Review

Calcium Signaling Series Donald M. Bers, Guest Editor

Calcium Signaling and Cardiac Arrhythmias

Andrew P. Landstrom, Dobromir Dobrev, Xander H.T. Wehrens

Abstract: There has been a significant progress in our understanding of the molecular mechanisms by which calcium (Ca2+) ions mediate various types of cardiac arrhythmias. A growing list of inherited gene defects can cause potentially lethal cardiac arrhythmia syndromes, including catecholaminergic polymorphic ventricular

Downloaded from tachycardia, congenital long QT syndrome, and hypertrophic cardiomyopathy. In addition, acquired deficits of multiple Ca2+-handling proteins can contribute to the pathogenesis of arrhythmias in patients with various types of heart disease. In this review article, we will first review the key role of Ca2+ in normal cardiac function—in particular, excitation–contraction coupling and normal electric rhythms. The functional involvement of Ca2+ in distinct arrhythmia mechanisms will be discussed, followed by various inherited arrhythmia syndromes caused 2+ http://circres.ahajournals.org/ by mutations in Ca -handling proteins. Finally, we will discuss how changes in the expression of regulation of Ca2+ channels and transporters can cause acquired arrhythmias, and how these mechanisms might be targeted for therapeutic purposes. (Circ Res. 2017;120:1969-1993. DOI: 10.1161/CIRCRESAHA.117.310083.)

Key Words: arrhythmias, cardiac ■ atrial fibrillation ■ calcium channels ■ cardiomyopathy ■ calcium release channel

2+ by guest on June 11, 2017 he bivalent cation calcium (Ca ) represents one of the Overview of Excitation–Contraction Tmost ubiquitous signal transduction molecules known.1 Coupling in the Heart It mediates a diverse array of biological functions including Regular contraction of the heart requires the conversion of , cellular exocytosis, neuronal activity, and electric activation (excitation) into mechanical force (con- triggering of programmed death. Since the first observa- traction). This process, known as excitation–contraction (EC) tion by Ringer in 1883 that Ca2+ was required for cardiac con- coupling, requires coordinated movement of Ca2+ ions at the traction, the role of Ca2+ as a signaling ion in the heart has cardiomyocyte level (Figure 1A). Each (AP), become increasingly appreciated.2 In addition, it has become triggered by influx of sodium (Na+) through the voltage-gated clear that abnormalities of Ca2+ homeostasis can play a key sodium channel (Nav1.5), thereby generating the INa current, role in the pathogenesis of common cardiovascular disorders, induces Ca2+ influx through voltage-activated L-type Ca2+ including cardiac arrhythmias. Human genetic studies of pa- 2+ channels (LTCCs, Cav1.2), creating the ICa,L current. This Ca tients with inherited arrhythmia syndromes have uncovered triggers a much larger Ca2+ release from the sarcoplasmic re- inherited mutations in various Ca2+ channels and Ca2+ trans- ticulum (SR), the principal intracellular Ca2+ storage organ- porters, directly implicating dysfunction of these proteins in elle.3 SR Ca2+ release is mediated by specialized Ca2+-release the disease mechanisms. Moreover, acquired modifications channels known as ryanodine receptor type-2 (RyR2).4 This of various Ca2+-handling proteins have been associated with process of Ca2+-sensitive RyR2-mediated SR release is known cardiac arrhythmias, including atrial fibrillation (AF) and ven- as Ca2+-induced Ca2+-release (CICR). The cytosolic Ca2+ binds tricular arrhythmias in failing hearts. In this review, we pro- to and activates cardiac C (TnC), the Ca2+-sensing vide a comprehensive overview of the potential contributions protein of the contractile apparatus and initiates myofilament of Ca2+ in arrhythmia mechanisms and highlight various gaps contraction. During diastole, relaxation occurs in knowledge and controversies in the field. when Ca2+ is removed from the cytosol either by sequestration

From the Section of Cardiology, Department of Pediatrics (A.P.L.), Cardiovascular Research Institute (A.P.L., X.H.T.W.), and Departments of Molecular Physiology and Biophysics, Medicine (Cardiology), Center for Space Medicine (X.H.T.W.), Baylor College of Medicine, Houston, TX; and Institute of Pharmacology, West German Heart and Vascular Center, University Duisburg-Essen, Essen, Germany (D.D.). Correspondence to Xander H.T. Wehrens, MD, PhD, Baylor College of Medicine, One Baylor Plaza, BCM335, Houston, TX 77030. E-mail wehrens@ bcm.edu © 2017 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.117.310083 1969 1970 Circulation Research June 9, 2017

each extruded Ca2+ ion, thereby creating a depolarizing tran- Nonstandard Abbreviations and Acronyms 2+ sient inward current (INCX). The rapid release of Ca from the AF atrial fibrillation SR into the cytosol, followed by rapid reuptake into the SR or AP action potential extrusion from the cell, creates a Ca2+ wave that runs through 2+ ARVC arrhythmogenic right ventricular cardiomyopathy the cardiomyocyte, and is known as the Ca transient. The 2+ CaM calmodulin amount of Ca released from the SR via RyR2 largely deter- 2+ CASQ2 -2 mines the Ca -transient amplitude, which correlates with the CaMKII Ca2+/calmodulin-dependent protein kinase II strength of systolic contraction. CICR Ca2+-induced Ca2+-release EC coupling occurs within specialized subcellular structures DCM dilated cardiomyopathy called junctional membrane complexes (JMCs), where LTCCs on transverse T-tubules—plasmalemmal invaginations that reach EC excitation–contraction deep into myocytes—are positioned in close proximity of the FKBP12.6 FK506-binding protein-12.6 RyR2 channels on the SR membranes (Figure 1B).5 The move- HRC histidine-rich Ca2+-binding protein ment of Ca2+ within these dyadic cleft domains is, in part, regu- iPSC induced pluripotent stem cells lated by JPH2 (junctophilin-2), a protein that provides a structural iPSC-CM iPSC-derived cardiomyocytes bridge between the plasmalemma and SR ensuring appropriate JCTN junctin proximity between the LTCC and RyR2 channels.6,7 JPH2 is also JMCs junctional membrane complexes necessary for BIN1 (bridging integrator 1) recruitment to develop Downloaded from JPH2 junctophilin-2 the T-tubule forming the dyad. There are important differences in LQTS long QT syndrome the organization of the JMC between atrial and ventricular cardio- LTCCs L-type Ca2+ channels myocytes.8 In ventricular myocytes, almost all Ca2+ release events NCX sodium-calcium exchanger type 1 (ie, sparks and transients/waves) are activated directly by LTCC PKA protein kinase A on T tubules, which leads to synchronized SR Ca2+ release and a http://circres.ahajournals.org/ SERCA2a SR Ca2+-ATPase type-2a rapid upstroke of the Ca2+ transient. In atrial cardiomyocytes, in SR which T tubules are relatively underdeveloped, the Ca2+ transient TnC troponin C begins with LTCC-triggered local SR Ca2+-release events at the TnI troponin I cell periphery that propagate slowly as Ca2+ waves toward the cell TnT troponin T center.9,10 In addition, atrial cardiomyocytes possess larger and TRDN triadin more heterogeneous axial tubules and much more Ca2+-buffering 11,12 RyR2 ryanodine receptor type-2 mitochondria than ventricular cardiomyocytes. Finally, anoth- 2+ WES whole-exome sequencing er class of Ca release channels known as inositol 1,4,5-trisphos- by guest on June 11, 2017 phate type 2 receptors may also contribute to CICR.13

