Phenotypical manifestations of mutations in the genes encoding subunits of the cardiac voltage-dependent L-type calcium channel

Abstract

The L-type Cardiac Calcium Channel (LTCC) plays a prominent role in the electrical and mechanical function of the heart. Mutations in the LTCC have been associated with a number of inherited cardiac arrhythmia syndromes, including Timothy, Brugada and Early Repolarization syndromes. Elucidation of the genetic defects associated with these syndromes has led to a better understanding of molecular and cellular mechanisms and the development of novel therapeutic approaches to dealing with the arrhythmic manifestations. This review provides an overview of the molecular structure and function of the LTCC, the genetic defects in these channels known to contribute to inherited disorders and the underlying molecular and cellular mechanisms contributing to the development of life-threatening arrhythmias.

Introduction

Transmembrane calcium current (I_Ca) in the human myocardium is conducted via a macromolecular complex that includes a pore-forming unit and a number of subunits that cooperate to control the entry of calcium ions. I_Ca is a relatively “new kid on the block” in the arena of inherited arrhythmogenic diseases. Recent studies have identified genetic variants that are associated with electrical abnormalities capable of causing a variety of clinical phenotypes, including Timothy, Brugada and Early Repolarization syndromes. These genetic defects have also guided us towards a better understanding of molecular and cellular mechanisms, which serve as a cornerstone for the development of novel therapies for our patients. In this review we will attempt to provide a comprehensive overview of the, molecular structure and function of the cardiac L-type calcium channel (LTCC), followed by a discussion of inherited disorders associated with genetic mutations in LTCC as well as a discussion of underlying molecular and cellular mechanisms contributing to the development of cardiac arrhythmias.

Molecular diversity and genomic organization of cardiac calcium channels in the heart

Voltage gated calcium channels (VGCC) are macromolecular complexes consisting of an ion conducting protein (the α₁ subunit), and additional “accessory” peptides: α₂δ, β_1–4, and γ subunits. These accessory subunits are not simply innocent bystanders. Indeed they modulate, gating, trafficking and response to neurohormonal stimuli. While the role of α₂ and γ subunits is less clear, the β subunit is known to increase the I_Ca density to enhance single channel open probability and to modulate inactivation;¹ it is also reported to activate Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) leading to Ca²⁺ overload.²

Classification of calcium channels

Calcium channels were first identified by Fatt and Katz in 1953³ and classified according to their biophysical and pharmacological (block by specific toxins) properties in the 1980’s⁴ (Table 1): neuronal (N-type) channel blocked by ω-conotoxin; resistant (R-type) channel; P/Q-type (P = Purkinje) channel blocked by ω-agatoxins; dihydropyridine-sensitive LTCC responsible for excitation-contraction coupling of in skeletal and cardiac muscle. Transient-type (T-type) calcium channel has been identified in neurons and pace-maker cells (Table 1).

Table 1.

Five Subclasses of calcium channels with distinct electrophysiological and pharmacological properties: L, T, N, P, Q and R.

Channel type	Gated By	Protein	Gene	Tissue Distribution	Drug Sensitivity
L	High Voltage	Cav1.1	CACNA1S	Skeletal muscle	DHP, PAA, BTZ
L	High Voltage	Cav1.2	CACNA1C	Heart, brain, aorta, lung, fibroblast	Dihyropyridines (DHP; nifedipine) Phenylalkylamines (PAA - verapamil) Benzothiazepine (BTZ - diltiazam)
L	High Voltage	Cav1.3	CACNA1D	Brain, endocrine, Atria and ventricle of fetal heart Supraventricular tissues of adult heart	DHP, PAA, BTZ
L	High Voltage	Cav1.4	CACNA1F
N	High Voltage	Cav2.2	CACNA1B	Brain	ω - CgTXGVIA (smaller 100 nM)
P/ Q	High Voltage	Cav2.1	CACNA1A	Brain, heart	ω - Aga-IVA (larger 100 nM) ω - CTX-MVIIC (larger 100 nM)
R	Intermediate Voltage	Cav2.3	CACNA1E	Brain	Ni2+ (smaller 30 mM)
T	Low Voltage	Cav3.1 Cav3.2 Cav3.3	CACNA1G CACNA1H CACNA1I	Heart, Brain. Bone (osteoclasts)	Ni2+, Mibefradil

The molecular architecture of VGCC is complex. There are eleven known alpha subunit (Ca_V) encoding genes in humans; these are divided into three major classes; Ca_V1.1 to 1.4 are those conducting the LTCC current expressed in smooth muscle, skeletal muscles and in the central nervous system. Ca_V 2.1 to 2.3 are responsible for P- N- and R-type Ca²⁺ current and are expressed in the central nervous system. Finally Ca_V 3.1 to 3.3 produce the T-type calcium current in the central nervous system, bones and peacemaking cells.

