Genetic Diseases: Catecholaminergic Ventricular Tachycardia

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Chapter 65 Genetic Diseases

Catecholaminergic Ventricular Tachycardia

Marina Cerrone, Carlo Napolitano, Silvia G. Priori

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is one of the most malignant inherited arrhythmogenic diseases, with a high incidence of sudden cardiac death among affected individuals. The first report of a patient with this disease was published in 1975, but the first systematic description came in 1978 with the work of Coumel et al and was further refined by the same group in 1995.¹^–³ In 2001, the authors of this chapter reported for the first time that the autosomal dominant form of the disease was caused by mutations in the gene of the cardiac ryanodine receptor.⁴ Shortly thereafter, the gene for an autosomal recessive form of CPVT was identified as the gene encoding cardiac calsequestrin⁵; overall, it became clear that CPVT is caused by abnormal calcium (Ca²⁺) homeostasis.

After the identification of the underlying genetic causes, basic science studies in cellular systems and animal models provided major advancements to the understanding of arrhythmogenic mechanisms in this disease.

In this chapter, the clinical and genetic aspects of CPVT will be reviewed together with the most important experimental observations on CPVT pathophysiology that provide the rationale for the development of novel therapies.

Clinical Presentation and Diagnosis

The initial presentation of CPVT is syncope or cardiac arrest occurring during adrenergic stress.^3,⁴ The mean age of onset of the first syncope is 12 years.⁶^–⁸ In the absence of treatment, the disease is highly lethal: The estimated incidence of sudden death is of 30% before age 40 years.⁹ Sudden death may be the first manifestation of the disease, thus making prevention of lethal events a difficult task.

The diagnostic challenge in CPVT is that in contrast to other inherited channelopathies such as long QT syndrome (LQTS) or Brugada syndrome, the surface electrocardiogram (ECG) is unremarkable. Since patients with CPVT often seek medical attention for the evaluation of unexplained syncope with a normal ECG and no structural abnormalities, they may be misdiagnosed as being affected by vasovagal syncope or epilepsy.

Minor, nondiagnostic features are resting sinus bradycardia and prominent U waves, but their diagnostic value has never been systematically addressed.^3,¹⁰ Furthermore, mild QT prolongation in some CPVT cases has been reported^2,³; thus the differential diagnosis for CPVT should include LQTS. Some patients with LQTS do have a mild phenotype (borderline QT interval and no symptoms), but their prognosis is completely different from that of patients with CPVT, which presents a higher incidence of sudden death and a limited response to β-blocker therapy.^6,¹¹

In the authors’ series of patients with CPVT, a positive history of sudden death, stress-induced syncope, or seizures was present in 30% of patients, suggesting that careful collection of family history plays an important role in the correct identification of this condition.⁶

Exercise stress testing is the single most important tool, since CPVT arrhythmias are often reproducible during graded exercise (Figure 65-1). The typical behavior of CPVT arrhythmias is that of a progressive worsening on increase in workload; isolated premature beats or couplets initially appear between 90 and 110 beats per minute (beats/min) followed by runs of nonsustained or sustained ventricular tachycardia (VT) when the heart rate further increases (see Figure 65-1).¹² Supraventricular arrhythmias are also a common finding and often precede the onset of ventricular arrhythmias.¹³ The morphology of VT is a hallmark of the disease: the so-called bidirectional VT, which is characterized by a 180-degree beat-to-beat rotation of the axis of the QRS complexes on the frontal plane.^4,⁶ Although this pattern is recognizable in the majority of patients, it is important to be aware that some patients also present with irregular polymorphic VT. Furthermore, the initial presentation of the disease may also be that of ventricular fibrillation (VF) triggered by sudden adrenergic activation that may be interpreted as idiopathic VF in the absence of documentation of typical CPVT arrhythmias.⁶ Holter monitoring and implantable loop recorders are helpful for diagnosis, especially in those patients in whom emotional triggers (alone or in combination with exercise) are more arrhythmogenic than exercise alone.^14,¹⁵

FIGURE 65-1 Continuous lead II electrocardiogram tracing of a patient affected by catecholaminergic polymorphic ventricular tachycardia during treadmill stress test. With the increase in the workload, the complexity of ventricular arrhythmias increases.

