Roles and Regulation of Voltage-gated Calcium Channels in Arrhythmias

DOI: 10.19102/icrm.2019.101006

JARED KUSHNER, MD,¹ XAVIER FERRER, BS,¹ and STEVEN O. MARX, MD¹

¹Division of Cardiology, Department of Medicine, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA

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ABSTRACT. Calcium flowing through voltage-dependent calcium channels into cardiomyocytes mediates excitation–contraction coupling, controls action-potential duration and automaticity in nodal cells, and regulates gene expression. Proper surface targeting and basal and hormonal regulation of calcium channels are vital for normal cardiac physiology. In this review, we discuss the roles of voltage-gated calcium channels in the heart and the mechanisms by which these channels are regulated by physiological signaling pathways in health and disease.

KEYWORDS. Arrhythmias, calcium, calcium channels, heart

The authors report no conflicts of interest for the published content. This work was supported by National Institutes of Health research grant nos. R01 HL113136 and R01 HL121253. Additionally, Dr. Kushner was supported by the National Institutes of Health research grant no. T32 HL007343 and the Glorney-Raisbeck Fellowship and Junior Faculty Award from the New York Academy of Medicine, while Mr. Ferrer was supported by the National Institutes of Health research grant no. 5T35HL007616-38.
Manuscript received September 12, 2018. Final version accepted March 4, 2019.
Address correspondence to: Steven O. Marx, MD, 622 West 168th Street, PH-3 Center, New York, NY 10032, USA. Email: sm460@cumc.columbia.edu.

Introduction

In the heart, calcium (Ca²⁺) entry through the voltage-gated Ca²⁺ channel initiates muscle excitation–contraction coupling. The influx of Ca²⁺ also contributes to the plateau phase of the action potential, pacemaker activity in nodal cells, and the modulation of critical cellular processes including metabolism and gene expression. Thus, a Ca²⁺ influx via voltage-gated Ca²⁺ channels in the heart links membrane depolarization to cellular functions. In this review, we will discuss mechanisms of Ca²⁺ handling in the heart and how dysfunctions of voltage-gated Ca²⁺ channels can lead to arrhythmias. Ca²⁺ channels are modulated by voltage, Ca²⁺, posttranslational modifications, and protein–protein interactions, which will also be reviewed. Finally, we will discuss existing pharmacological therapies that target voltage-gated Ca²⁺ channels.

Structure and cellular electrophysiological function

Six classes of voltage-gated Ca²⁺ channels exist that can be classified by membrane voltage activation (low versus high), susceptibility to pharmacologic antagonists, and rate of inactivation (Table 1); these include the T-, L-, N-, P-, Q-, and R-type channels. Of these, only long-lasting (L)- and transient (T)-type Ca²⁺ channels are expressed in cardiomyocytes.^1,2 In Ca²⁺ channel nomenclature, the chemical symbol of calcium, Ca, is followed by a subscript “V,” denoting voltage as the primary regulator, and two numerical identifiers corresponding to the α₁ subunit gene subfamily and the order of discovery within that subfamily, respectively.^3,4 Ca_V1.1, Ca_V1.2, Ca_V1.3, and Ca_V1.4 exhibit relatively long-lasting currents and are referred to as L-type Ca²⁺ channels. Ca_V3.1, Ca_V3.2, and Ca_V3.3, which are T-type Ca²⁺ channels, exhibit transient Ca²⁺ currents and are activated at more negative potentials relative to L-type Ca²⁺ channels (Table 1).

Voltage-gated Ca²⁺ channels are composed of the pore-forming α₁ subunit and several auxiliary subunits including β and α₂δ (Figure 1). Four homologous domains, each with six transmembrane helices and a pore loop between S5 and S6 forming the α₁ subunit. Alternating, positively charged arginine or lysine residues at every third or fourth position in S4 of each domain impart voltage sensitivity.⁵ The α₁ subunit contains binding sites for most regulators and drugs, whereas the β, α₂δ, and γ subunits contribute to trafficking, anchoring, and regulatory functions. The pore region contains binding sites for all major L-type Ca²⁺ channel-blocking agents including dihydropyridines, phenylalkylamines, and benzothiazepines (Table 1).⁶