into the SR by the SR Ca2+-ATPase type-2a (SERCA2a) or Regulation of Intracellular Calcium Handling out into the extracellular space by the Na+/Ca2+-exchanger The activity of Ca2+ channels and exchangers involved in EC type-1 (NCX). In addition, there is a minor contribution by coupling is regulated by several mechanisms and signaling the PMCA (plasmalemmal Ca2+-ATPase). Na+/Ca2+-exchanger pathways in response to changing demands for cardiac out- type-1 is electrogenic, as it imports 3 Na+ ions into the cell for put. For example, the fight-or-flight response activates the

Figure 1. Role of calcium-handling in excitation–contraction (EC) coupling. A, Schematic overview of key Ca2+-handling proteins involved in EC coupling. B, Schematic diagram of Ca2+ release unit and major components of the JMC (junctional membrane complex). The transverse tubule (TT) and sarcoplasmic reticulum (SR) membranes approximate to form the dyad. BIN1 indicates bridging integrator 1; Cav1.2, L-type Ca2+ channel; CAV3, caveolin-3; JPH2, juncophilin-2; NCX1, Na+/Ca2+ exchanger type-1; PM, plasma membrane; PMCA, plasmalemmal Ca2+- ATPase; RyR2, ryanodine receptor type-2; and SERCA2a, sarco/endoplasmic reticulum ATPase type-2a.* Landstrom et al Calcium Signaling and Cardiac Arrhythmias 1971

sympathetic portion of the autonomous nervous system with The LTCC, responsible for voltage-dependent Ca2+ entry downstream effects on Ca2+ signaling (recently reviewed).14 into the cells, consists of a macromolecular protein complex Activation of the β-adrenoceptor (βAR) causes a rise in the comprised of a pore-forming Cav1.2 (α subunit) and various intracellular concentration of the second messenger cAMP. auxillary subunits (α2, β, δ, and γ) that modulate channel Downstream effectors of cAMP include cAMP-dependent function (Figure 3). Similar to RyR2, the LTCC is regulated protein kinase A (PKA), which in turn can phosphorylate Ca2+ by protein kinases such as CaMKII and PKA as well as pro- transporters including LTCC, RyR2, and SERCA2a regulatory tein phosphatase type 1, phosphatase type 2A, and calcineu- proteins like phospholamban and sarcolipin. In addition, the rin (also known as protein phosphatase type 2B), which can Ca2+/calmodulin-dependent protein kinase II (CaMKII) can modulate channel gating. In addition, regulatory subunits, like modulate Ca2+ homeostasis in response to changes in heart rate, CaM, are embedded within the channel complex.26 LTCC are cellular oxidation levels, and persistent βAR stimulation.4,15 localized to rafts of other sarcolemmal ion channels and mem- Each of the Ca2+ channels and transporters consists of pore- brane-limited proteins.27,28 A critical mediator of this mem- forming proteins and various accessory subunits that modu- brane clustering is CAV3 (caveolin 3) that binds and interacts late the amount of Ca2+ that is moved through the pore. These with the N-terminal part of JPH2.29 channels and exchangers have been extensively reviewed else- Finally, SERCA2a is a macromolecular complex required where.16,17 Perhaps one of the most well-studied multiprotein for Ca2+-reuptake into the SR (Figure 4). It is allosterically complexes is the RyR2 macromolecular complex. A diverse regulated by phosphorylation-mediated conformational shifts array of RyR2-interacting proteins directly regulate RyR2 of its regulatory subunit phospholamban that can be phos- Downloaded from channel activity by binding to the pore subunit (ie, FKBP12.6 phorylated by PKA and CaMKII.30,31 These post-translational [FK506-binding protein-12.6], CaM [calmodulin], CASQ2 modifications can relieve the phospholamban -mediated inhi- [calsequestrin-2], JCTN [junctin], TRDN [triadin], βII-spectrin; bition of SERCA2a, allowing for rapid Ca2+-reuptake. HRC Figure 2).18,19 CASQ2 binds to RyR2 via both JCTN and TRDN. (histidine-rich Ca2+-binding protein) has been shown to bind RyR2 is strongly regulated by luminal (within the SR) Ca2+ lev- the SR luminal side of SERCA2a and to interact with TRDN, http://circres.ahajournals.org/ els, either by direct Ca2+ binding to RyR2 or by luminal Ca2+ potentially coordinating Ca2+-reuptake with SR Ca2+-release interacting with CASQ2, JCTN, and TRDN.20 Other proteins in along with S100A1.32,33 Moreover, CALR (calreticulin) may the RyR2 macromolecular complex regulate the level of RyR2 play a role in the inactivation and degradation of SERCA2a post-translational modification. Examples include the protein ki- under oxidative stress.34 nases PKA, CaMKII, and newly discovered SPEG (striated pref- erentially expressed protein kinase), and protein phosphatase Fundamental Arrhythmia Mechanisms type-1 and type-2A (PP1 and PP2A, respectively) that regulate The mechanisms responsible for cardiac arrhythmias are gen- the actual level of RyR2 phosphorylation.21–24 The RyR2 channel erally divided into 2 major categories: enhanced or abnormal

by guest on June 11, 2017 is also regulated by S-nitrosylation and oxidation.25

Figure 2. Ryanodine receptor type-2 (RyR2) macromolecular complex. Cartoon representing RyR2 pore-forming subunits with accessory proteins that bind to and/or modulate channel Figure 3. L-type Ca2+ channel (LTCC) macromolecular function. CaM indicates calmodulin; CaMKII, Ca2+/calmodulin- complex. Cartoon representing Cav1.2 pore-forming α subunit dependent protein kinase II; CASQ2, calsequestrin-2; FKBP12.6, with accessory α2, β, γ, δ subunits. CaMKII indicates Ca2+/