(Modified from Liu N, Ruan Y, Priori SG: Catecholaminergic polymorphic ventricular tachycardia, Prog Cardiovasc Dis 51:23–30, 2008.)

Programmed electrical stimulation does not contribute to clinical assessment; conversely, epinephrine infusion may often induce the typical pattern of VT, although its diagnostic sensitivity does not appear to be higher than that of exercise stress test.

Genetic Bases

The majority of familial CPVT cases present an autosomal dominant pattern of inheritance. In 1999, Swan et al identified a significant linkage with some microsatellite markers at locus 1q42-q43.¹⁶ On the basis of this information, the authors of this chapter undertook a candidate gene screening in the critical region and identified the cardiac ryanodine receptor (RyR2) as the mutant CPVT gene.⁴ In the same year, Lahat et al described an autosomal recessive transmission pattern on the chromosomal locus 1p13-21 in a large Bedouin CPVT family.¹⁷ Subsequently, they also succeeded in identifying a mutation on the cardiac calsequestrin (CASQ2) gene, encoding for cardiac calsequestrin.⁵

Autosomal dominant (RyR2) CPVT is, by far, the most frequent variant, and CASQ2 mutations represent 1% to 2% of all genotyped CPVT. More recently, on the basis of the evidence that the patients with Andersen-Tawil syndrome may have bi-directional VT, it has been suggested that some CPVT cases can be explained by KCNJ2 mutations.^18,¹⁹ A detailed discussion of the link between KCNJ2 mutations and CPVT is beyond the scope of this chapter, but it is relevant to note here that preliminary experimental studies on adenoviral-infected cardiac myocytes with KCNJ2-CPVT mutations show a cellular phenotype very similar to that observed in RyR2-mutant myocytes.²⁰

In 2007, a new autosomal recessive form of CPVT mapping on the chromosomal locus 7p14-22 was reported by Bhuiyan et al, but the gene disease has not yet been discovered.²¹ So far, more than 70 different mutations have been associated with CPVT, and they all are single–base pair substitutions causing the replacement of an amino acid. As expected with regard to autosomal recessive disorders, the number of families with CPVT linked to CASQ2 mutations is fairly small. At present, only seven mutations have been discovered, and they can be inherited in homozygous or compound heterozygous form. A recent analysis from the authors’ group has demonstrated that genetic screening on the RyR2 gene can identify at least 62% of probands who present with a clinical picture suggestive for the disease²²; genetic testing helps identify silent mutation carriers and for reproductive counseling (Figure 65-2). Furthermore, RyR2-negative and CASQ2-negative patients should undergo KCNJ2 screening. KCNJ2 mutations are associated with a favorable outcome (sudden death is an exceptional event in this case).

FIGURE 65-2 Yield of genetic testing in the autosomal dominant form of catecholaminergic polymorphic ventricular tachycardia (CPVT). FSM, Fondazione S. Maugeri Laboratories, Pavia, Italy; IVF/CA, idiopathic ventricular fibrillation/cardiac arrest; FM, family members; SD, sudden death.

(Modified from Rong B, Napolitano C, Bloise R, et al: Yield of genetic screening in inherited cardiac channelopathies: How to prioritize access to genetic testing, Circ Arrhythmia Electrophysiol 2:6–15, 2009.)

Mechanisms of Arrhythmias in Autosomal Dominant CPVT: From Cellular Studies to Engineered Murine Models

Summary of RyR2 Physiology

The cardiac ryanodine receptor, RyR2, is a tetrameric channel that regulates the release of Ca²⁺ from the sarcoplasmic reticulum (SR) to the cytosol during the plateau phase of the cardiac action potential. Clearly, this is an important physiological function that allows coupling the electrical activation with the contraction of myofibers (so-called electro-mechanical coupling). Abnormal RyR2 function leads to changes in the amount of Ca²⁺ released from the SR and consequently both SR and cytosolic Ca²⁺ concentrations may vary (the other important player for intracellular Ca²⁺ control is the adenosine triphosphate [ATP]–dependent Ca²⁺ pump [SERCA], which, by controlling the uptake of Ca²⁺ from the cytoplasm to the SR, has an impact on the amount of “releasable” Ca²⁺). RyR2 malfunction often causes increased SR release and cytosolic Ca²⁺ overload; this induces compensatory phenomena that tend to restore the balance, such as the activation of the sodium-calcium exchanger (NCX). Unfortunately, such compensatory mechanisms may be arrhythmogenic (see below).