There are four β-subunit genes (Ca_Vβ_1–4). The β₂ subunit is the predominantly expressed isoform in the adult heart. In all of the different β subunits, the guanylate kinase (GK) and Src-homology 3 (SH3) domains are very similar, whereas the N-termini (variable region 1), the linker between SH3 and GK (variable region 2), and the C-termini (variable region 3) are quite different.^7–9 All β subunits interact with the pore-forming α subunit via the intracellular loop between transmembrane domains I and II (Figure 1). In a cell-specific manner, β subunits can increase trafficking of the channel to the plasma membrane and modulate both activation and inactivation. In mice, global or cardiac-specific deletion of the Cacnb2 gene leads to abnormal heart development and embryonic death.¹⁰ Conditional deletion of the β₂ subunit in adult mouse cardiomyocytes causes a 96% or so reduction in β₂ protein expression, but surprisingly only a 29% reduction in Ca²⁺ current, with no obvious cardiac impairment,¹¹ implying that, in adult hearts, the β₂ expression may be expendable. However, interpretation of this result is ambiguous, as it is complicated by the remnant (~4%) β₂ expression as well as the presence of other Ca_Vβ isoforms expressed in adult cardiomyocytes.⁹ Moreover, a contrasting viewpoint was provided by a study in which short hairpin RNA–mediated knockdown of β₂ in adult rat myocytes substantially diminished Ca²⁺ current.¹²

To definitively address the controversies regarding the role of β subunits in mediating the trafficking and regulation of Ca²⁺ channels in the heart, we created transgenic mice lines with three mutations in the α-interaction domain in the I–II loop of the α_1C subunit, which renders the pore-forming α_1C subunit incapable of binding β subunits. With this new model, we definitively demonstrate in vivo that the β subunit binding to α_1C is not required for trafficking and that the basal function of β-less Ca⁺² channels is only minimally altered.¹³

The α₂δ subunit is a 175-kDa single transmembrane protein encoded by four genes (Cacna2d1, Cacna2d2, Cacna2d3, and Cacna2d4) with multiple splice variants. Although the messenger RNAs of α₂δ 1 through 3¹⁴ have been identified in human myocardium, only α₂δ-1 is known to bind with Ca_V1.2 (Figure 1).

Ca²⁺ channels are inactivated in both voltage- and Ca²⁺-dependent manners, but Ca²⁺-dependent inactivation is the dominant mechanism. L-type Ca²⁺ channels associate with calmodulin (CaM) (Figure 1), which modulates both Ca²⁺-dependent inactivation and Ca²⁺-dependent facilitation, where Ca²⁺ currents increase after repetitive stimulation.¹⁵ The importance of CaM regulation of L-type Ca²⁺ channels in the heart has been demonstrated by overexpressing in adult cardiac myocytes a mutated CaM protein that cannot bind Ca²⁺, leading to very long action potentials because of the loss of Ca²⁺-dependent inactivation.¹⁶ Although Ca²⁺ entry via the Ca²⁺ channel can contribute to Ca²⁺-dependent inactivation, remarkably, it is specifically the Ca²⁺ released from the sarcoplasmic reticulum via the ryanodine receptors that is the primary determinant of Ca²⁺-dependent inactivation.

Roles of L- and T-type calcium channels in the heart

Understanding the regulation of myocyte Ca²⁺ regulation is essential to understanding cardiac arrhythmogenesis. Ca_V1.2 is situated on the transverse tubules in close proximity with ryanodine receptors (RyR2), which are intracellular Ca²⁺ release channels located on the sarcoplasmic reticulum. Ca²⁺ entry through the L-type Ca²⁺ channels triggers ryanodine receptors to release Ca²⁺ from the sarcoplasmic reticulum into the cytoplasm as part of a process known as Ca²⁺-induced Ca²⁺ release (Figure 2).¹⁷ Ca²⁺ influx via Ca²⁺-channel current and Ca²⁺ release via ryanodine receptors are required for myofilament activation (Figure 2). Ca²⁺ binds to troponin C on the thin filament, allowing the myosin heads to bind to actin.¹⁸ The strength of contraction is proportional to the concentration of Ca²⁺ surrounding the myofilaments. In order to fully relax myocytes in preparation for the next heartbeat, the amount of Ca²⁺ that enters the cardiac cell during steady state must equal the amount of Ca²⁺ that leaves the cell.¹⁸ The reduction in cellular Ca²⁺ concentration is driven by Ca²⁺ transport via the sarcoplasmic reticulum Ca²⁺–adenosine triphosphate pump (SERCA) and the sarcolemmal Na⁺/Ca²⁺ exchanger (Figure 2). During the action potential, voltage-gated Ca²⁺ channels open and allow Ca²⁺ to flow down its electrochemical gradient, causing the plateau phase (phase 2) of the action potential (Figure 3). Outside of the t-tubules, L-type Ca²⁺ channels are also localized to caveolae—membrane invaginations important for concentrating proteins that are essential for the coordinating responses to extracellular signals—wherein Ca²⁺ influx can control signal transduction pathways. In ventricular myocytes, the Ca²⁺ current is mediated nearly entirely by the L-type Ca²⁺ channel, Ca_V1.2. In atrial and especially pacemaker cells, the L-type Ca²⁺ channel isoform Ca_V1.3, which activates at more negative potentials, contributes to the late phase 4 depolarization that underlies these cells’ automaticity (Table 1).

