CPVT is a genetic stress-induced cardiac channelopathy characterized by adrenergically mediated polymorphic ventricular tachyarrhythmias that may lead to SCD, particularly in pediatric and young adult cases [6,7,8]. As the name implies, CPVT is triggered by increased catecholamines during exercise or emotional stress in healthy individuals with structurally normal hearts and a regular resting ECG [6].

The prevalence of CPVT is about 1:10,000 in the general population [8,9]. The average age of onset of clinical symptoms is between 7 and 9 years [7,10].

Clinically, CPVT is diagnosed in the absence of structural cardiac anomalies, normal ECG, and unexplained catecholamine-induced or stress-induced bidirectional VT, polymorphic premature ventricular beats, or VT in persons younger than 40 years [11].

Usually, a definite CPVT diagnosis is made after an average delay of 2 years from the first arrhythmogenic event due to a primary suspicion of vasovagal discomfort or a neurological etiology [12]. The early detection of CPVT is crucial to prevent SCD. Hence, molecular genetic screening is necessary to confirm an uncertain clinical diagnosis of CPVT and identify asymptomatic family members.

The life-threatening arrhythmias in CPVT are mainly caused by unregulated Ca²⁺ release from the SR [13]. Indeed, the electrocardiographic pattern of the ventricular tachycardia observed in CPVT patients is very similar to the arrhythmias linked to intracellular Ca²⁺ overload and the delayed afterdepolarizations (DADs) detected in digitalis toxicity [14,15]. Thus, DADs and triggered physical activity have been proposed as the underlying arrhythmogenic mechanism in CPVT [14,15]. Consistently, mutations in calcium-handling proteins implicated in the release of Ca²⁺ from the SR are associated with the CPVT phenotype, namely the RYR2 and CASQ2 proteins [3,16]. Of note, RYR2 and CASQ2 are parts of the multimolecular Ca²⁺ release channel complex located in the SR. Although RYR2 serves as a Ca²⁺ release channel, the SR Ca²⁺-binding protein, CASQ2, plays a dual role by regulating RYR2 function and serving as a buffer for SR Ca²⁺ [13].

The ryanodine receptor 2 (RYR2) gene encodes the cardiac ryanodine receptor in the SR. The encoded RYR2 protein is the major Ca²⁺ channel protein in the membrane of the SR, which acts as intracellular Ca²⁺ storage in cardiomyocytes. Ca²⁺ can be released from the SR to the cytosol by the RYR2 channels [17]. Ca²⁺ release in cardiomyocytes is triggered by increased Ca²⁺ levels due to the activation of CACNA1C channels. A dysfunctional RYR2 channel leads to diastolic Ca²⁺ leak from the SR and contributes to the development of DADs [15].

During normal cardiac contraction, RYR2 is activated by cytosolic Ca²⁺, whereas under the conditions of storage overload, RYR2 opening is regulated by SR Ca²⁺ [18]. In addition to RYR2 activation, SR luminal Ca²⁺ also determines the RYR2 channel closing [18].

During exercise, RYR2 phosphorylation by protein kinase A partially dissociates FK-binding protein 12.6 (FKBP12.6) from the RYR2 channel, leading to an increase in intracellular Ca²⁺ release and cardiac contractility [19]. Functional studies using Fkbp12.6 −/− mice showed exercise-induced cardiac ventricular arrhythmias resulting in SCD. Indeed, RYR2 mutations linked to exercise-induced arrhythmias in patients with CPVT reduced the affinity of FKBP12.6 for RYR2 channels and increased single-channel activity during exercise. These findings suggested that ‘leaky’ RYR2 channels can trigger malignant arrhythmias, likely causing CPVT [19].

Gain-of-function mutations in RYR2 are found in approximately 79% to 95 % of CPVT1 cases with an autosomal dominant pattern of inheritance [11,20]. Loss-of-function RYR2 mutations are less frequent and linked to other ventricular arrhythmia syndromes [7].

CPVT-linked RYR2 mutations increase the likelihood of spontaneous RYR2 openings and Ca²⁺ leak from the SR during diastole, triggering malignant arrhythmias [20]. To date, over 150 RYR2 mutations have been associated with CPVT [16]. The majority of RYR2 mutations are located in four well-conserved domains including the pore, pseudo-voltage sensor, and central domains. Indeed, these domains are implicated in channel activation and gating. A potential link between mutation localization and phenotype severity has been emphasized [18,21]. Therefore, mutations located in the C-terminus of the RYR2 protein have been correlated with sudden death during sleep [22].