Since Ca²⁺ is not only important for electrical and mechanical activation but also for a wide range of enzymatic and metabolic pathways, it is clear that RyR2 abnormalities may exert additional (and, so far, not thoroughly investigated) consequences on cell physiology.²³

RyR2 function (SR Ca²⁺ release) is regulated by several accessory proteins such as calsequestrin, triadin, junctin, and FKBP12.6. Furthermore, the adrenergic tone controls the RyR2 channel through phosphorylation, which is a crucial function determining the amount of Ca²⁺ released from the SR. Catecholamines activate protein kinase-A (PKA) and calcium-calmodulin–dependent kinase (CaMKII) that phosphorylate RyR2 at different sites (e.g., serine 2008, 2808, and 2914) and act as a throttle on the Ca²⁺ release process.

RyR2 Mutations and Catecholaminergic Polymorphic Ventricular Tachycardia

The effects of RyR2 mutations have been studied in vitro and in vivo by using different experimental models (expression in lipid bi-layers, heterologous cell expression, and knock-in murine models).

RyR2 mutations can affect both the activation and the inactivation of the channel in several manners. It is also likely that different mutations have different consequences (mutation-specific effects) similar to what was demonstrated for plasmalemmal ion channel causing other inherited arrhythmogenic conditions. Of note, independently from subcellular mechanisms, the final common effect of CPVT mutations (both RyR2 and CASQ2) is similar to that of digitalis intoxication: Ca²⁺ overload, activation of NCX in the forward mode, generation of transient inward current (I_ti), and delayed after-depolarizations (DADs) (see below).

The proposed “primum movens” for RyR2-CPVT mutations to lead to Ca²⁺ overload is uncontrolled release (leakage) during diastole, which could be detected, according to different authors, only on adrenergic activation (phosphorylation) or already in the unstimulated condition.²⁴^–²⁶ Given the complexity of the SR Ca²⁺ release process, the leakage could, in principle, be caused by several mechanisms.^24,^25,²⁷ The hypotheses so far advanced are summarized in the following paragraphs (Figure 65-3).

FIGURE 65-3 Different hypotheses of RyR2 dysfunction caused by catecholaminergic polymorphic ventricular tachycardia (CPVT)–related mutations. A, FKBP12.6 disassociation caused by CPVT mutations and its rescue by drugs as K201. B, The threshold of store overload–induced calcium release (SOICR) is reduced in CPVT mutants. C, CPVT mutations destabilize interdomain interactions, affecting proper folding of the protein that ultimately causes Ca²⁺ leakage.

Subcellular Mechanisms for RyR2 Dysfunction in Catecholaminergic Polymorphic Ventricular Tachycardia

FKBP12.6 Dissociation with RyR2 Mutants

FKBP12.6, also known as calstabin, is thought to play a role as a “stabilizer” of the channel in the closed state during diastole. Hints for the possible role of FKBP12.6 in CPVT were initially collected in experimental studies in heart failure. During congestive heart failure, chronic adrenergic hyperactivation may lead to weaker FKBP12.6 binding that can be the cause of Ca²⁺ overload-mediated arrhythmogenesis in this common disease.²⁸ The FKBP12.6 hypothesis, originally developed by Andrew Marks and co-workers, was subsequently extended to CPVT pathophysiology; these authors demonstrated that some RyR2 mutants may cause a reduced binding affinity for FKBP12.6, which is further increased by sympathetic stimulation (see Figure 65-3).²⁷ More recently, the same group performed an FKBP12.6 binding assay in a RyR2 knock-in mouse engineered with the R2474S mutation which has a CPVT-like phenotype²⁹; they confirmed their initial hypothesis by demonstrating reduced FKBP12.6 binding in the presence of adrenergic stimulation. In parallel with these studies, Marks’ group also tried to pharmacologically treat the binding defect by using K201, a benzothiazepine derivative that was expected to increase FKBP-RyR2 binding. Long-term administration of this drug (or of its novel derivative, S107) was able to restore binding and to prevent the occurrence of arrhythmias both in the FKBP12.6 knock-out murine model and in the RyR2^R2474S/WT knock-in murine model.^29,³⁰