Three types of calcium channels are expressed in the human heart: T-type, P-type, and L-type. T-type calcium channels encompass three different homolog α₁ subunits: Ca_V3.1 (gene: CACNA1G), Ca_V3.2 (gene: CACNA1H) and Ca_V3.3 (gene: CACNA1L). Ca_V3.1 and Ca_V3.2 are expressed in the sinus node and contribute to peacemaking activity⁵ but also in ventricular myocytes during fetal life.⁶ The P-type calcium channels are found in Purkinje cells in cerebellum but also in low levels in the heart, where its expression is altered during heart failure. ^{7, 8} Ca_v1.3 is an L-type calcium channel expressed in both atria and ventricle of the fetal heart, but only in supraventricular tissues (SA node, AV node and atria) of the adult heart. ⁹ Although no mutations of Ca_v1.3 have as yet been associated with human disease, deletion of Ca_v1.3 has been reported to cause sinus bradycardia, AV block as well as hearing impairment. ¹⁰

Of specific interest to this review, is the L-type calcium channel and more specifically the Ca_V1.2 channel encoded by the CACNA1c gene, which maps to chromosomal locus 12p13.3. Ca_V1.2 is the most widely expressed calcium channel in the heart where it plays a prominent role in defining the electrical milieu.¹¹

Genetic diversity of the cardiac calcium channel CACNA1c subunit

CACNA1c has a very complex genomic architecture and transcriptional profile. It spans > 500 kb and undergoes extensive alternative splicing. There are 12 alternative splicing loci that generate 42 different variants. Alternative splicing occurs in the N-terminal, DI-II and DII-DIII linkers, between S5 and S6 in DI and DII, and in the C-terminal region.¹² Interestingly some of these splice variants are physiologically expressed and have been associated with a diversity of biophysical properties: (1) depolarizing shift of inactivation for alternatively spliced exons 31–33 encoding the DIV S3–S4 linkers;¹² (2) faster inactivation kinetics, strong voltage-dependence, and no Ca²⁺-dependent inactivation¹³ for exon 41–42 variants in the C-terminal; (3) dihydropirydine sensitivity for exon 21–22 variants in the DIII-S2 variant¹³ and for exon 9 in IS6;¹⁴ (4) protein kinase C sensitivity for the N-terminal variant resulting from alternative splicing of exon 1–2.¹⁵ CACNA1c splice variant relative abundance presents inter-individual variability¹⁶ and disease-specific variability. The splice variant containing exon 31 is more abundant in normal human hearts with a switch to the exon 32 variant in failing hearts.¹⁷ Preferential deletion of exon 9 and inclusion of exon 33 has been observed in rats with myocardial infarction.¹⁸

These observations support the notion that Ca_v1.2 is responsible for a significant portion of the electrical heterogeneity under physiological and pathophysiological conditions. It follows also that the identification of CACNA1c mutations may bring about interpretation hurdles both at the pathophysiological and clinical level.

Structure-function of Cardiac L-type channels

Cardiac calcium channels are composed of a central pore-forming Ca_V1.2 subunit (α1) and a set of ancillary subunits (α2, δ, and β) (Figure 1). The α1 subunit contains four homologous domains (I–VI) of six transmembrane segments (S1 to S6), each connected by intracellular linkers (I–II, II–III, and III–IV loops) and to the intracellular N- and C-termini. Ca_V1.2 channels are clustered in the t-tubules in close proximity to the sarcoplasmic reticulum Ca²⁺ release channels ryanodine receptor 2 (RyR2) to form the so-called “calcium-release units”,¹⁹ This spatial interaction allows optimal coupling for the calcium-induced-calcium release process.

Figure 1.

Schematic of L-type calcium channel. SH3-Src homology domain; GK-guanylate kinase like domain; VWA- von Willebrand factor-A domain

I_Ca activates in response to voltage but the biophysics of the current is not completely understood because of the complexity of molecular and genomic components. I_Ca activation at physiological extracellular calcium concentration is around–30 mV and it has slower kinetic than I_Na.²⁰ I_Ca inactivation has two components, voltage- (VDI) and Ca²⁺ (CDI)-dependent. The Ca²⁺ mediated component of inactivation is linked to intracellular concentration of calcium ([Ca2+]i). The interdomain loop I–II of Ca_v1.2 is important in controlling this process via Ca_Vα1/β interaction. Interestingly, Timothy syndrome mutations (see below), are located in this region. Additional elements that affect inactivation and channel kinetics are in the S6 segments and in the C-terminus.²¹ Regional and sex-related differences in I_Ca density have been reported in animal models ²² and may affect the propensity for early afterdepolarization development. Interestingly, higher current density in apex vs. base in females but not in males²² has been postulated to account for the higher propensity of increased risk of cardiac events among females with long QT syndrome (LQTS) type 2(LQT2).²³