FK506-binding protein-12.6; JCTN, junctin; JPH2, juncophilin-2; calmodulin-dependent protein kinase II; CAV3, caveolin-3; ICa,L, PKA, protein kinase A; PM, plasma membrane; PP, protein L-type Ca2+ current; JPH2, juncophilin-2; PKA, protein kinase phosphatase; SR, sarcoplasmic reticulum; TECRL, trans-2,3- A; PM, plasma membrane; PP, protein phosphatase; and SR, enoyl-CoA reductase-like protein; and TRDN; triadin. sarcoplasmic reticulum. 1972 Circulation Research June 9, 2017

regions where EADs reach the threshold to propagate, they generate triggers that initiate reentry. DADs typically occur during diastole and conditions of el- evated cellular Ca2+-loading (Figure 5B). They are caused by spontaneous rises in cytoplasmic Ca2+-concentration, which

activate NCX, generating forward mode INCX, although other Ca2+-sensitive currents (nonselective cationic currents and chloride currents) might also contribute to DAD formation.44 The amplitude of the DAD depends on the size of the resting K+ conductance, mainly determined by the inward-rectifier K+-

current IK1, relative to INCX amplitude. When IK1 is low, the same 45 INCX will produce a larger DAD and vice versa. When DADs

reach excitation threshold, INa is activated and spontaneous APs can arise. DAD-mediated triggered activity contributes to ar- rhythmogenesis associated with catecholaminergic polymor- Figure 4. SR Ca2+-ATPase type-2a (SERCA2a) phic ventricular tachycardia (CPVT), HF, and AF. macromolecular complex. Cartoon representing SERCA2a complex required for reuptake of Ca2+ from the cytosol to the SR. Reentry can occur around a fixed anatomic obstacle or CALR indicates calreticulin; CaMKII, Ca2+/calmodulin-dependent in a substrate in which functional properties permit initia- Downloaded from protein kinase II; HRC, histidine-rich Ca2+ binding protein; PKA, tion and maintenance of reentrant circuits.46 The likelihood protein kinase A; PP, protein phosphatase; ROS, reactive oxygen of reentry formation is determined by the tissue properties species; RyR2, ryanodine receptor type-2; SERCA2a, sarco/ endoplasmic reticulum ATPase type-2a; SR, sarcoplasmic of conduction and refractoriness, with abnormal conduction reticulum; and TRDN, triadin. (slowing and local block) and refractoriness (abbreviated or prolonged) making reentry more likely (Figure 5C and 5D). http://circres.ahajournals.org/ impulse generation (ie, focal activity) and conduction dis- Refractory period depends on APD, whereas conduction ve- turbances (ie, reentry).35,36 Focal activity includes enhanced locity largely depends on INa, expression and localization of automaticity and triggered activity. Automaticity causes gap-junction proteins, and composition of extracellular ma- spontaneous generation of APs that do not require induction trix (ie, fibrosis). When the refractory period decreases (like by previous beats. Healthy myocardium is not normally au- in AF), the circuits are smaller and more numerous, simulta- tomatic, but disease conditions (ie, heart failure [HF]) can neous termination of all circuits is unlikely and the arrhyth- lead to resting membrane potential depolarization to more mia is sustained. When the refractory period is prolonged positive values causing abnormal automaticity. The most (like in HF), the heterogeneity (dispersion) of refractoriness

by guest on June 11, 2017 common causes of focal arrhythmias are early afterdepolar- is increased and the occurrence of reentry promoting conduc- izations (EADs) that precede full repolarization (typically tion block is more likely. The reentry-promoting substrate corresponding to phase 2 and phase 3 repolarization of the can be caused by disease-related cardiac remodeling or pre- human AP) and delayed afterdepolarizations (DADs) that oc- disposing genetic factors, but can also be produced by altered cur after full repolarization. restitution dynamics and subcellular Ca2+-alternans (SR Ca2+ EADs cause focal firing by depolarizing surrounding load and release alternans).47 Altered Ca2+ signaling can con- tissue to excitation threshold EADs and are most character- tribute to the formation of a reentry substrate by 2 mecha- istic of Purkinje-fiber tissue and ventricular tachyarrhyth- nisms: promoting dispersion of excitability, and promoting mias associated with HF and long QT syndrome (LQTS; dispersion of refractoriness.35 For example, DADs that do not Figure 5A). EADs are usually, but not exclusively, associ- reach the threshold to trigger an AP can cause resting mem- ated with excessive AP prolongation (ie, by increased in- brane potential depolarization, increasing Na+-channel inac- 37 + 38,39 ward ICa,L and late Na -current INa,L or INCX or by reduced tivation and promoting dispersion of excitability. The latter + K -currents [IK]), allowing ICa,L to recover from inactivation might lead to regional conduction block of impulses arising and depolarize the cardiomyocyte by allowing Ca2+ to en- from regions with suprathreshold DADs, thereby promoting 40 ter. CaMKII-dependent ICa,L phosphorylation slows inac- reentry initiation. EADs that remain below the threshold to tivation and accelerates recovery from inactivation, further propagate may increase dispersion of refractoriness, also enhancing the likelihood of EADs.37 At membrane potentials creating a reentry substrate. The rapid rates during DAD- 2+ negative to the threshold of ICa,L activation, spontaneous SR induced triggered activity can promote Ca transient alter- Ca2+ release–activated NCX favors the nonequilibrium reac- nans, which can cause spatially discordant APD alternans, 41,42 tivation of INa, driving phase 3 EADs induction. Finally, thereby enhancing the dispersion of refractoriness and the EADs have also been associated with AP duration (APD) likelihood of reentry.48 Thus depending on the cellular and shortening, occurring late in phase 3 of the AP.43 If the in- tissue context, EADs, DADs, and Ca2+ alternans can provide tracellular Ca2+ concentration is still high (ie, because of a the trigger and may contribute to the formation of the reen- large Ca2+ transient) when the membrane potential is nega- try-promoting substrate. A deep understanding of the detailed 2+ tive to the equilibrium potential for NCX, INCX can be activat- molecular mechanisms by which abnormal Ca -signaling in- ed leading to membrane depolarization. This type of EADs creases the susceptibility to cardiac arrhythmias is key for the typically occurs after termination of ventricular tachycar- development of novel therapeutic options for prevention and dia (VT), ventricular fibrillation (VF), and AF. Overall, in treatment of cardiac arrhythmias. Landstrom et al Calcium Signaling and Cardiac Arrhythmias 1973 Downloaded from http://circres.ahajournals.org/ by guest on June 11, 2017

Figure 5. Key electrophysiological mechanisms leading to cardiac arrhythmias. Ectopic (triggered) activity is primarily caused by (A) early afterdepolarizations (EADs) that occur mainly during bradycardia or after a pause, and (B) delayed after depolarizations (DADs) that occur usually during tachycardia. Reentry requires a vulnerable substrate, which can be caused by (C) action potential shortening or (D) 2+ 2+ + + + 2+ dispersion of refractoriness. ICa,L indicates L-type Ca current; IK,Ca, Ca dependent K current; INa,L, late Na current; and INCX, Na /Ca exchanger current.