Despite the large amount of evidence provided by the Andrew Marks group, other authors either were not able to replicate their results or provided conflicting evidence when they used alternative approaches. George et al and Jiang et al reported normal FKBP12.6-RyR2 interaction in vitro.^24,³¹ Chronic treatment with K201 did not prevent arrhythmias in vivo or the occurrence of DADs in isolated myocytes in a CPVT knock-in mouse RyR2^R4496C/WT in a study by the authors of this chapter (Figure 65-4).³² Furthermore, in this model, Western blot experiments also demonstrated physiological interaction between RyR2 and FKBP12.6.³² Along these lines, Zissimopulos et al demonstrated that FKBP12.6 binding is either unaltered or even increased in the presence of CPVT-RyR2 mutations.³³ Another report by Hunt et al provided evidence that K201 may modulate RyR2 independently from FKBP12.6.³⁴ For example, these authors showed that K201 abolished spontaneous Ca²⁺ release in cardiac myocytes in the presence of FK506 that dissociates FKBP12.6 from RyR2.

FIGURE 65-4 Patch-clamping recording in RyR2^R4496C/WT ventricular myocytes. A, Delayed after-depolarizations in baseline and triggered activity elicited by superfusion with isoproterenol. B, K201 fails to prevent the occurrence of DADs. C, I_ti current recording in an RyR2^R4496C/WT cardiomyocyte in the presence of isoproterenol and after addition of K201 or ryanodine.

(Modified from Liu N, Colombi B, Memmi M, et al: Arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia: Insights from a RyR2 R4496C knock-in mouse model, Circ Res 99:292–298, 2006.)

Overall, considering the limited availability of effective medical therapy for patients with CPVT, the possibility of exploring new treatment options such as K201 and S107 remains appealing; the fact that K201 might be effective independently from its action on FKBP12.6 justifies further studies on this drug.

Defective Interdomain Interaction

RyR2 is a large molecule with a complex tertiary and quaternary structure, in which interactions between domains are important for channel function. Indeed, domain-domain interactions ensure proper folding during the opening and closing of the channel.³⁵

Several RyR2-CPVT mutations occur in the N-terminal (residue 70-466) and the central domains (residues 2246-2543).³⁶ The N-terminal and central domains interact, and their relative conformational change from zipped (tight interaction) to unzipped (loose interaction) is involved in the channel gating process.³⁵ Recent data have highlighted how some CPVT-related mutants may affect this mechanism by inducing a shift toward a mostly unzipped interaction that destabilizes the channel (see Figure 65-3). To test this hypothesis, Oda et al generated a synthetic peptide, DPc10, encompassing the Gly2460-Pro2495 region of RyR2 that mimics an RyR2 mutant: The peptide destabilizes the N-terminal–central region interaction through competitive activity and reproduces the biophysical effect of CPVT mutations.³⁷ The same group engineered several other peptides, mimicking mutations located either in the N-terminal or in the central region of the protein, demonstrating that the mutations caused Ca²⁺ leakage through the disruption of interdomain interaction.³⁸ Independent support to the hypothesis of unzipping has been provided by George et al by means of high-resolution confocal microscopy and fluorescence resonance energy transfer analysis.³⁹