Genetic disorders associated with cardiac calcium channel mutations

Timothy Syndrome

Cardiologists consider Timothy syndrome (TS) a rare variant of the Long QT syndrome (LQT8) but its complex phenotype involving central nervous system and metabolic abnormalities justifies a separate classification. In 1992, Reichenbach²⁴ reported a case of intrauterine bradycardia secondary to AV block caused by pronounced QT interval prolongation. The male infant also showed hand and feet syndactyly. He died suddenly at 5 months and the authors proposed the clinical phenotype to be a novel clinical entity: the “heart and hand syndrome”.

Phenotype and Natural History

TS is characterized by multisystem dysfunction and developmental defects causing dysmorphic facial features, webbing of the toes and fingers (syndactyly) and autism. ^{25, 26} The initial cardiac rhythm disturbances often occurs early on in life: fetal bradycardia, marked QT prolongation at birth and 2:1 functional atrioventricular conduction block. In such instances recognition of hand and feet syndactyly (with few exceptions – see below) facilitates the diagnosis (Figure 2).

Figure 2.

Clinical manifestations of Timothy Syndrome (TS). TS is characterized by multisystem dysfunction and developmental defects causing dysmorphic facial features including round face, flat nasal bridge, receding upper jaw, and thin upper lip (A–C) and webbing of the toes and fingers (syndactyly) (D and E). Electrocardiogram (ECG) shows severe QT interval prolongation causing 2:1 atrioventricular block seen as two atrial beats (P-waves) for each ventricular beat (QRS complex). Right panel ECG shows alternating T-wave polarity (arrows), indicating severe cardiac repolarization abnormality (F). Ventricular tachycardia recorded from a TS patient by an implanted automatic defibrillator (G). (from Splawski et al, Cell:119,19,2004, with permission).

QT interval is usually in the upper range of values found in LQTS patients. In our cohort (n=21, one of the largest available), QTc interval range measured at resting electrocardiogram (ECG) is from 510ms to 700ms.^{25, 27} The shape of repolarization is also recognizable as a hallmark: long ST segment with small T wave (LQT3-like pattern) but also with giant negative T wave in the precordial leads. Macroscopic T wave alternans is often observed during Holter monitoring (Figure 2). TS presentation also includes congenital cardiac defects (60% of patients) and hypertrophic or dilated cardiomyopathy in 25% of cases.²⁷

Autism and autism spectrum disorder is a common manifestation of TS (83% in our series).²⁸ It is well known that CACNA1c is expressed in the central nervous system; recent data suggest it is implicated in brainstem function²⁹ behavioral control such as “fear learning”,³⁰ while population genetics studies have identified an association of specific single nucleotide polymorphisms (SNPs) (e.g. rs7959938 and rs1006737) and the risk of bipolar disorders and schizophrenia.^{29, 31} Although there is no direct evidence for CACNA1c to cause autism, it is conceivable that, when mutated, it may cause a wide range of neuro-developmental and psychiatric disorders.

Few TS patients have survived to puberty thus far; average survival is 2–3 years; this figure partially explains the low prevalence of the disease, which is almost entirely due to the sporadic mutations with few exceptions (see below).

The onset of cardiac tachyarrhythmia (ventricular tachycardia (VT) or fibrillation (VF)) is the cause of death in 79% of cases. No specific triggers for cardiac arrhythmias are known. However, life-threatening arrhythmias have been frequently reported during anesthesia.^{25, 26} Non-arrhythmic death is also possible in TS: sepsis possibly due to reduced immunity and intractable hypoglycemia have been anecdotally reported as possible causes of death.²⁷

Genetics of Timothy syndrome

The lack of familial clustering of the disease seemed to be a peculiar feature of the disease. For this reason the initial hypothesis was that of chromosomal rearrangements. However, extensive karyotype investigations did not identify any significant alterations, therefore sporadic mutations or recessive inheritance was postulated. Through careful scrutiny of the ST-T wave morphology we hypothesized the involvement of an inward current and undertook a collaborative genetic screening program in TS patients with Mark Keating’s group. In 2004, we identified the G1216A transition in an alternatively spliced exon (8A), which causes the G406R missense mutation in DI/S6 segment (Figure 3),²⁵ The same nucleotide variation was detected in all patients included in this initial cohort, suggesting an unusual molecular homogeneity. In 2005 Splawski et al. reported two additional cases (G406R and G402S) associated with the typical multifaceted phenotype but without syndactyly.²⁶ One patient was carrier of the G406R mutation while a novel variant, G402S was found in the second case. Interestingly, both variants were present in exon 8 at variance with the first reported G406R occurring in the alternatively spliced exon 8A. The lack of syndactyly was explained by the differential expression of the two alternative exons.