Arrhythmias Caused By Heritable Defects VT or VF. The majority of CPVT cases present in childhood in Calcium-Handling Genes and have normal cardiac repolarization on ECG measure- The discovery of the first inherited mutations in genes encod- ment of the QT interval.52 The estimated prevalence of CPVT ing Ca2+-regulatory proteins has provided the best evidence is around 1:5000 to 1:10 000 depending on the population to date that defects in intracellular Ca2+-handling can directly studied.53 cause different cardiac arrhythmias (Figure 6). In the follow- RYR2-Encoded Ryanodine Receptor Type-2 (CPVT-1) ing section, we will review several inherited arrhythmia syn- In the first major case series of children described to have dromes that are often caused by mutations in genes encoding CPVT, the authors noted the presence of bidirectional VT as Ca2+ channels, transporters, or related proteins.49 an arrhythmia previously associated with digitalis toxicity.52 Catecholaminergic Polymorphic Ventricular This led to the hypothesis that the cause of CPVT may be Tachycardia because of DADs induced by increased SR Ca2+ load exac- The inherited arrhythmia disorder CPVT is one of the most- erbated by catecholamines.54 A genetic locus associated with deadly arrhythmias known, and it classically manifests with CPVT was first mapped to 1q42-43 of the genome in a large βAR-induced syncope or sudden cardiac death (SCD).50,51 study of 2 unrelated families with a heritable, autosomal-dom- CPVT was first described in 1978 as a distinct syndrome inant syndrome manifesting as stress-induced polymorphic associated with syncope and arrhythmia in the setting of a VT, syncope, and SCD with structurally normal hearts.55 Two structurally normal heart. This condition can present with years later, inherited mutations in the RYR2 gene were identi- premature ventricular contractions at rest, or with exercise, fied as the most common genetic subtype of CPVT (CPVT-1; and this ventricular ectopy can degenerate into bidirectional Table 1).56,57 1974 Circulation Research June 9, 2017

Figure 6. Diagram showing which genes have been linked to genetic arrhythmia disorders. Yellow fll indicates gene that encodes a Ca2+-sensitive or Ca2+-handling protein. CPVT indicates catecholaminergic polymorphic ventricular tachycardia; and IVF, idiopathic ventricular fbrillation. Downloaded from

Subsequent studies rapidly expanded the spectrum of stabilizing property of FKBP12.6 was not universally ob- RYR2 mutations in CPVT which account for ≈50% to 60% served.65 As this line of exploration was developing, a separate http://circres.ahajournals.org/ of all cases.56 Pathogenic mutations most often alter a single body of evidence was emerging that RyR2 phosphorylation at amino acid (missense mutations) and are inherited in auto- serine 2808 (S2808) by PKA could increase channel opening somal-dominant pattern. CPVT-associated mutations in RYR2 probability as part of the fight-or-flight mechanism.66,67 These almost always result in increased SR Ca2+ leak, which is am- studies converged with the observation that PKA-mediated in- plified in the setting of increased sympathetic drive.58 This creased channel sensitivity to Ca2+ was based on partial disso- increased propensity to SR Ca2+ leak can be detected as an ciation of FKBP12.6 binding after S2808 phosphorylation and increase in the frequency of elementary Ca2+-release events identified lethal exercise-induced arrhythmias in FKBP12.6 (ie, Ca2+ sparks).59 It is thought that diastolic SR Ca2+ leak can knockout mice (Fkbp12.6-/-).58 This observation was expanded 2+

by guest on June 11, 2017 lead to increased intracellular Ca that activates NCX dur- to other forms of cardiac disease, including HF, whereby el- ing diastole, leading to DADs and triggering of ventricular ar- evated βAR signaling through PKA resulted in hyperphos- rhythmias.60 Several aspects of the pathophysiology of CPVT phorylated S2808 and dissociation of FKBP12.6.68,69 These caused by RyR2 mutations remain controversial, including the findings have not been universally observed by other investi- potential role of reduced binding of FKBP12.6 to RyR2, chan- gators have catalyzed many follow-up studies that have intro- nel gating deficits in the absence of βAR stimulation, and the duced debate in the field.70,71 Some have argued that reduced potential involvement of SR Ca2+ overload as an additional Ca2+ reuptake into the SR led is the predominant mechanism mechanism. For example, the role of FKBP12.6 in regulating underlying HF72 or that phospholamban activity and increased RyR2 Ca2+-release and the role of PKA-mediated phosphory- SR Ca2+ load is involved.73 There is also evidence that CaMKII lation on RyR2 in cardiac arrhythmia and HF are subjects of phosphorylation of RyR2 may contribute to the development ongoing debate.61 of HF and arrhythmogenesis through increased Ca2+ leak.74 Early studies demonstrated that FKBP12.6 was expressed For in-depth review of this topic, please refer to previous ar- in the heart, associated with RyR2, and modulated CICR.62 ticles.75–77 Overall, these studies highlight the complexity of Furthermore, studies found that FKBP12.6 directly bound Ca2+ release regulation in the cardiac myocyte. RyR2 and stabilized the closed conformational state of Studies of several knockin mouse models of human the protein such that removal caused SR Ca2+ leak.63,64 This RYR2 mutations have provided additional insights into the

Table 1. Summary of CPVT-Associated Genes

Type MIM* Gene Protein Genetic Locus Frequency Inheritance CPVT 1 604772 RYR2 1q42.1-q43 50%–60% AD CPVT 2 611938 CASQ2 Calsequestrin 2 1p13.1 Rare AR CPVT 3 614021 TECRL Trans-2,3-enoyl-CoA reductase-like 7p22-p14 Rare AR CPVT 4 614916 CALM1 Calmodulin 1 14q31-q32 Rare AD CPVT 5 603283 TRDN Triadin 6q22.31 Rare AR AD indicates autosomal dominant; AR, autosomal recessive; and CPVT, catecholaminergic polymorphic ventricular tachycardia. *Phenotype MIM number. Landstrom et al Calcium Signaling and Cardiac Arrhythmias 1975

pathogenesis of CVPT.59,78–80 Based on some of these studies, cytosolic Ca2+ levels and reduced SR-store Ca2+ that was fur- it has been proposed that Purkinje cells in a mouse model of ther exacerbated by βAR stress.95 CASQ2 is part of the RyR2 CPVT exhibited a higher frequency and amplitude of spon- macromolecular complex that also involves the SR proteins taneous SR Ca2+-release events, suggesting that focal ar- TRDN and JCTN.96,97 The levels of these proteins are often rhythmias might originate from the specialized conduction dramatically altered when CASQ2 is genetically ablated or system.81 More sophisticated genetic studies are needed to mutated.96,98 Moreover, increased levels of CALR and RyR2, confirm whether Purkinje cells are truly the source of triggered which increases SR Ca2+ leak, have been reported in mice with arrhythmias in CPVT mutant mice and in patients with this mutant CASQ2.95 Therefore, it cannot be excluded that some condition. Finally, recent studies in human induced pluripo- of the RyR2 functional changes in CASQ2 mutant mice are, at tent stem cells (iPSC) have confirmed previous studies on re- least in part, mediated by changes in TRDN, JCTN, or CALR combinantly expressed channels and studies in mouse models, levels. Finally, the mechanisms of increased arrhythmogen- while providing additional mechanistic insights. For example, esis have been confirmed in human iPSC-CM. For instance, it has been shown that iPSC-derived cardiomyocytes (iPSC- the βAR agonist isoprenaline caused DADs, oscillatory ar- CM) from patients with CPVT exhibit an increased suscepti- rhythmic prepotentials, and aftercontractions in cardiomyo- bility to DADs because of abnormal SR Ca2+-release events.82 cytes derived from CPVT patients with CASQ2 variants but Overall, these studies demonstrate that exacerbation of DADs not from individuals with normal CASQ2.99,100 after sympathetic stimulation is the key mechanism and that TECRL-Encoded Trans-2,3-Enoyl-CoA Reductase-Like