The calsequestrin 2 (CASQ2) gene encodes the calsequestrin protein localized in the SR of cardiac and slow skeletal muscle cells. CASQ2 is Ca²⁺-binding protein that stores Ca²⁺ for muscle function. Ca²⁺ ions are bound by clusters of acidic residues at the protein surface [23]. CASQ2 plays a pivotal role in triggering muscle contraction by regulating the release of lumenal Ca²⁺ through the RYR2 channel. Thus, CASQ2 significantly contributes to the cardiac ECC and regulates the heartbeat rate [23].

Mutations in CASQ2 cause approximately 2 to 5% of CPVT with an autosomal recessive pattern of inheritance. Rarely is an autosomal dominant model associated with CASQ2 mutations [8]. CASQ2 c.539A>G; p.(Lys180Arg) was the first reported mutation associated with an autosomal-dominant inheritance of CPVT [24].

Mutations in CASQ2 result in a lack of control of the RYR2 channel and, consequently, a constant release of Ca²⁺ into the cytoplasm, leading to arrhythmias [13]. Indeed, to regulate Ca²⁺ release, calsequestrin is anchored to RYR2 by triadin and junction proteins. It has been proposed that the interaction between CASQ2 and RYR2 may contribute to the refractory period of Ca²⁺ release occurring after each physiological CICR but the mechanism is not fully understood [13,20]. The most plausible CPVT mechanism linked to nonsense CASQ2 mutations is impaired Ca²⁺ buffering. However, missense mutations, such as the CASQ2 c.98G>A; p.(Arg33Gln), have been linked to Ca²⁺ buffering decrease and an alteration of CASQ2/RYR2 interaction [20].

Casq2-null mice exhibited normal Ca²⁺ release from the SR and contractile function under basal conditions. However, mutant mice had an increase in SR volume and an absence of Casq2-binding proteins such as triadin-1 and junction [25]. Exposure to catecholamines in Casq2-null myocytes induced increased diastolic SR Ca2+ leak, which resulted in premature spontaneous SR Ca²⁺ release and triggered arrhythmias [25].

The trans-2,3-enoyl-CoA reductase-like (TECRL) gene encodes the trans-2,3-enoyl-CoA reductase protein, which is an endoplasmic reticulum protein mainly expressed in the heart and skeletal muscle [26]. The TECRL protein consists of 363-amino acid with an N-terminal ubiquitin-like domain, 3 transmembrane regions, and a C-terminal 3-oxo-5-alpha steroid 4-dehydrogenase domain and plays a crucial role in intracellular Ca²⁺ homeostasis [27]. Indeed, the concentration of the expressed TECRL protein is critical for Ca²⁺ regulation of major cardiac proteins such as RYR2, CASQ2, and CALM [26,27].

Bhuiyan et al. in 2007 described the first CPVT phenotype associated with the TECRL gene according to an autosomal recessive pattern of inheritance, mapped to chromosome locus 7p22–p14 [28]. The members of this family were diagnosed with an early-onset and highly malignant form of CPVT with a history of SCD during physical activity [27,28]. Using exome sequencing, Devalla et al. (2016) identified a homozygous loss-of-function mutation in the TECRL gene in all the affected members of this same family [27].

To assess the functional consequence of the TECRL c.331+1G>A mutation, human induced pluripotent stem cells (hiPSCs) from a 5-year-old homozygous patient (TECRLHom-hiPSCs), his heterozygous father (TECRLHet-hiPSCs), and a non-carrier family member (CTRL-hiPSCs) were generated [27]. hiPSCs were differentiated into cardiomyocytes (CMs) and analyzed in vitro. Using this in vitro model, the authors showed that the c.331+1G>A mutation in TECRL leads to the skipping of exon 3. Moreover, the TECRLHomhiPSC-CMs closely replicated the disease phenotype and the mutant cells showed an increase in triggered electrical activity upon catecholaminergic stimulation [27].