Figure 3.

Predicted topology of the Ca_v1.2 (α1c) subunit with associated β2 and α2δ subunits showing the location of mutations associated with Brugada syndrome (BrS), Brugada syndrome with shorter than normal QTc intervals (BrS+SQTS), early repolarization syndrome (ERS), Timothy Syndrome and idiopathic ventricular fibrillation (IVF) probands. AID = alpha subunit interaction domain; BID=beta subunit interaction domain. SH3-Src homology domain; GK-guanylate kinase like domain. Larger symbols with numbers denote multiple probands with the same mutation. Modified from ²⁰, with permission. (Illustration Credit: Cosmocyte/Ben Smith).

The rare familial recurrence of TS has been linked to parental mosaicism. A mosaic is defined as an organism carrying cells with variable genotype at one or more loci. Thus apparently healthy parents may harbor a mutation only in the germline (or in few tissues) and be able to generate an affected offspring. If a CACNA1c mutation carrier expresses a low percentage of mutant cells, the clinical manifestation are variable and can be mild and atypical (depending upon the specific tissue expressing the mutant channel).

Mosaicism was initially suggested in the original publication from Reichenback et al.²⁴ and genetically demonstrated by Splawski et al. in 2004 and 2005.^{25, 26} The demonstration of mosaic CACNA1c mutation carriers has important implications: first of all there is the possibility of incomplete penetrance or mild/atypical manifestations, second phenotypically healthy parents may generate multiple affected offspring due to germline mutations.

Thus, genetic testing should be carried out on DNA extracted from multiple tissues (at least those easily testable: blood, sperm, saliva, skin) even if the proband’s parents are clinically unaffected. Finally, it is important to consider that the combination of syndactyly and QT prolongation may occur in a different inherited arrhythmogenic disorder, such as the Andersen-Tawil syndrome (ATS). This disorder (also referred to as LQT7) has autosomal dominant inheritance and it is characterized by QT prolongation, ventricular arrhythmias, periodic paralysis and facial dimorphisms. Differential diagnosis is important since ATS is usually a benign condition that rarely causes sudden death.³²

J Wave Syndromes

Because they share a common arrhythmic platform related to amplification of I_to -mediated J waves, and because of similarities in ECG characteristics, clinical outcomes and risk factors, congenital and acquired forms of Brugada (BrS) and early repolarization (ERS) syndromes have been grouped together under the heading of J wave syndromes.³³ Recent studies have implicated loss of function mutations in all three subunits of the cardiac LTCC in the generation and accentuation of electrocardiographic J waves associated with these syndromes, as discussed below.^{34, 35}

Brugada Syndrome

BrS, an inherited cardiac arrhythmia syndrome associated with a relatively high risk of VF, was first described as a new clinical entity in 1992.¹⁰ The ECG features of the Brugada patient includes an accentuated J wave displaying a real or apparent right branch bundle block (RBBB) and ST segment elevation in the right precordial leads (V₁–V₃).³⁶

The ECG characteristics and clinical outcomes of the BrS are influenced by heart rate, autonomic tone as well as other conditions and agents that accentuate the manifestation of the J wave, which is inscribed as a result of a transmural voltage gradient caused by a prominent action potential notch in epicardium but not endocardium.^{33, 37} Sodium channel blockers like procainamide, pilsicainide, propafenone, and flecainide unmask the prominent J waves via an outward shift in the balance of currents active in the early phases of the action potential.^37–39 Overall rate of cardiac arrest / VF in BrS is 8–12%. Few variables have proven to be useful metrics for risk stratification; the presence of a spontaneous coved-type ST segment elevation and history of syncope appear to be clearly associated with risk of events.⁴⁰ Inducibility with programmed electrical stimulation, QRS fragmentation and the type of underlying mutation are still under debate.^40–43

Genetics of Brugada syndrome

BrS has been associated with mutations in eight distinct genes, which encode cardiac ion channels or proteins that modulate ion channel activity. Mutations in SCN5A (Na_v1.5, BrS1) have been reported in 11–28% of BrS probands, CACNA1C (Ca_v1.2, BrS3) in 6.7%, CACNB2b (Ca_vβ2b, BrS4) in 4.8% and mutations in Glycerol-3-phophate dehydrogenase 1-like enzyme gene (GPD1L, BrS2), SCN1B (β₁-subunit of sodium channel, BrS5), KCNE3 (MiRP2; BrS6) and SCN3B (β3-subunit of sodium channel, BrS7) are much more rare.^{34, 44–50} These genetic defects lead to either a loss of function of sodium (I_Na) or I_Ca, or a gain of function of transient outward current (I_to). It is noteworthy that mutations in the calcium channel genes often gives rise to BrS associated with shorter than normal QT interval, in some cases qualifying them for a combined diagnosis of BrS and short QT (BrS+SQT).³⁴