Store Overload–Induced Calcium Release

The open-state probability of RyR2 increases as the SR Ca²⁺ increases. If SR Ca²⁺ reaches a critical threshold, spontaneous Ca²⁺ leakage may occur even in the absence of membrane depolarization (i.e., calcium-induced calcium release). Chen et al proposed the hypothesis that some RyR2 mutants increase channel sensitivity to luminal Ca²⁺ (in other words, reduce the threshold for spontaneous Ca²⁺ release) (see Figure 65-3).^25,^26,³¹ This phenomenon was termed store overload–induced calcium release (SOICR). A reduced SOCIR threshold was shown for nine RyR2 mutations. Interestingly, while luminal (i.e., inside the SR) Ca²⁺ sensitivity was increased, no evidence for an increased sensitivity to cytosolic Ca²⁺ was observed. On the contrary, Thomas et al reported increased sensitivity of the receptor to cytosolic Ca²⁺ in L433P and R176Q/T2504M RyR2 mutants.⁴⁰ While the role of cytosolic Ca²⁺ sensitivity has still to be determined, it appears evident that a reduced threshold for release has an important role, since, as shown by Venetucci et al, increasing ryanodine receptor open probability alone does not produce arrhythmogenic diastolic Ca²⁺ release because of the accompanying decrease of SR Ca²⁺content (and reduced driving force).^41,⁴²

Insights from RyR2-CPVT Murine Models

Knock-in murine models have been pivotal to the understanding of the cellular and whole-heart pathophysiology of CPVT.^29,^43,⁴⁴ In 2005, the chapter authors’ group provided initial evidence that by engineering a RyR2-CPVT mutation in the mouse genome (at variance with what observed for several other murine models of inherited arrhythmias), it is possible to reproduce the phenotype observed in the clinical setting. By homologous recombination, a conditional knock-in mouse harboring the R4496C mutation (a frequent mutation found in CPVT families) was created.⁴³ The typical CPVT bi-directional VT can be generated in this mouse with adrenergic stimulation in the absence of structural abnormalities.⁴³ Furthermore, it was demonstrated that the presence of adrenergic-dependent DADs increased NCX-transient inward current (I_ti) and triggered activity as the cellular mechanisms for CPVT (see Figure 65-4).³² In a subsequent study, the same group observed the onset of abnormal Ca²⁺ waves during diastole, which paralleled the occurrence of DAD development both at baseline and during isoproterenol superfusion.⁴⁵ Increased propensity to DAD development in RyR2-R4496C mice was demonstrated also in isolated Purkinje cells by Cerrone et al (Figure 65-5).⁴⁶

FIGURE 65-5 Patch-clamping recording in isolated RyR2^R4496C/WT Purkinje cells. A, Confocal and transillumination images of a ventricular myocyte and a Purkinje cell from the same heart. Di-8- ANEPPS staining remarks the lack of transverse T-Tubule system in the Purkinje cell. B, Spontaneous pacemaker activity recorded in an isolated Purkinje cell. C, Delayed after-depolarizations and triggered activity in baseline and after perfusion with isoproterenol in an RyR2^R4496C/WT Purkinje cell.

(Modified from Cerrone M, Noujaim SF, Tolkacheva EG, et al: Arrhythmogenic mechanisms in a mouse model of catecholaminergic polymorphic ventricular tachycardia, Circ Res 101:1039–1048, 2007.)

Finally, additional data supporting the concept that DADs are the arrhythmogenic mechanisms in CPVT were provided in an elegant study by Paavola et al in patients with CPVT; they used monophasic action potential recordings that showed DAD-like humps originating from the right ventricular septal wall and preceding the occurrence of ventricular arrhythmias.⁴⁷

Transgenic models of CPVT have also provided information on the mechanisms of CPVT arrhythmias at the whole-heart level in studies using the optical mapping technique. In collaboration with José Jalife, the authors of this chapter showed that the arrhythmic beats of both polymorphic and bi-directional VT have a focal origin.⁴⁶ Epicardial optical mapping demonstrated that during bi-directional VT, the beats alternately originate from the right ventricle and from the left ventricle and arise from an area coincident with the anatomic insertion of the major bundle branches of the conduction system (Figure 65-6).⁴⁶ Additionally, administration of Lugol’s solution to chemically ablate the Purkinje network in the right ventricle converts bi-directional VT to monomorphic left-sided VT (see Figure 65-6).⁴⁶ Endocardial optical maps have also shown that during polymorphic VT, the site of origin of the beats mapped on the endocardial right ventricular wall corresponded to free-running Purkinje fibers (Figure 65-7).⁴⁶ Overall, these experiments support the relevant role of the Purkinje network in the pathogenesis of CPVT arrhythmias.