In contrast to L-type Ca²⁺ current, T-type Ca²⁺ current has little effect on cardiomyocyte excitation–contraction coupling in the heart.¹⁹ The high density of T-type Ca²⁺ current in nodal cells²⁰ and embryonic cardiomyocytes,²¹ however, is consistent with their putative role in pacemaker function. T-type Ca²⁺ channels contribute to triggered or pacemaker activity because they activate at even more negative potentials than L-type Ca²⁺ channels (Table 1). However, they produce smaller peak Ca²⁺ currents and cannot substitute for L-type Ca²⁺ channels because T-type Ca²⁺ channels do not target to the sarcolemmal–sarcoplasmic reticulum junctions and therefore cannot initiate sarcoplasmic Ca²⁺ release.

Pharmacology

There are three main chemical classes of organic Ca²⁺ channel drugs, specifically dihydropyridine (prototype: nifedipine), phenylalkylamines (prototype: verapamil), and benzothiazepines (prototype: diltiazem). All three classes of drugs bind within a single overlapping region close to the pore and the proposed activation gate.^22,23 These drugs interfere with the voltage-dependent cycling of the channel.^24–26 The uncharged dihydropyridines, which possess higher affinity for the inactivated channel conformation (voltage- or use-dependent block), induce and stabilize inactivated channel states.^24–27 Smooth-muscle Ca_V1.2 channels are more sensitive to inhibition by dihydropyridines than cardiac Ca_V1.2 channels because the inactivated channel states are favored in arterial smooth muscle cells due to the relatively depolarized membrane potential of these cells and the splice variant of the S6 segment of domain 1, which is specifically expressed in this tissue.^25,28,29

Ca_V1.3 is less sensitive to dihydropyridines than Ca_V1.2 is. Phenylalkylamines and benzothiazepines bind to the open and inactivated states with high affinity and stabilize the inactivated channel states, slowing recovery from inactivation, leading to use-dependent inhibition.^30,31 Therefore, inhibition increases with higher heart rates, rationalizing the use of verapamil for tachyarrhythmias. Whereas verapamil and diltiazem always reduce inward Ca²⁺ currents, some dihydropyridines, such as (−)-BAY-K-8644 and (+)-SDS-202-791, are gating modifiers that act as agonists, increasing current amplitudes, tail currents, and single-channel open probability.²⁷

Disease states/channelopathies

Prolongation of the action-potential duration increases the loading of Ca²⁺ within the cells due to prolonged Ca²⁺ entry and a reduced diastolic interval for Ca²⁺ efflux. Moreover, some L-type Ca²⁺ channels become available again during the prolonged action-potential duration, and the channels reactivate, creating an inward Ca²⁺ influx, which can cause early afterdepolarizations.³² Increased L-type Ca²⁺ current also contributes to delayed afterdepolarizations and Ca²⁺-evoked arrhythmias, which occur after repolarization is complete and are exacerbated by sarcoplasmic reticulum Ca²⁺ overload. Mutations in L-type Ca²⁺ channels have been associated with inherited arrhythmia syndromes.

Timothy syndrome, a multisystem disorder characterized by a prolonged QT interval and syndactyly as well as variably penetrant phenotypes of autism spectrum disorders, craniofacial abnormalities, and hypoglycemia,³³ is caused by the loss of voltage-dependent inactivation.³³ The heterogeneous phenotype reflects the distribution of expression in the heart, brain, kidney, gastrointestinal tract, immune system, smooth muscle, testis, and pituitary and adrenal glands (Table 1). The diagnosis is typically made within the first few days of life due to fetal bradycardia caused by functional 2:1 atrioventricular block. When completely expressed, Timothy syndrome is typically lethal within the first years of life. Repolarization is markedly prolonged in most patients with Timothy syndrome, with the corrected QT interval often exceeding 550 ms to 600 ms.³⁴ Congenital cardiac defects are present in 60% of patients and cardiac hypertrophy and ventricular dilatation have been reported to occur in 50% of patients.^33–35 Ventricular arrhythmias are the most frequent cause of death. There are no systematic studies assessing the best therapeutic strategy for patients with Timothy syndrome published to date. The available evidence supports the use of β-blockers and late Na⁺ channel blockers, and the use of implantable cardioverter-defibrillators for primary prevention is also reasonable.^36–39 Close monitoring of glucose levels is essential, however, since β-blockers can mask the hypoglycemia caused by Timothy syndrome.