Recent studies have identified CACNA2D1 (Ca_Va2δ) as a new BrS-susceptibility gene.⁵¹ Missense mutations predicting p.S709N and p.D550Y alteration of the a2δ subunit of the LTCC were identified in three BrS probands. Functional expression studies indicate that the p.D550Y mutation coupled with a p.Q917H rare polymorphism in CACNA2D1 reduces I_Ca to 25% of normal (Barajas et al., unpublished observation). Our most recent yields in a cohort of 205 probands diagnosed with a J wave syndrome indicate that 12.3% BrS and BrS+SQT probands display mutations in α1, β2 or α2δ subunits of LTCC. With inclusion of rare polymorphisms, the yield increased to 17.9%. The genotype of approximately 60–65% of BrS probands remains to be identified.

Although loss-of-function mutations in the LTCC are known to predispose to a phenotype consisting of BrS with an abbreviated QTc, the majority of BrS probands thus far identified with calcium channel mutations present with normal QTc intervals. This apparent paradox has been attributed to the co-presence of genetic variations in other ion channel genes that are known to lead to prolongation of QTc in 86% (12 of 14) of patients displaying characteristics of BrS and a normal QTc.⁵¹

Early Repolarization Syndrome

An early repolarization (ER) pattern, consisting of a J point elevation, a notch or slur on the QRS (J wave), and tall/symmetric T waves, is generally found in healthy young males and is traditionally regarded as totally benign.^{52, 53} The pathogenticity of an ER pattern and its association with potentially life-threatening arrhythmias emerged from observations in the coronary-perfused wedge preparation in 2000.^{33, 54} Many case reports and experimental studies have long suggested a critical role for the J wave in the pathogenesis of idiopathic ventricular fibrillation (IVF) (see ³³ for references). Several recent studies have provided a definitive association between ER and IVF.^55–59

Because of its high prevalence in the general population ER cannot be considered a specific marker for sudden cardiac death (SCD), but the high incidence of ER in patients with IVF suggests that ER is a predisposing factor. As will be discussed in the next section, a transient J wave augmentation secondary to a shift of the balance of currents active in the early phases of the action potential due to any one of a number of risk factors may set the stage for the development of VF in patients with ER, thus precipitating the early repolarization syndrome (ERS).

A classification scheme for ERS recently proposed on the basis of available data, associates risk with spatial localization of the ER pattern.³³ Type 1 is associated with ER pattern predominantly in the lateral precordial leads; this form is very prevalent among healthy male athletes and is thought to be largely benign. Type 2, displaying an ER pattern predominantly in the inferior or infero-lateral leads, is associated with a moderate level of risk; In a community-based general population study of 10,864 middle-aged subjects, Tikannen et al. reported that J-point elevation of > 0.2 mV in the inferior leads is associated with a markedly elevated risk of death from cardiac arrhythmia (adjusted relative risk, 2.92; 95% CI, 1.45 to 5.89; P = 0.01).⁵⁶ Type 3, displaying an ER pattern globally in the inferior, lateral and right precordial leads, appears to be associated with the highest level of risk and is often associated with electrical storms.³³ BrS represents a fourth variant in which ER is limited to the right precordial leads.

The dynamic nature of J wave manifestation in ERS is well recognized. The amplitude of J waves, which may be barely noticeable during sinus rhythm, may become progressively accentuated with increased vagal tone and bradycardia and still further accentuated following successive extrasystoles and compensatory pauses giving rise to short long short sequences that precipitate VT/VF.^{33, 58, 60}

Genetics of Early repolarization syndrome

Data relative to the genetic and molecular basis for ERS is very limited. Haissaguerre and co-workers were the first to associate a missense mutation in KCNJ8 (S422L) with ERS.⁶¹ Functional expression data in support of this as a disease-causing genetic variant was recently provided by Medeiros-Domingo and co-workers.⁶² However, the gold standard for demonstrating a change in sensitivity to adenosine triphosphate (ATP) involves the study of inside-out patches with the internal membrane exposed to increasing concentrations of ATP. These data unfortunately are still not available. The prospect of a gain of function in I_K-ATP as the basis for ERS is supported by the observation that pinacidil, an I_K-ATP opener, induces both the electrocardiographic and arrhythmic manifestation of ERS in LV wedge preparations.³³

Our recent study designed to identify mutations in the LTCC identified 4 individuals with ERS among 205 J wave syndrome probands with mutations in highly conserved residues of CACNA1C, CACNB2 and CACNA2D1.⁵¹ Preliminary expression studies indicate that these mutations are associated with a loss of function of I_Ca supporting the thesis that all three are ERS-susceptibility genes (Barajas et al., unpublished observation).