Downloaded from β-blockers, dantrolene, CaMKII inhibitors like KN-93s, and Protein (CPVT-3) RyR2-inhibiting compounds such a S107 all represent po- A third genetic subtype of CPVT is the gene encoding TECRL 82–84 tential therapeutic options for CPVT. Subsequent clinical (trans-2,3-enoyl-CoA reductase-like protein). Initially identi- studies in patients with CPVT confirmed the antiarrhythmic fied by linkage analysis in a consanguineous Sudanese family 85 potential of dantrolene. Thus, iPSC-CM from patients with with multiple SCDs among children while playing, subse- CPVT may represent a valuable system for preclinical drug http://circres.ahajournals.org/ quent whole-exome sequencing (WES) identified mutations screening. in a handful of families and probands in TECRL.101,102 Each pa- CASQ2-Encoded CASQ2 (CPVT-2) tient demonstrated VT and VF, particularly with exertion, and A second rare genetic subtype of CPVT (CPVT-2) is caused had SCD. Interestingly, although the subjects had normal QT by autosomal-recessive variants in CASQ2-encoded CASQ2, intervals at baseline, adrenergic stimulation caused QT inter- the most abundant Ca2+-buffering protein in the SR. These val prolongation. As such, mutations in the TECRL gene seem mutations are relatively rare among CPVT cases, accounting to cause an overlap syndrome with features clearly associated for only ≈3% to 5% of all patients with CPVT.86 This genetic with not only CPVT but also congenital LQTS (see below). Creation of a mouse model of TECRL mutations is neces- by guest on June 11, 2017 subtype, initially identified in 7 families of a Bedouin tribe in northern Israel, is characterized by resting bradycardia and sary to examine arrhythmia mechanisms in the experimental VT by treadmill or βAR activation with isoprenaline infu- setting. Studies of iPSC-CM generated from the Sudanese pro- 2+ sion.87 Recently, a family with a unique autosomal-dominant band demonstrated reduced systolic Ca -transient amplitudes 2+ form of CPVT was found to be caused by a CASQ2 muta- and reduced caffeine-stimulated Ca transient amplitudes (an 2+ tion.88 CASQ2 is the cardiac-specific isoform of a family of index of SR Ca content) along with elevated resting cyto- 2+ 2+ proteins that directly and indirectly regulate SR Ca2+ storage solic Ca levels, consistent with presence of SR Ca leak as 82,99 and release.89 CASQ2 has a high-binding capacity (40–50 seen in CPVT-1 and CPVT-2. In addition, mutant iPSC- 2+ mol of Ca2+/mol) but a moderate affinity (Kd of 1 mmol/L) CMs demonstrated slower Ca -transient upstroke velocity 2+ for free Ca2+ and serves as molecular sink for Ca2+ that has and impaired SR Ca -reuptake when compared with both been sequestered into SR after cardiac contraction.90 With heterozygous and wild-type controls. Interestingly, stimula- tion with norepinephrine resulted in an increased propensity an increased prevalence of acidic amino acid residues, it is for DADs, which was suppressed by flecainide. AP recordings thought that the negatively charged CASQ2 directly binds revealed prolonged APD also suggesting a clinical overlap free Ca2+.91 between TECRL mutation-positive individuals with features All CASQ2 mutations identified to date are missense, dele- of both CPVT and LQTS.102 At present, the mechanisms by tion, or nonsense mutations that lead to a severe reduction, or which loss of TECRL function alters SR Ca2+-handling or complete loss of, the CASQ2 protein.92 RyR2 channels that ionic currents resulting in prolonged APD remain unknown. lack CASQ2 open spontaneously without being triggered by 2+ 93 ICa,L-mediated Ca influx. Studies in isolated rat cardiomyo- CALM1-Encoded Calmodulin Type-1 (CPVT-4) cytes transfected with mutant CASQ2 protein revealed a re- A fourth subtype of CPVT (CPVT-4) is caused by inherited duced SR store Ca2+ capacity with spontaneous Ca2+ transient mutations in CALM1-encoded CaM. The locus for this vari- generation and evidence of DADs.94 This effect was abrogated ant, 14q31-q31, was initially found by linkage analysis in a by addition of citrate, a low-affinity Ca2+ buffer, suggesting large, multigenerational Swedish family.103 Family members that mutant CASQ2 destabilizes SR-store Ca2+ capacity that demonstrated multiple episodes of syncope and sudden death, alters the Ca2+-sensitivity of RyR2 resulting in proarrhyth- particularly with exercise and exertion. On clinical evalua- mic DADs.94 Other studies utilizing knockin mice carrying tion, affected individuals demonstrated ventricular ectopy and missense or radical loss-of-function human mutations dem- evidence of VT/VF that was suppressed by β-blockers. The onstrated reduced CASQ2 expression with elevated resting genetic haplotype was inherited in an autosomal dominant 1976 Circulation Research June 9, 2017