Analysis of intracellular calcium dynamics of the TECRLHom-hiPSCs revealed altered Ca²⁺ properties, including a high diastolic Ca²⁺, smaller amplitude and slower decay of cytosolic Ca²⁺ transients, and a prolonged action potential duration [26,27].

The TECRL c.331+1G>A mutation was subsequently reported by Jaouadi et al. (2020) in a consanguineous family with three deceased children, each at 8 years old [29]. The three SCD events occurred during normal daily activities (playing, slow walking, and at school). Exome sequencing of the family revealed the presence of the homozygous TECRL c.331+1G>A mutation in the last deceased child. Both parents were found to be heterozygous for the variant. The father was asymptomatic with a structurally normal heart and no history of cardiac arrhythmias, whereas the mother had a history of syncopes and a clinical suspicion of Brugada syndrome [29]. Intriguingly, no CPVT or LQTS features were noted in this family [29].

Although the first reported patients with TECRL mutations displayed strict CPVT features or CPVT-specific features combined with a long QT interval but not an isolated LQTS [27,30], the newly identified cases carrying TECRL mutations displayed divergent cardiac phenotypes within a single genetic locus [30].

Moscu-Gregor et al. (2020) have identified four additional mutations in the TECRL gene in CPVT patients with severe and early-onset clinical presentation at the homozygous and compound heterozygous state (c.415C>T; p.(Gln139*), c.893T>C; p.(Val298Ala), c.926C>A; p.(Ser309*), and c.869C>A; p.(Pro290His)). The authors concluded that variants in TECRL may be causative of up to 5% of CPVT patients [31].

Overall, patients with TECRL mutations presented a highly lethal form of arrhythmias, with a median age of symptom onset at 8 years of age [30,32].

Triadin (TRDN) is one of the major transmembrane proteins located in the junctional SR playing a role in ECC regulation and Ca²⁺ influx in the calcium release complex [33]. Mutations in the TRDN gene lead to a significant decrease in protein expression causing Ca²⁺ overload in the SR, which may explain the development of CVPT [34,35].

From a cohort of 97 CPVT patients with no mutations in the RYR2 and CASQ2 genes, Roux-Buisson et al. (2012) identified three mutations in the TRDN gene, which cosegregated with the disease according to an autosomal recessive pattern in two families: a c.del53_56ACAG; p.(Asp18Alafs*13) homozygous deletion in the first family and compound heterozygous mutations c.176C>G; p.(Thr59Arg) and c.613C>T; p.(Gln205*) in the second family [35]. Thereafter, Rooryck et al. (2015) identified compound heterozygous pathogenic mutations (c.613C>T; p.(Gln205*) and c.22 + 29 A>G) in two sisters with CPVT [36]. Overall, the TRDN gene was associated with less than 1% of CPVT5 cases [35].

Calmodulin proteins (CALM) are members of the EF-hand calcium-binding protein family and play an essential role in Ca²⁺ sensing and signal transducing. Three distinct calmodulin genes (CALM1, CALM2, and CALM3) are distributed within the human genome that encode the identical protein but differ at the nucleotide level. The three calmodulin genes share about an 80% identity within their coding regions. Calcium-induced activation of calmodulin modulates the function of cardiac ion channels including CACNA1C, SCN5A, and RYR2 [37,38].

Mutations in CALM1, CALM2, and CALM3 genes have been associated with CPVT.

Nyegaard et al. (2012) identified a heterozygous CALM1 c.161A>T; p.(Asn53Ile) mutation that segregated with the disease in 10 affected family members [38]. A de novo missense mutation in CALM1, c.293A>G; p.(Asn97Ser), was subsequently identified in a 23-year-old woman with a history of resuscitated cardiac arrest at 4 years of age due to ventricular fibrillation while running. Both substitutions showed compromised calcium binding [38].

Makita et al. (2014) identified two heterozygous missense CALM2 mutations in two patients with overlapping features of LQTS and CPVT [39]. The c.396T>G; p.(Asp132Glu) mutation was identified in a 29-year-old woman who was initially diagnosed with neonatal LQTS and later with exercise-induced polymorphic ventricular ectopy. The second CALM2 variant, c.407A>C; p.(Gln136Pro), was identified in an 8-year-old girl with a presumptive diagnosis of LQTS and CPVT who died suddenly during exercise despite treatment with β-blockers [39]. The two mutations were de novo. The encoded mutant calmodulin proteins impaired C-domain Ca²⁺-binding affinity, likely causing Ca²⁺ signaling dysfunction [39].