Association of Site of Mutation with Channel Dysfunction

The predicted topology of the three subunits of LTCC showing the location of the mutations thus far identified is illustrated in Figure 3. Six of the 9 mutations in Ca_v1.2 α1 subunit associated with the J wave syndromes were in either the N or C terminus. It is interesting that no mutations were detected in any of the transmembrane regions of Ca_v1.2. Consistent with this finding is the demonstration by Soldatov and co-workers of voltage-gated mobility of the C-and N-cytoplasmic tails of Ca_v1.2 and their important regulatory role in voltage- and Ca²⁺⁻ dependent inactivation.^{63, 64} In addition, it has been shown that cleavage of the C terminus of native Ca_v1.2 channels results in a proteolytic fragment that is able to act as a repressor of Ca_v1.2 promoter activity.^{65, 66} Thus, mutations in the C terminus could have very significant effects on the regulation of expression level and on function of the Ca_v1.2 channel. Another mutation of great interest is p.E1115K because it is located in the region of the calcium ion selectivity site, giving rise to multiple SCD in the family.⁵¹

Most recently, Navedo et al.⁶⁷ demonstrated that a cluster of Ca_v1.2 channels organize to facilitate coordinated openings and closings. Fluorescence resonance energy transfer (FRET) analysis of enhanced green fluorescent protein (EGFP)- and red fluorescent protein (RFP)-tagged Ca_v1.2 channels suggest that coupled gating between these channels may involve transient interactions between 2–6 adjacent channels of adjacent channels via their C termini. This finding provides further insight into the mechanisms underlying channel dysfunction associated with C terminus mutations.

Molecular and cellular pathophysiology of calcium channel mutations

Timothy Syndrome

Timothy syndrome arises from sporadic missense mutations (G406R and G402R) affecting the I–II linker of Ca_v1.2, which is believed to act as the inactivation gate. By disrupting inactivation, these mutations lead to a gain of function of ICa, which is responsible for the clinical phenotype.²⁵ Barrett and Tsien found that the TS mutation G406R selectively slows VDI while sparing or slightly speeding the kinetics of CDI.⁶⁸ CDI did not proceed to completeness but was observed to level off at approximately 50%, consistent with a change in gating modes. Thiel et al.,⁶⁹ found a VDI loss in the cells infected with the mutant channels when intracellular Ca²⁺ was buffered. In the presence of normal Ca²⁺ solution, CaMKII activity was increased and the cells exhibit prolonged action potentials and (ER). Inhibition of CaMKII activity reversed the proarrhythmic phenotypes to normal.⁶⁹ G406R may create a CaMKII consensus site at Ser-409 and that Ser-409 phosphorylation leads to an increased channel activity (“mode 2” gating). It is unknown however whether or not Ser.409 is a CaMKII target.

J Wave Syndromes

Transmural differences in early phases of the action potential (phases 1 and 2) are responsible for inscription of the electrocardiographic J wave.^{70, 71} The ventricular epicardium commonly displays action potentials with a prominent I_to -mediated notch or spike and dome. A prominent I_to -mediated action potential notch in ventricular epicardium but not endocardium produces a transmural voltage gradient during ERS that registers as a J wave or J point elevation on the ECG.⁷²

Conditions and agents that influence I_to kinetics or ventricular activation sequence can modify the manifestation of the J wave on the ECG. Because of its slow recovery from inactivation, I_to is increased following slowing of heart rate, resulting in an increase in the magnitude of the J wave.^{73, 74}

Augmentation of net early repolarizing current, due either to a decrease of inward current or an increase of outward current, accentuates the notch leading to augmentation of the J wave or appearance of ST segment elevation. A further increase in net repolarizing current can result in partial or complete loss of the action potential dome, leading to a transmural voltage gradient that manifests as greater ST segment elevation.^73–75

Brugada Syndrome

In regions of the myocardium exhibiting a prominent I_to, such as the right ventricular outflow tract epicardium, accentuation of the action potential notch by a reduction of calcium or sodium channel current or an increase in outward current, results in a transmural voltage gradient that leads to coved ST segment elevation (Figure 4B). Under these conditions, there is little in the way of an arrhythmogenic substrate. However, a further outward shift of the currents active during the early phase of the action potential can lead to loss of the action potential dome, thus creating a dispersion of repolarization between epicardium and endocardium as well as within epicardium, between the region where the dome is lost and regions at which it is maintained (Figure 4C).

Figure 4.