FIGURE 65-6 Bi-directional ventricular tachycardia (VT) elicited in an RyR2^R4496C/WT mouse. A, Activation map in isolated Langendorff-perfused mouse heart during sinus rhythm and its corresponding volume-conducted electrocardiogram (ECG). B, Activation maps in the same heart during bi-directional VT elicited by perfusion with calcium and isoproterenol and corresponding volume-conducted ECG. Map 1 and 2 correspond to the two alternating QRS complexes indicated on the ECG. C, Volume-conducted ECG after injection of Lugol’s solution in the right ventricular cavity in one anesthetized RyR2^R4496C/WT mouse after induction of bi-directional VT. The rhythm converts into monomorphic VT after ablation of the right bundle branch. RV, Right ventricular; VT, ventricular tachycardia.

(Modified from Cerrone M, Noujaim SF, Tolkacheva EG, et al: Arrhythmogenic mechanisms in a mouse model of catecholaminergic polymorphic ventricular tachycardia, Circ Res 101:1039–1048, 2007.)

FIGURE 65-7 Polymorphic ventricular tachycardia in an RyR2^R4496C/WT mouse. A, Each color corresponds to the earliest activation of each beat on the volume-conducted electrocardiogram shown, superimposed on an image of the right endocardial wall. B, Magnification of the boxed areas, emphasizing the correspondence of each beat with Purkinje fibers. Beat 5 originates outside the field of view.

(Modified from Cerrone M, Noujaim SF, Tolkacheva EG, et al: Arrhythmogenic mechanisms in a mouse model of catecholaminergic polymorphic ventricular tachycardia, Circ Res 101:1039–1048, 2007.)

Mechanisms of Altered Ca²⁺ Homeostasis in CPVT Linked to CASQ2 Mutations

Mutations in cardiac calsequestrin (CASQ2) that cause the autosomal recessive CPVT represent a minority of cases. Current evidence based on limited experience shows no major phenotypic difference between CASQ2-CPVT and RyR2-CPVT. The few CASQ2 mutations reported so far have been extensively studied in vitro and through transgenic models.

Summary of CASQ2 Physiology

CASQ2 has been initially described as a Ca²⁺ buffering protein resident in the SR lumen. It exists in monomeric and polymeric form; polymerization occurs at high Ca²⁺ and the polymerized protein presents higher Ca²⁺ buffering capacity. When luminal Ca²⁺ is low, CASQ2 binds to junctin and triadin and inhibits RyR2. In the presence of a rise in luminal SR Ca²⁺, the binding among CASQ2, triadin, and junctin is weakened by the replacement of the binding sites by Ca²⁺, and this process gradually increases the open probability for the RyR2 channel.⁴⁸ Thus, CASQ2 is both a Ca²⁺ buffer molecule and a RyR2 modulator.

Subcellular Mechanisms for CASQ2 Dysfunction in Catecholaminergic Polymorphic Ventricular Tachycardia

Seven CASQ2 mutations have been reported so far, four leading to a truncated protein (nonsense or frameshifts) and three missense mutations. In vitro studies have highlighted three classes of effects: (1) impaired CASQ2 polymerization, (2) altered buffering properties, and (3) altered CASQ2-RyR2 interaction.

Terentyev et al suggested that a reduction or absence of CASQ2, as it happens with the truncation mutants, leads to a decrease of the time necessary to re-establish Ca²⁺ storage, thus facilitating a premature activation of the RyR2 and, as a consequence, diastolic Ca²⁺ leakage.⁴⁹

The D307H was the first missense mutation to be linked to the disease.⁵ In in vitro studies, the mutant protein was not able to form properly oriented dimers, and this affected the Ca²⁺ binding capacity.⁵⁰ Adenoviral-mediated overexpression of D307H in rat cardiomyocytes showed that Ca²⁺ binding capacity is impaired despite the presence of endogenous native protein (dominant-negative effect)^51,⁵²; similar results have been obtained in CASQ2^D307H myocytes from a transgenic murine model.⁵³ In contrast, CASQ2-R33Q increases diastolic SR Ca²⁺ release, without affecting Ca²⁺ storing capacity, and it may cause CPVT through a loss of CASQ2-inhibiting effect on RyR2 (Figure 65-8).^54,⁵⁵ The L167H mutation was identified in a patient with compound heterozygosity (Figure 65-9), and it appears to cause a complex phenotype including abnormal (increased) Ca²⁺ buffering properties, impaired polymerization, and loss of RyR2 inhibitory properties.^55,⁵⁶