fashion and was completely penetrant. Subsequent genetic For example, a homozygous frameshift mutation, TRDN- analysis of the ≈70 known genes within the locus demonstrat- D18Afs*13, was noted in a proband with cardiac arrest at ed a heterozygous CaM-N53I mutation that segregated with age of 2 years who was found to have polymorphic VT.112 incidence of disease within the family. A second mutation, A second independent proband hosted 2 mutations, TRDN- CaM-N97S, was identified in an unrelated proband from Iraq Q205X and TRDN-T59R, and demonstrated proximal muscle who was diagnosed with CPVT and was negative for muta- weakness, syncope with exertion and bidirectional ventricu- tions in RYR2. lar ectopy.112 Thus, TRDN mutations can cause CPVT in an Calmodulin is a ubiquitously expressed Ca2+-sensitive sig- autosomal-dominant manner. naling molecule that is found in all eukaryotic cells.104 There As discussed above, TRDN is a transmembrane protein are 3 CAM genes in humans, CALM1, CALM2, and CALM3, on the SR that forms a macromolecular complex with RyR2, which all encode a single protein—CaM. CaM is a relatively CASQ2, and JCTN.113 TRDN is a multiprotein family arising small, 148-amino acid α-helical protein with 4 classical Ca2+ from alternative splicing of a single TRDN gene. Two isoforms binding EF hands that each bind to a single Ca2+ cation. This di- are exclusively expressed in , whereas a third rect Ca2+-binding property allows conformational shifts in the isoform (also known as Trisk 32 or CT1) is expressed mainly N- and C-terminal domains of the protein that mediate a vari- in cardiac muscle.114 Interestingly, all 3 TRDN mutations local- ety of interactions with a large number of intracellular binding ized to a region of the protein that is common to all isoforms, targets.105 A dumbbell-shaped molecule, CaM, can sense both including skeletal muscle isoforms.112 The link between TRDN local and global Ca2+ levels, which allows for exquisite sensi- mutations and skeletal myopathy remains unknown. In vitro Downloaded from tivity to a variety of Ca2+-signaling events with downstream functional analysis of the TRDN-T59R mutation in nonmuscle regulation of many Ca2+-handling proteins.106 Within the heart, COS-7 cells demonstrated intracellular retention and degrada- CaM plays a key role in EC coupling and is critical for the tion of the mutation protein. Furthermore, viral transduction SR Ca2+ release and subsequent Ca2+ reuptake into the SR. of TRDN-T59R mutant protein into Trdn-/- mice demonstrated The LTCC and RyR2 are both important binding partners of no expression of the protein by immunofluorescence of iso- http://circres.ahajournals.org/ CaM.107 Ca2+ entering the cardiomyocyte via LTCCs binds to lated cardiomyocytes.112 Thus, functionally CPVT-associated CaM which, in turn, binds to the C-terminal IQ domain of the mutations lead to a severe TRDN function in cardiomyocytes. -/- Cav1.2 channel α pore subunit (α1C) of LTCC. This process Electron microscopy studies of cardiomyocytes from Trdn allows Cav1.2 channels to cluster and interact with each other, mice revealed fragmentation and overall reduction in contacts allowing for sufficient Ca2+ entry to initiate EC coupling.108 between the junctional SR and T-tubules.115 The function of CaM also binds to RyR2, and binding of CaM reduces the CRU channels was impaired with reduced negative feedback 2+ 2+ open probability of RyR2. Conversely, impaired binding of of SR Ca release on ICa,L. This uninhibited sarcolemmal Ca 2+ CaM to RyR2 caused by a mutated binding domain on RyR2 influx via ICa,L likely caused SR Ca overload leading to spon- by guest on June 11, 2017 leads to a variety of cardiac pathologies.109 taneous SR Ca2+-release events on βAR stimulation. In vitro experiments have revealed that mutations in the gene encoding CaM compromise Ca2+-binding and result in an Congenital LQTS aberrant interaction with the CaM-binding domain of RyR2.103 Congenital LQTS refers to a distinct group of cardiac chan- Subsequent studies revealed that the CaM-N97S mutation in nelopathies characterized by delayed cardiac repolarization, the C domain reduced Ca2+-binding affinity of the C-domain which places affected individuals at risk for syncope, seizures, and impaired binding to RyR2 at low Ca2+ concentrations, and SCD. A relatively common arrhythmia syndrome, affect- which was predicted to lead to an increased RyR2 open state. ing as many as 1 in 2500 patients, this delay in cardiac repo- larization occurs in the absence of an underlying syndrome This impaired inhibitory gating regulation was confirmed or structural heart disease.116,117 Approximately 75% of LQTS by subsequent studies of RyR2 single-channel recordings in cases are because of mutations in 3 genes: KCNQ1-encoded the presence of mutant CaM and functionally resulted in an I potassium channel (Kv7.1, LQTS-1), KCNH2-encoded I increased susceptibility for RyR2-mediated store overload– Ks Kr potassium channel (Kv11.1, LQTS-2), and SCN5A-encoded induced Ca2+ release.110,111 In contrast, the CaM-N53I variant, I sodium channel (NaV1.5, LQTS-3).118 These ion chan- which localized to the opposing N domain, demonstrated a Na nels play key roles in the cardiac AP and genetic defects in small yet significant increase in the Ca2+-saturation of the C these channels delay repolarization. Several channel interact- domain with an alteration to RyR2 binding affinity. These ing proteins, such as ANK2-encoded ankyrin B (LQTS-4), findings demonstrated that mutations in CALM1 are associ- KCNE1-encoded min-K (LQTS-5), and KCNE2-encoded ated with CPVT through 2 distinct mechanisms of RyR2 dys- min-K-related protein 1 (LQTS-6), among others, interact regulation and support a model whereby the Ca2+-saturated with these major channels and have been implicated as rare C-lobe is constitutively bound to RyR2 while the N lobe sens- causes of LQTS.119,120 To date, hundreds of mutations have es fluctuations in cellular Ca2+.111 been identified in 17 LQTS-susceptibility genes (Table 2). TRDN-Encoded Triadin (CPVT-5) In addition, large population-based GWAS (genome-wide Finally, a fifth subtype of CPVT (CPVT-5) is caused by muta- association study) analysis exploring common genetic vari- tions in TRDN-encoded TRDN. Mutations in TRDN were first ants associated with QT prolongation have identified many identified by candidate gene approach, and a small number loci that encode Ca2+-signaling proteins that were associated of probands were identified with either homozygous loss-of- with longer QT durations.121 Although the majority of the ac- function mutations or compound heterozygous mutations. cepted LQTS genes encode proteins that govern the flux of Landstrom et al Calcium Signaling and Cardiac Arrhythmias 1977