Cellular basis for electrocardiographic and arrhythmic manifestation of Brugada Syndrome. Each panel shows transmembrane action potentials from one endocardial (top) and two epicardial sites together with a transmural ECG recorded from a canine coronary-perfused right ventricular wedge preparation. A: Control. B: Combined calcium and sodium channel block with terfenadine (5 µM) accentuates the epicardial action potential notch creating a transmural voltage gradient that manifests as a ST segment elevation or exaggerated J wave in the ECG. C: Continued exposure to terfenadine results in all-or-none repolarization at the end of phase 1 at some epicardial sites but not others, creating a local epicardial dispersion of repolarization (EDR) as well as a transmural dispersion of repolarization (TDR). D: Phase 2 reentry occurs when the epicardial action potential dome propagates from a site where it is maintained to regions where it has been lost giving rise to a closely coupled extrasystole. E: Extrastimulus (S1–S2 = 250 msec) applied to epicardium triggers a polymorphic VT. F: Phase 2 reentrant extrasystole triggers a brief episode of polymorphic VT. (From Antzelevitch and Yan ³³, with permission)

When _Ito is prominent, as it is in the right ventricular epicardium,^74–76 an outward shift of current causes phase 1 of the action potential to progress to more negative potentials at which the I_Ca fails to activate, leading to an all-or-none repolarization and loss of the dome (Figure 4C). Because loss of the action potential dome is usually heterogeneous, the result is a marked abbreviation of action potential at some sites but not others. The epicardial action potential dome can then propagate from regions where it is maintained to regions where it is lost, giving rise to a very closely coupled extrasystole via a mechanism that we have termed phase 2 reentry (Figure. 4D).⁷⁷ The extrasystole produced via phase 2 reentry often result in an R-on-T phenomenon precipitating life-threatening arrhythmias (Figures. 4E and F).

Early Repolarization Syndrome

An outward shift of current may extend beyond the action potential notch thus leading to an elevation of the ST segment akin to early repolarization. Activation of the ATP-sensitive potassium current (I_K-ATP) or depression of I_Ca can effect such a change.⁵¹ Figure 5 shows an example of ER produced by calcium channel current inhibition. Transmural gradients generated in response to I_Ca block could manifest in the ECG as a diversity of ER patterns including J point elevation, slurring of the terminal part of the QRS and mild ST segment elevation. The ER pattern could facilitate loss of the dome and lead to the development of ST segment elevation, phase 2 reentry, and VT/VF.

Figure 5.

Verapamil-induced early repolarization and phase 2 reentry mediated extrasystole. Each panel shows simultaneous recordings from two epicardial and one endocardial site of a canine ventricular coronary-perfused wedge preparation together with a pseudo-ECG recorded across the perfusion bath. A: Control B: Recorded in the presence of 1 µM verapamil. Phase 2 reentry gives rise to a closely coupled extrasystole (Courtesy of Jeffrey Fish, DVM).

The ability to recapitulate the ECG and arrhythmic manifestations of ERS using agents that inhibit I_Ca or augment I_K-ATP is consistent with the fact that ERS has been found to be associated with mutations in genes encoding the I_K-ATP (KCNJ8) as well calcium channels (CANCA1c, CACNB2, CANCA2D1).

Morrissey and co-workers showed that ventricular Kir6.1 (pore-forming unit of the I_K-ATP channel) is more strongly expressed in epicardial ventricular myocytes.⁷⁸ This heterogeneous transmural distribution of Kir6.1, if present in humans, can contribute to the development of ST segment elevation observed in patients with BrS and ERS carrying mutations in KCNJ8 or SUR2A. The presence of an additional repolarization force during the early phases of the epicardial action potential, due to a gain of function of I_K-ATP, can generate an early repolarization pattern in the ECG by causing depression of the epicardial action potential dome.³³ The heterogeneous distribution of I_K-ATP channels may be unmasked by a reduction in calcium channel activity (due to mutations or calcium channel blocking drugs).The transmural distribution of I_to, may also accentuate the effects of reduced I_Ca. Moreover, a further outward shift in the balance of current due to augmented vagal influence (I_K-ACh activation), bradycardia (greater availability of I_to), or mild ischemia (more I_K-ATP), can lead to heterogeneous loss of the action potential dome, thus creating a dispersion of repolarization that can facilitate the development of phase 2 reentry and polymorphic VT.^{33, 79}

Approach to Therapy of Timothy and J waves Syndromes Associated with Calcium Channel Mutations

Timothy Syndrome

Ventricular tachyarrhythmias (VT or VF) is the leading cause of death in TS and should be a primary target for therapy. Although no data are specifically available for the TS cohort, β-blockers are used based on the evidence that they are effective in most LQTS.⁸⁰ Additional drug therapy (mexiletine, calcium channel blockers) has been proposed in an attempt to abbreviate ventricular repolarization, restore 1:1 conduction, and thus reduce the risk of arrhythmias, but their use has to be considered investigational. Ranolazine has been reported to suppress atrial and ventricular arrhythmias associated with TS in experimental studies as well as in one case report.^{81, 82} Overall the lack of firm data on effectiveness of drug therapy suggest the use of an implantable cardioverter defibrillator (ICD) in all patients with a confirmed diagnosis and QTc >500ms. Careful monitoring and aggressive therapy of infections (altered immune response) and blood glucose levels is also mandatory.