FIGURE 65-8 Effect of the CASQ2^R33Q mutation. A, Single-channel recording of RyR2 activity and its disruption by the CASQ2^R33Q mutation compared with wild type (WT). B, Immunostaining of CASQ2^R33Q/R33Q and WT cardiomyocytes emphasizing the decreased amount of CASQ2 in the mutant. Bars: 20 µm. C, Electron microscopy showing the lack of electrondense chain-like CASQ2 molecules clustered parallel to the CASQ2^R33Q/R33Q sarcoplasmic reticulum (SR) membrane (the arrow shows their presence in the WT). Bars: 0.1 µm. D, Spontaneous bi-directional-VT in the CASQ2^R33Q/R33Q mouse during acoustic stimulation. TT, Transverse tubule.

(Modified from Terentyev D, Nori A, Santoro M, et al: Abnormal interactions of calsequestrin with the ryanodine receptor calcium release channel complex linked to exercise-induced sudden cardiac death, Circ Res 98:1151–1158, 2006; and Rizzi N, Liu N, Napolitano C, et al: Unexpected structural and functional consequences of the R33Q homozygous mutation in cardiac calsequestrin: A complex arrhythmogenic cascade in a knock in mouse model, Circ Res 103:298–306, 2008.)

FIGURE 65-9 Effect of two CASQ2 mutations inherited in compound heterozygosis. A, Pedigree of the family. Black circle indicated by the arrow is the case-index who inherited two mutations, one from each of the parents. B, Baseline electrocardiogram and ventricular tachycardia elicited by exercise in the case-index. C, Ca²⁺ sparks recorded in rat ventricular myocytes infected with wild-type G112+5X or L167H CASQ2 protein. Top: Surface plot of averaged Ca²⁺ sparks. Bottom: Representative line-scan images.

(Modified from di Barletta MR, Viatchenko-Karpinski S, Nori A, et al: Clinical phenotype and functional characterization of CASQ2 mutations associated with catecholaminergic polymorphic ventricular tachycardia, Circulation 114:1012–1019, 2006.)

Insights from CASQ2-CPVT Murine Models

As in the case of RyR2-CPVT, the mouse models reproducing the autosomal recessive CASQ2-CPVT have provided important pathophysiological information but also are of great value in the unraveling of some molecular mechanisms of cardiac Ca²⁺ regulation.

Knollmann et al created a CASQ2 knock-out murine model, in which VT and VF could be induced by β-adrenergic stimulation (isoproterenol).⁵⁷ In isolated CASQ2 null myocytes, increased diastolic Ca²⁺ leakage leading to DADs and triggered activity was observed, thus proving the similarities between RyR2-CVPT and CASQ2-CVPT. Interestingly, analysis of this model proved that even in the absence of CASQ2, the SR Ca²⁺ storage capacity was not changed. The authors of this study pointed to the ultrastructural abnormalities involving the increase of the SR volume, which may represent a compensatory phenomenon to preserve SR Ca²⁺ storing capacity.⁵⁷

More recently, the chapter authors’ group developed the CASQ2^R33Q/R33Q knock-in model (R33Q was found in a severe case of recessive CPVT), which reproduces the CPVT phenotype.⁵⁸ In contrast to the RyR2-R4496C model, in this model arrhythmias occur even spontaneously in the presence of mild stressors (i.e., loud sudden noise) (see Figure 65-8). CASQ2^R33Q/R33Q cardiomyocytes showed DADs and triggered activity not only during adrenergic stimulation but also in resting conditions.⁵⁸ In contrast to the CASQ2 knock-out model, SR Ca²⁺ is reduced (see Figure 65-8). Rather unexpectedly for a missense mutation, this model is also characterized by reduction of CASQ2 protein (both in the total cell homogenate and in the microsomal fraction) in the presence of normal expression at the mRNA level. Further investigations showed that the mutant R33Q protein, compared with the wild type, is significantly more prone to proteolysis.⁵⁸