Na+ and K+ about the sarcolemma, there is mounting evidence arrests with markedly elevated QTc intervals. They were each that Ca2+ fluxes and intracellular Ca2+ signaling are associated found to host a heterozygous mutation—CaM-D130G and with prolonged cardiac repolarization and LQTS. CaM-D96V mutations, respectively.134 Subsequent validation genotyping in a cohort of LQTS patients yielded an unrelated CACNA1C-Encoded L-Type Calcium Channel (LQTS-8) proband with CaM-D130G and a second subject with CaM- The CACNA1C gene encodes the Cav1.2 (α ) channel sub- 1C F142L. These mutations were all found to localize either with- unit of the LTCC, a macromolecular channel complex respon- in, or immediately adjacent to, the third and fourth EF hand sible for I and EC coupling.3 The Cav1.2 protein comprised Ca,L domains of the C-terminal lobe, resulting in impaired Ca2+- 4 homologous domains (DI through DIV) that are connected binding of the domain.134 Interestingly, subsequent biochemi- by intracellular linker regions (I–II, II–III, and III–IV loops) cal investigations have elucidated 2 distinct mechanisms of and 6 transmembrane segments (S1 through S6).122 Mutations CaM and RyR2 dysregulation. CaM-D130G and CaM-D96V in CACNA1C have been associated with many human diseas- both impaired CaM-dependent inhibition of RyR2, resulting es that have cardiac manifestations. Classically, mutations in in an increased open state when single channels were recorded CACNA1C have been associated with Timothy syndrome—a and an increased propensity for store overload–induced Ca2+ disease characterized by extreme QT interval prolongation, release.110 In contrast, although CaM-F142L demonstrated syndactyly, neurodevelopmental delay, and SCD predisposi- reduced Ca2+ binding, it was unexpectedly found to enhance tion.123–126 Expansion of clinical genetic testing has identified CaM-dependent gating inhibition of RyR2 and related RyR2- many CACNA1C mutations in individuals demonstrating only mediated store overload–induced Ca2+ release. Specialized Downloaded from cardiac abnormalities (QT prolongation, structural heart dis- thermodynamic and NMR spectral analysis of the interaction ease, and cardiomyopathy), without extracardiac abnormali- between CaM-F142L and the reciprocal binding domain of ties, so-called cardiac-only TS.127 Individuals with only QT RyR2 demonstrated unique alterations in the protein–protein prolongation, and a diagnosis of LQTS, have been identified interface suggesting that the mutation does not disrupt the in a large number of independent cohorts. negative regulatory role of CaM despite an impaired ability to http://circres.ahajournals.org/ Many CACNA1C mutations have been characterized in bind free Ca2+.110 In addition to mutations identified in CALM1 vitro through heterologous expression in cell lines such as and CALM2, the first reports of LQTS-associated mutations HEK293 and TSA201 cells and demonstrate either increased in CALM3 have been recently reported. Specifically, a CaM- peak I , decreased current density with increased window Ca,L D130G mutation was identified in a neonate with a profoundly current, or negative activation/positive inactivation shifts.128 elevated QTc interval.135 To date, mutations in CALM3 have Experimental and modeling studies have demonstrated that not been widely identified and there have been no robust mutant CACNA1C can lead to enhanced I , and DAD- Ca,L mechanistic studies to evaluate the role of CaM in LQTS. mediated triggered activity.129 In addition, they can steepen the Taken together, these studies identify divergent mechanisms

by guest on June 11, 2017 APD restitution curve, disrupt rate-dependent cardiac excita- of disease pathogenesis that can, nonetheless, result in altered tion dynamics, and promote the development of alternans.130 RyR2 inhibition by CaM. Finally, CACNA1C mutations can amplify dispersion of repo- As described previously, the loss of RyR2 gating inhibi- larization across the tissue, which produces T-wave alternans tion is classically associated with the development of CPVT, and T-wave inversion on the ECG.130,131 and the link between these mutations and LQTS remains un- Although the overall functional impact of these mutations explored. One explanation for this dichotomy is that there are is the prolongation of phase 2 of the AP causing delayed repo- additional molecular effects to impaired CaM activity, such as larization, there does not seem to be a clear mechanistic differ- increased I , which can prolong the APD. This possibility ence between the CACNA1C mutations that lead to Timothy Ca,L is supported by early studies in guinea pig cardiomyocytes, syndrome (TS), cardiac-only TS, or LQTS. Indeed, this is re- which demonstrate reduced CaM-dependent inactivation of flected in the recent identification of a CACNA1C-I1166T in I with expression of LQTS-associated CAM mutations. In a proband with TS and independently identified CACNA1C- Ca,L addition, LQTS-associated CAM mutations result in electric I1166V mutation, localizing to the identical residue, in a alternans in a high dispersed manner across and within cells patient with LQTS.132,133 Given the lack of robust mechanis- consistent with the electric remodeling observed in canonical tic studies, it remains unclear how a near-identical genetic LQTS-associated mutations.136 Given the clear role of these substrate can lead to variable expressivity and severity of a mutations on RyR2 gating, it is likely that there is a significant disease phenotype. It is likely that genetic modifiers contrib- molecular overlap between the LQTS- and CPVT-associated ute to the differential phenotype manifestations. Additional mutations. However, the effect of CPVT-associated mutations mechanistic studies, perhaps utilizing iPSC-CM derived from in other sarcolemmal ionic currents that shape APD and cardi- Timothy syndrome, cardiac-only TS, and LQTS patients with ac repolarization are largely unexplored although the Nav1.5 CACNA1C mutations may yield insight into genomic, epig- and delayed rectifying I currents are strong candidates. enomic, molecular, and biophysical changes that are specific K Recently, the first attempts to derive patient-specific thera- to each disease presentation. pies to mitigate the abnormally prolonged repolarization have CALM1-, CALM2-, and CALM 3-encoded calmodulin been reported. In 2017, human iPSC-CMs were derived from 1, 2, and 3 (LQTS-14–16) a subject who was diagnosed with LQTS shortly after birth af- In 2013, the first mutations in the CALM1 and CALM2 genes ter a cardiac arrest with a markedly elevated QTc of 740 who were associated with LQTS.134 Two unrelated infant probands hosted a CaM-D130G mutation.137 Human iPSC-CMs derived were described with a severe phenotype of recurrent cardiac from dermal fibroblasts demonstrated prolonged APD and 1978 Circulation Research June 9, 2017

Table 2. Summary of LQTS-associated genes

Type MIM* Gene Protein Genetic Locus Frequency Inheritance LQTS 1 192500 KCNQ1 Kv7.1 11p15.5-p15.4 30–35 AD LQTS 2 613688 KCNH2 KV11.1 7p36.1 25–30 AD LQTS 3 603830 SCN5A NaV1.5 3p22.2 5–10 AD LQTS 4 600919 ANK2 Ankyrin B 4q25-q26 Rare AD LQTS 5 613695 KCNE1 MinK 21q22.12 Rare AD LQTS 6 613693 KCNE2 MinK related protein 1 21q22.12 Rare AD LQTS 7 170390 KCNJ2 Kir2.1 17q24.3 Rare AD LQTS 8 601005 CACNA1C CaV1.2† 12p13.33 Rare AD LQTS 9 611818 CAV3 Caveolin 3 3p25.3 Rare AD LQTS 10 611819 SCN4B Sodium channel β4 11p23 Rare AD LQTS 11 611820 AKAP9 Yotiao 7p21.2 Rare AD LQTS 12 612955 SNTA1 Syntrophin α1 20q11.21 Rare AD Downloaded from LQTS 13 613485 KCNJ5 Kir3.4 11q24.3 Rare AD LQTS 14 616247 CALM1 Calmodulin 1† 14q32.11 Rare AD LQTS 15 616249 CALM2 Calmodulin 2† 2p21 Rare AD LQTS 16 114183‡ CALM3 Calmodulin 3† 19q13.32 Rare AD http://circres.ahajournals.org/ LQTS 17 603283‡ TRDN Triadin† 6q22.31 Rare AR AD indicates autosomal dominant; AR, autosomal recessive; and LQTS, long QT syndrome. *Phenotype MIM number. †Ca2+-sensitive proteins or involved in Ca2+-signaling. ‡Gene MIM number.