Gomez-Hurtado et al. (2016) identified a heterozygous CALM3 mutation, c.308C>T; p.(Ala103Val), in a 31-year-old woman among a cohort of 12 CPVT patients with no mutations in the other known CPVT genes. The CALM3 mutation was shown to activate RYR2 Ca²⁺ release channels, generating Ca²⁺ waves and depleting the SR Ca²⁺ store [40,41]. Moreover, it has been shown that CPVT calmodulin mutants tend to bind to RYR2 with higher affinity than wild-type, which can explain their autosomal-dominant mode of action [40,41].

Thus, both de novo and inherited mutations have been reported and patients harboring CALM mutations may present overlapping features of LQTS and CPVT.

The plakophilin-2 (PKP2) gene encodes the desmosomal plakophilin-2 protein. Mutations in the PKP2 gene were associated primarily with arrhythmogenic right ventricular cardiomyopathy (ARVC) [42]. Subsequently, Cerrone et al. (2017) have used conditional mouse deletion of Pkp2 in cardiomyocytes to demonstrate that the lack of Pkp2 reduces expression levels of Ryr2, Ank2, Cacna1c, Trdn, and Casq2 proteins, leading to disruption of intracellular Ca²⁺ homeostasis and isoproterenol-induced arrhythmias, even in the absence of overt structural heart disease [43]. Tester et al. (2019) screened the PKP2 gene in genotype-negative patients with CPVT. PKP2 mutations were found in 27.7% of CPVT cases and 5.3% of exercise-related sudden unexplained death in the young cases. Cardiac imaging or autopsy demonstrated a structurally normal heart in all patients [44].

The ankyrin 2 (ANK2) gene encodes an ankyrin-B protein that is located mainly in the transverse-tubule SR sites of the cardiomyocytes [45]. This protein is a crucial part of the Na⁺/Ca²⁺ exchanger, Na⁺/K⁺ ATPase, and inositol trisphosphate (InsP3) receptor. Thus, ankyrins have key roles in membrane trafficking and regulation of different ion channels in the heart [46]. A loss-of-function mutation of ankyrin-B was initially associated with LQTS 4 [45]. Subsequently, Mohler et al., (2004 and 2007), identified nine ANK2 loss-of-function mutations in patients with variable expressivity of cardiac dysfunction including bradycardia, sinus arrhythmia, idiopathic ventricular fibrillation, and CPVT [47,48]. The CPVT patients carried the ANK2 c.4864C>A; p.(Leu1622Ile), c.5437G>A; p.(Glu1813Lys), and c.T4547A; p.(Val1516Asp) mutations [47,48]. The ANK2 mutations were shown to abolish the ability of ankyrin-B to restore defective Ca²⁺ dynamics. The authors also noted abnormal localization and expression of the Na⁺/Ca²⁺ exchanger, Na⁺/K⁺ ATPase, and InsP3 [47].

HCM is a primary cardiac disorder characterized by an increased left ventricular wall thickness in the absence of other loading conditions [61]. HCM is the most common inherited heart disease, with a prevalence of 1/200 to 1/500, and is mostly inherited in an autosomal dominant manner [62,63]. Patients with HCM have a higher risk of developing clinical complications such as progressive heart failure, arrhythmia, and SCD [63]. Molecular genetic studies have demonstrated that HCM is mainly caused by mutations in sarcomeric genes encoding contractile myofilament proteins [62,64,65,66]. The frequency of mutations within sarcomeric genes varies from 25% to 65% of patients [67].

Increased calcium buffering has been proposed as the causal mechanism leading to the alteration of intracellular Ca²⁺ cycling and triggering Ca²⁺-dependent hypertrophy [68]. In vivo experiments using LV guinea pig cardiomyocytes expressing mutations in the TNNT2, TNNI1, and TPM1 genes demonstrated increased diastolic Ca²⁺ and Ca²⁺ reuptake [68]. Moreover, mutations in the MYBPC3 gene may increase myofilament Ca²⁺ sensitivity and promote cardiac hypertrophy due to the inability to release Ca²⁺ and relax from contraction [69].