J Wave Syndromes

The mainstay of therapy for the J wave syndromes is the implantation of an ICD.^{49, 83–85} However, ICD implantation is problematic in infants and young children. A pharmacologic approach to therapy, based on a rebalancing of currents active during the early phases of the epicardial action potential so as to reduce the magnitude of the action potential notch and/or restore the action potential dome, has been a focus of basic and clinical research. Because the presence of a prominent transient outward current, I_to, is central to the mechanism underlying the J wave syndromes, the most rationale approach to therapy, regardless of the ionic or genetic basis for the disease, is to partially inhibit I_to.⁸⁶ The only agent on the market with significant I_to blocking properties is quinidine.

Brugada Syndrome

Experimental studies have shown quinidine to be effective in restoring the epicardial action potential dome, thus normalizing the ST segment and preventing phase 2 reentry and polymorphic VT in experimental models of BrS.⁷⁵ Clinical evidence of the effectiveness of quinidine in normalizing ST segment elevation in patients with the BrS has been reported.^87–89 The effects of quinidine to prevent inducible and spontaneous VF has been reported by Belhassen and coworkers.⁸⁸ A prospective Registry for asymptomatic Brugada patients has been created with the aim of tracking the effectiveness of empiric therapy with quinidine.⁹⁰ The development of a more cardioselective and I_to-specific blocker would be a most welcome addition to the limited therapeutic armamentarium currently available.

Agents that boost I_Ca, such as β adrenergic agents like isoproterenol or the phosphodiesterase III inhibitor cilostazol, are useful as well.^{75, 91, 92} Isoproterenol, sometimes in combination with quinidine, has been shown to be effective in normalizing ST segment elevation in patients with the BrS and in controlling electrical storms, particularly in children.^{87, 89, 93–95}

Early Repolarization Syndrome

Data relative to an approach to therapy of arrhythmic complications of early repolarization syndrome are scarce owing to the recent emergence of this syndrome. Experimental studies suggest an approach to therapy similar to that used in BrS.³³ Quinidine and β adrenergic agonists have recently been shown to be effective in suppressing arrhythmic events in patients with ERS.⁹⁶

Conclusions

The LTCC is widely distributed and plays a critical physiological role in many organs, including the heart. Once thought to be rare, genetic mutations in the subunits of the LTCC are now recognized to be relatively common and to be associated with a wide variety of cardiac arrhythmic syndromes including Timothy, Brugada and early repolarization syndromes as well as other clinical phenotypes. Mutations of LTCC genes were identified only recently. Over the past 6 years, over 25 mutations in the α1, β2 and α2δ subunits have been identified. Although, we have made significant progress in the identification of genetic variations contributing to sudden death syndromes and the ionic and cellular mechanisms responsible for the associated arrhythmias, we are clearly at the tip of the iceberg. With further advances in our understanding of the function of LTCC in health and disease, we hope to develop better therapies for these syndromes.

Non-standard Abbreviations and Acronyms

ATP

adenosine triphosphate

ATS

Andersen-Tawil syndrome/LQT7

BrS

Brugada syndrome

BrS+SQT

Brugada syndrome and short QT

[Ca2+]i

intracellular concentration of calcium

CaMKII

Ca²⁺-calmodulin-dependent protein kinase II

Ca_V

voltage-activated calcium channel

Ca_V3.1

gene: CACNA1G

Ca_V3.2

gene: CACNA1H

Ca_V3.3

gene: CACNA1L

CDI

Ca2+ dependent

ECG

electrocardiogram

EGFP

enhanced green fluorescent protein

ER

early repolarization

ERS

early repolarization syndrome

FRET

fluorescence resonance energy transfer

I_Ca

transmembrane calcium current

ICD

implantable cardioverter defibrillator

I_K-ATP

adenosine triphosphate sensitive potassium current

IVF

idiopathic ventricular fibrillation

LTCC

L-type cardiac calcium channel

LQTS

long QT syndrome

N-type

neuronal type calcium channel

P/Q-type

Purkinje type calcium channel

RBBB

right branch bundle block

RFP

red fluorescent protein

SCD

sudden cardiac death

SNP

single nucleotide polymorphism

R-type

resistant type calcium channel

T-type

Transient type calcium

TS

Timothy syndrome/LQT8

VDI

voltage dependent

VF

ventricular fibrillation

VGCC

voltage gated calcium channels

VT

ventricular tachycardia