Mutations in 19 genes have been identified as associated with the Brugada phenotype, causing either a decrease in inward Na⁺ or Ca²⁺ currents or an increase in outward K⁺ currents.⁴⁰ The resultant outward shift in the balance of currents active during phases 1 and 2 of the epicardial action potential allows for the already prominent transient outward K⁺ current to augment phase 1 repolarization. If the membrane potential is repolarized too much, L-type Ca²⁺ channels fail to activate, leading to a reduction in the action potential plateau predominantly in the right ventricular subepicardial cells in which the transient outward K⁺ current is most prominent. Loss-of-function mutations in the pore-forming α_1C subunit, the β_2b subunit, and the α₂δ₁ subunit have also been linked to Brugada, early repolarization, and short-QT syndromes.^41–44 Agents that augment L-type Ca²⁺ currents, such as β-adrenergic agonists, have been shown to have therapeutic efficacy in Brugada syndrome.^40,45–47

Short-QT syndrome⁴⁸ is one of the rarest inheritable cardiac channelopathies, characterized by an accelerated cardiac repolarization. It is an autosomal-dominant disease with five identified causative genes, including three that encode for K⁺ channels (KCNH2, KCNQ1, and KCNJ2) and two that encode for subunits of the L-type Ca²⁺ channels (CACNA1C and CACNB2).^49–51 Mutations in the Ca_V1.2 genes CACNA1C and CACNB2b have also been associated with both idiopathic ventricular fibrillation and early repolarization syndrome.⁴³

A mutation in CACNA1D, which encodes Ca_V1.3, was identified in a Pakistani family with pronounced bradycardia resulting from nonconducting Ca_V1.3 channels.⁵² A loss of Ca_V1.3 reduces automaticity in pacemaker cells. Taken together, mutations of the core subunits of the L-type Ca²⁺ channels cause various cardiac syndromes and arrhythmias, including long-QT syndrome, Timothy syndrome, Brugada syndrome, short-QT syndrome, early repolarization, and bradycardia.

Posttranslational regulation of calcium channels

Epinephrine and norepinephrine bind to β-adrenergic receptors in cardiomyocytes, the activation of which augments inotropy, lusitropy, and chronotropy.¹⁸ The activity of both protein kinase A (PKA) and Ca²⁺-CaM-activated protein kinase (CaMKII) increases with β-adrenergic stimulation, and both kinases provoke a rise in Ca_V1.2 activity. The heightened activation of Ca_V1.2, in turn, triggers increased contractility and Ca²⁺-responsive signaling pathways, which contribute to the pathogenesis of heart failure and hypertrophy.^53,54

The molecular mechanism responsible for the β-adrenergic regulation of cardiac Ca_V1.2 has remained a mystery. Experiments expressing recombinant Ca_V1.2 in cultured cells (which have been, up until recently, the primary means of studying Ca_V1.2 regulation) have not given a clear answer, since β-adrenergic regulation is not reliably reconstituted in standard cell lines and cardiomyocytes are irrevocably altered when cultured ex vivo.^55,56 Thus, studies are required in native systems. The failure thus far to identify any single site as essential for β-adrenergic modulation led us to propose an alternative hypothesis: that a combination of phosphorylation sites in α_1C is required for β-adrenergic stimulation of Ca_V1.2. Since β-adrenergic regulation of cardiac Ca_V1.2 is conserved in vertebrates, we identified conserved PKA consensus sequences in the α_1C subunit of five species: mouse, rat, rabbit, guinea pig, and human. We then generated α_1C transgenic mice in which we replaced the 17 conserved consensus PKA phosphorylation sites that were not previously studied and the five conserved PKA/CaMKII phosphorylation sites that were known to be nonessential.^57–60 Surprisingly, we found that none of these PKA consensus phosphorylation sites were necessary.⁶¹ Instead, we found that β-subunit binding to the Ca_V1.2 α_1C subunit, but not PKA phosphorylation of β, is absolutely essential for the augmentation of Ca²⁺ current and cardiac contractile response to β-adrenergic−related PKA stimulation.¹³ These findings identify the key regulatory mechanisms impacting β-adrenergic regulation of Ca²⁺ influx and contractility in the heart.

Ca_V1.2 is also a major target of CaMKII, and the resulting Ca²⁺-dependent facilitation of Ca_V1.2 current, observed as a positive “staircase” of Ca²⁺ current in which current amplitude increases and inactivation slows over a series of repetitive pulses, is a powerful feed-forward effect on Ca²⁺ signaling in the heart. It is likely that phosphorylation of both α_1C and β₂ subunits are required for CaMKII potentiation.^62,63

Conclusions

Ca²⁺ channels are absolutely essential regulators of intracellular Ca²⁺, automaticity, and contractility. The channels are regulated by a macromolecular complex consisting of core subunits and kinases, phosphatases, cytoskeletal proteins, and adaptor proteins. The dysfunction of the channels, caused by either genetic or acquired factors, is associated with heart failure, hypertrophy, or arrhythmias. Future ideal goals include providing a greater molecular understanding of the mechanisms underlying Ca²⁺ channel subcellular targeting, function, and modulation in cardiomyocytes in health and disease.

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