Nevertheless, there is a lack of strong evidence to show whether these alterations are a causal factor of HCM or consequential clinical manifestations. With the advent of patient-specific iPSC models, Lan et al. (2013) have provided evidence that the elevation of intracellular Ca²⁺ is the initial factor in HCM development [5]. Indeed, time-based gene expression data analysis of single iPSC-CMs carrying the MYH7: Arg663His mutation revealed that the downstream effectors of cardiac hypertrophy (e.g., GATA4 and MEF2) were expressed in a Ca²⁺-dependent manner [5]. Furthermore, the authors concluded that elevated cardiomyocyte Ca²⁺ loading seems to contribute to both cardiac hypertrophy and arrhythmogenesis [5].

Troponin is a central regulatory protein of striated muscle contraction located on the actin filament. The cardiac troponin complex is a heterotrimeric myofilament composed of three subunits: an elongated troponin T subunit (TNNT2), an inhibitory troponin I subunit (TNNI3), and a Ca²⁺-sensitive troponin C subunit (TNNC1). TNNC1 is a sarcomeric Ca²⁺ sensor that binds to the cytosolic divalent cation at the specific Ca²⁺ binding site to enhance its interaction with TNNI3. This complex reduces the inhibitory function of TNNI3, releasing it from actin, and causes the troponin–tropomyosin complex to move into the actin groove, exposing myosin binding sites [72]. Accordingly, TNNC1 plays a critical molecular role in the initiation of myofilament contraction [67].

Mutations in the TNNC1 gene are rare, occurring in ~0.4% of HCM patients [73]. The first HCM-associated mutation in TNNC1, c.86T>A; p.(Leu29Gln), was identified by Hoffmann et al. in 2001 [70]. Using in vitro and in situ models, this mutation was found to affect the Ca²⁺-dependent structural change in cardiac TnC in trabeculae under basal conditions and abolish the effect of force-generating myosin cross-bridges. [74]. Six additional mutations in the TNNC1 gene (c.23C>T; p.(Ala8Val), c.91G>T; p.(Ala31Ser), c.251G>A; p.(Cys84Tyr), c.402G>T; p.(Glu134Asp), c.435C>A; p.(Asp145Glu), and c.363dupG; p.(Gln122Alafs*30)) have been associated with HCM by the screening of the TNNC1 gene in a cohort of 1025 HCM patients [73,75,76,77].

Functional studies showed an increased Ca²⁺ sensitivity of force development for c.23C>T, c.251G>A, and c.435C>A and force recovery for c.23C>T and c.435C>A mutations. The TNNC1 c.402G>T mutation showed no changes in these parameters [73]. The frameshift mutation TNNC1 c.363dupG is located in the EF-hand 3 domain and was found to destroy the H-helices of troponin C that are required for the interaction with troponin I [78]. Moreover, the functional analysis suggested that the TNNC1 c.91G>T mutation directly affects Ca²⁺ sensitivity and may alter Ca²⁺ handling, leading to arrhythmogenesis [67].

Mutations in the ryanodine receptor type 2 (RYR2) gene are typically associated with CPVT, ventricular arrhythmias due to calcium release deficiency syndrome, and arrhythmogenic right ventricular cardiomyopathy/ dysplasia. In 2006, Fujino et al. reported the first RYR2 mutation potentially involved in HCM: c.3320C>T; p.(Thr1107Met) [79]. This mutation was subsequently identified in exome-sequencing cohorts with a relatively high frequency (MAF=0.0004) [80] and in CPVT patients [81], questioning its involvement in HCM. Recently, Alvarado et al. (2019) identified a novel RYR2 mutation c.3372G>A; p.(Pro1124Leu), in an HCM patient who did not have a sarcomeric mutation [82]. Functional studies have shown that homozygous mice for this mutation presented mild cardiac hypertrophy combined with an increase in the expression of calmodulin, a classical inhibitor of RYR2 [82].

The alpha kinase 3 (ALPK3) gene encodes the alpha-protein kinase 3 that may act as a transcriptional regulator through the phosphorylation of cardiac transcription factors [83]. The ALPK3 gene is early expressed in the cardiac crescent and later remains highly expressed in cardiomyocytes throughout life [84,85]. Recently, ALPK3 was identified as an important cardiac pseudokinase that inserts into the nuclear envelope and M-band of the sarcomere [85].

