited in their efficacy due to their lack of specificity. In contrast, a CaMKII inhibitory peptide was delivered in a cardiomyocyte-specific way by AAV9 and found to be effective in reducing arrhythmias burden *in vivo* in the CPVT model of RyR2 (R176Q+/<sup>−</sup>) [107]. Collectively, these studies provide strong evidence supporting AAV-based gene therapy as a promising future therapy for CPVT patients.

#### **9.3 Targeting sinus node dysfunction**

CPVT patients also present sinus node dysfunction and bradycardia, which are recapitulated in the mouse models of CPVT. The pathophysiological role and underlying mechanism for sinus node dysfunction are discussed in details elsewhere [22]. Targeting the impaired sinus node dysfunction has been proposed as a therapeutic approach for CPVT. It has been shown that increasing heart rate by (1) pharmacological intervention (atropine), (2) atrial overdrive pacing, or (3) re-expressing CASQ2 in the CASQ2 KO mouse all appear to reduce arrhythmia burden [83, 108]. Further, atropine has been tested in a small group of 6 CPVT patients and was found to be effective in reducing exercise-induced arrhythmic events [109].

#### **9.4 Other potential therapies**

Tremendous effort has been expended on identifying and developing small molecules that specifically target DCR of RyR2, since DCR is implicated in a spectrum of cardiac disorders. Dantrolene, a drug used clinically to treat a skeletal muscle condition of malignant hyperthermia, exhibited partial protection for a subset of CPVT patients [110]. The recently discovered ent-(+)-verticilide, an unnatural verticilide enantiomer, appears to be a potent and selective RyR2 inhibitor [87]. It reduced DCR, triggered activity in cells, and arrhythmias *in vivo* when tested with the CPVT model of CASQ2 KO. It seems to exert a stronger antiarrhythmic effect when compared with dantrolene or flecainide. More details on the current state of therapeutic small molecule development are reviewed elsewhere [111].

#### **10. Conclusion**

Great progress has been made in the past few decades to help us better understand CPVT and develop therapeutics for this deadly arrhythmia syndrome. These efforts will continue in both basic science and clinical studies and will provide deeper mechanistic insight on the molecular, cellular, and tissue mechanisms of CPVT. Since DCR is implicated in a spectrum of human diseases, knowledge obtained from these studies will also benefit the development of therapies for other cardiac dysfunctions including heart failure and metabolic heart disease.

#### **Acknowledgements**

We want to thank the American Heart Association for providing funding and National Institutes of Health (1R15HL154073).