Song et al studied two additional murine models, one carrying a null mutation (knock-out) and the other with the D307H mutation.⁵³ No significant phenotypic differences between the two models, including increased levels of RyR2 and calreticulin, were detected.⁵³ Calreticulin is a fetal protein that shows Ca²⁺ binding capacity but is not present in adult hearts; the authors thus inferred that its increase in their model should be interpreted as a compensatory mechanism to balance the lack of CASQ2.

Neither the CASQ2 knock-out model generated by Knollmann nor the CASQ2^R33Q/R33Q mouse showed altered expression of RyR2 or calreticulin.^57,⁵⁸ Therefore, until further data are available, it remains unclear why these two different models present a common compensatory mechanism that is not evident in other models, in particular in the knock-out model.

In summary, cellular and animal studies seem to agree in concluding that CASQ2 mutations cause CPVT by a common final pathway: a critical reduction in protein levels, with increased propensity for diastolic calcium leak.

Current Therapy and Future Directions

Catecholaminergic Polymorphic Ventricular Tachycardia Therapy in the Clinical Setting

On the basis of the clear evidence of the critical role of adrenergic stimulation as a trigger for arrhythmias in CPVT, β-blockers were proposed as a reasonable therapeutic approach following the initial reports on this disease.^2,³ β-Blockers should be started immediately when CPVT is diagnosed; agreement in the scientific community seems to indicate nadolol as the first choice among the available options—for its once-daily dosing and nonselective inhibition of adrenergic stimuli. Asymptomatic bradycardia in these patients should not be used as a reason to reduce the dosage of β-blocker therapy. In fact, the demonstration that DAD-induced arrhythmias are facilitated by faster heart rates provides the rationale to consider the bradycardic effect of β-blockers as an additional antiarrhythmic benefit, along with their inhibitory effect on the sympathetic drive.⁵⁹

More recently, larger epidemiologic studies showed that β-blockers may fail to provide protection from life-threatening events in a relevant proportion of patients.^6,⁸ In the Italian CPVT Registry, the incidence of recurrent arrhythmia while on therapy is as high as 30%.⁶ In case of recurrences of syncopal episodes or VT while on therapy, an implantable cardioverter-defibrillator (ICD) should be considered. Obviously, after ICD implantation, β-blocker treatment should be maintained to minimize the risk of device interventions. Indeed, in the series by the authors of this chapter, 50% of implanted patients received an appropriate ICD therapy during a 2-year follow-up.⁹

The incomplete protection afforded by β-blockers calls for the need of identifying adjunctive effective therapies. Calcium channel blockers, in particular verapamil, have been studied in a limited patient series as a possible alternative to β-blocker therapy by Swan et al and Sumitomo et al.^60,⁶¹ The study of Rosso et al evaluated the efficacy of a combined association between β-blockers and verapamil. In their series, the combination of therapies reduced or even suppressed the recurrences of exercise-induced arrhythmias, ICD shocks, or both.⁶²

Preliminary data on the possible efficacy of flecainide have been recently published.⁶³ Flecainide has been shown to prevent stress-induced arrhythmias in CASQ2 knock-out mice and spontaneous SR Ca²⁺ release in isolated myocytes. It was also tested in two patients with CPVT who were implanted with an ICD and were refractory to conventional therapy, and it prevented arrhythmias during exercise test. Although presented only in an anecdotal report, these data are opening encouraging paths to be explored in the attempt to find new therapeutic strategies for treating CPVT.

Wilde et al provided preliminary evidence for the long-term effectiveness of left sympathetic cardiac denervation (LCSD) in three patients with CPVT. However, patients on β-blockers and LCSD who still experience recurrences of sustained VT and syncope do also exist (Priori SG, personal communication).⁶⁴ Thus, caution is needed before considering this approach as an effective therapy for high-risk CPVT patients.