Homozygous ALPK3-truncating mutations were initially associated with early-onset cardiomyopathy [84]. Pediatric cases reported thereafter consistently displayed a severe clinical presentation with irregular cardiac remodeling and an inconsistent syndromic pattern [84,86,87,88]. Indeed, pediatric cases with biallelic ALPK3-truncating mutations showed a variable HCM phenotype with atypical distribution of hypertrophy (concentric/ apical/ asymmetric septal hypertrophy/right ventricular dysfunction) and variable facio-thoraco-skeletal features [84,85,86,87,88,89]. Notably, homozygous ALPK3 carriers diagnosed in utero or at birth presented a mixed DCM/HCM phenotype with progression to an HCM phenotype with age [87,89]. Although the majority of heterozygous parents or relatives of the reported cases were found to be healthy or with mild HCM, recent case reports and studies extended the phenotype and genotype spectrum of ALPK3 mutations to include (i) missense mutations, (ii) compound-heterozygous and autosomal-dominant patterns of inheritance, and (iii) adult-onset HCM with a less severe clinical presentation [89,90].

The ALPK3 gene appears to be essential for the normal formation of the intercalated disc and the organization of cardiomyofibril in humans and mice [86,88,91]. It has been shown that hiPSCs-derived cardiomyocytes containing a homozygous ALPK3- truncating mutation displayed defective Ca²⁺ handling in addition to sarcomeric disorganization and impaired intercalated disc integrity [84,88].

A functional study of the Ca²⁺ flux during contraction demonstrated that ALPK3-mutant cardiomyocytes displayed several changes in intracellular Ca²⁺ including significantly increased irregular Ca²⁺-transients, which may explain the cellular hypertrophy [88]. Indeed, using multi-electrode array analysis, ALPK3–hiPSCs-derived cardiomyocytes demonstrated an extended extracellular field potential duration, indicating that the loss of ALPK3 disrupts membrane repolarization [88].

These results suggest that intracellular Ca²⁺ is elevated in ALPK3-deficient cardiomyocytes, agreeing with what has been observed in other hiPSC models of HCM [84,88].

The junctophilin 2 (JPH2) gene encodes junctophilin 2, a major component of the junctional membrane complex involved in Ca²⁺ homeostasis and ECC [92]. Junctophilins are a family of proteins found in all excitable cells [93]. JPH2 plays a crucial role in maintaining the proper structure of the cardiac dyad, which is necessary for effective CICR [94]. The implication of the JPH2 gene in HCM was first reported by Landstrom et al. (2007) with the identification of three mutations (c.301A>C; p.(Ser101Arg), c.421T>C; p.(Tyr141His), and c.494C>T; p.(Ser165Phe), in three unrelated patients with HCM negative for sarcomeric or Z-disc mutations [95]. The characterization of these mutations using an in vitro model of myocyte culture showed a decrease in CICR amplitude and disruption of cellular ultrastructure. The c.421T>C and c.494C>T mutations were found to induce cellular hypertrophy [95]. Of note, mutations in JPH2 are considered a rare cause of HCM and are found in less than 1% of index cases [67].

The phospholamban (PLN) gene encodes phospholamban, a major substrate for the cAMP-dependent protein kinase in cardiac muscle. In its unphosphorylated state, PLN is an inhibitor of cardiac muscle SERCA2a. This inhibition is abolished upon phosphorylation of the PLN protein. The consecutive activation of the Ca²⁺ pump results in enhanced muscle relaxation rates. Thus, PLN is a key regulator of cardiac diastolic function [96,97,98].

Mutations in the PLN gene are known to cause DCM [99]. However, rare promoter mutations have been identified in multiple independent cohorts of HCM patients [100,101]. Nevertheless, only the rare truncating PLN c.116T>G; p.(Leu39Ter) mutation is recognized as a causative HCM mutation [102]. This mutation cosegregated with HCM in a multigenerational family and the truncated protein is likely to impair PLN and SERCA2a interactions [103]. The authors also estimated an overall yield of PLN–HCM mutations of 0.65% by comparing different studies reporting PLN mutations in HCM cohorts [102].