larger Ca2+ transients with slower rise and decay kinetics when involving LQTS-associated TRDN mutations, and while Trdn- compared to wild-type iPSCs from an unrelated ostensibly /- mice have a known propensity for arrhythmogenesis with by guest on June 11, 2017 healthy donor.138 Furthermore, CaM-D130G imparted a sig- βAR stimulation, QT prolongation has not been detected.115,140 nificant decrease to CaM-dependent inactivation of the LTCC. Given the previously described possibility that Trdn-/- mice The authors utilized CRISPR-mediate interference of the likely demonstrate reduced negative feedback of RyR2- 2+ transcription of CALM2, which specifically reduced expres- mediated SR Ca -inhibition of ICa,L, an interesting possibility sion of the mutant protein without altering expression of either is that the increased Cav1.2 current might lead to APD prolon- CALM1 or CALM3. This selective expression inhibition res- gation. This would be an indirect mechanism of QT prolonga- cued the prolonged APD in iPSC-derived cardiomyocytes.138 tion that is analogous to the CACNA1C mutations described in

Although this study represents a major step forward in gene LQTS-8 that produce an increased ICa,L current. Further exten- therapy approaches to altering monogenic disease expression, sive studies are needed to delineate these hypotheses. translating this technique to an in vivo model of arrhythmia remains an active and challenging area of exploration. Idiopathic VF Idiopathic VF (IVF) is a genetic disease characterized by TRDN-Encoded Triadin (LQTS-17) a documented VF event that is otherwise unexplained. The most recent gene associated with LQTS is TRDN- Comprising ≈1% of out-of-hospital cardiac arrest survivors encoded TRDN, which has been previously also linked to presenting with a shockable rhythm, IVF can often be chal- CPVT-5. Identified after WES of probands negative for the lenging to diagnosis.141,142 Furthermore, in the setting of a known LQTS-associated genes, a handful of TRDN null vari- normal ECG, the affected status of an individual can only be ants were identified. As with CPVT-5, each mutation-positive known after an arrhythmic event, which makes genetic stud- proband demonstrated either homozygous inheritance of ies difficult to interpret. Traditionally associated with muta- loss-of-function allele or a compound heterozygous muta- tions in the SCN5A-encoded Nav1.5, the first IVF-associated tion with a loss-of-function allele.112,139 Both entities clinically mutations were often described in sporadic cases presenting manifest as SCD with either QT prolongation (LQTS-17) or with VF and had significant clinical overlap with a group of signs of ventricular ectopy in the absence of QT prolonga- Nav1.5-mediated channelopathies known as Brugada syn- tion (CPVT-5) diagnosed at an early age. This combination drome.143,144 New genetic testing platforms have allowed for of clinical findings, in addition to the skeletal muscle weak- the identification of other IVF genes implicated in families ness occasionally noted with CPVT-5, and the TRDN genet- with the arrhythmia (Figure 5), and recent advances in WES ic substrate has been labeled the so-called triadin knockout have identified the first genes encoding Ca2+-handling proteins syndrome. To date, there have been no mechanistic studies in children with IVF. Landstrom et al Calcium Signaling and Cardiac Arrhythmias 1979

In 2014, a family with a history of VF and SCD with Ca2+ dysregulation plays a significant role in the pathological normal ECG and echocardiograms was subjected to WES remodeling and hypertrophy. Furthermore, abnormal Ca2+- after kindred were found to be genotype negative for the signaling and the myofilament sensitivity to Ca2+ are both major LQTS, CPVT, and arrhythmogenic right ventricular known triggers for ventricular arrhythmias. Sarcomeric HCM cardiomyopathy (ARVC)–associated genes. This identified a genes are divided into subgroups based on the location of the CALM1-F90L mutation in a proband who experienced out- encoded protein in the cardiac sarcomere consisting of the of-hospital arrest because of VF at the age of 16 years with no thick, intermediate, and thin myofilaments. Mutations in genes clinical evidence of LQTS, CPVT, cardiomyopathy, or other encoding the thick myofilament (MYH7-encoded beta myosin SCD-predisposing cause.145 Subsequent functional evaluation heavy chain, MYL2-encoded regulatory myosin light chain, of the CALM1-F90L mutation demonstrated impaired CaM and MYL3-encoded essential myosin light chain), the interme- stability and impaired Ca2+ binding cooperativity.109 It was diate myofilament (MYPBC3-encoded cardiac myosin bind- concluded that the F90L mutation likely perturbs the position ing protein C), and the thin filament proteins (ACTC-encoded of 2 Ca2+ EF hands within the C-lobe relative to each. As a actin, TPM1-encoded α-tropomyosin, TNNT2-encoded car- result, the ability of the first occupied site to induce a favor- diac troponin T [TnT], TNNI3-encoded cardiac troponin I able conformational shift in the second, which is needed to [TnI], and TNNC1-encoded TnC) have been linked with de- facilitate Ca2+-binding, is impaired. The authors concluded velopment of HCM.154–160 Mechanisms of sarocomeric HCM that this creates a relatively insensitive CaM protein that is not pathogenesis have been extensively reviewed.161,162 responsive over small changes in Ca2+ concentration.109 Downloaded from Although the impact of the F90L on the function of CaM Arrhythmia Predisposition in Sarcomeric HCM is known, the ultimate effect of this perturbation on RyR2 gat- Early in the exploration of the sarcomeric gene association ing or other Ca2+-handling proteins is still unknown. While with HCM, it was proposed that the arrhythmia burden, man- there has been some incremental progress in identifying the ifest in SCD, might be higher with certain mutations. For genetic and molecular causes of IVF, mutations remain rare example, early studies identified individuals in large families http://circres.ahajournals.org/ and IVF remains enigmatic as a disease entity. As with the de- of HCM hosting either the MYH7-R403Q or MYH7-R453C velopment of CPVT and LQTS, one possibility is that altered missense mutations with increased sudden deaths compared 163 CaM function associated with IVF selectively impairs some to those hosting a MYH7-V606M mutation. Furthermore, ion channels while leaving other channels unaltered. A tempt- early genotype–phenotype studies of TNNT2 suggested an ing target is Nav1.5, which contains many IVF-associated association with decreased life expectancy and a high inci- 164 mutations. SCN5A-associated IVF and Brugada syndrome dence of SCD despite minimal cardiac hypertrophy. These mutations demonstrate a diverse array of biophysical effects studies proposed that individual mutations, or mutations in in heterologous cell line overexpression models. For example, specific genetic loci, may predispose to lethal arrhythmic by guest on June 11, 2017 some SCN5A IVF/Brugada syndrome mutations create depo- events in HCM. As the field has matured, these associations larizing shifts in channel inactivation, whereas others create were not universally observed, and there is significant het- hyperpolarization shifts in both activation and inactivation, all erogeneity in the expression and penetrance of sarcomeric 165–168 with the ultimate effect of loss-of-function effect on Nav1.5 HCM disease. This controversy has been reviewed 169,170 and VF predisposition.143,146 It is possible that IVF-associated previously. Overall, these genotype–phenotype correla- CAM mutations results in loss of Nav1.5 depolarizing current tions did not have mechanistic support for the arrhythmia and dispersion of excitability—a known molecular substrate burden observed in some cases; however, a growing body of 2+ for reentry-mediated VF. This possibility is supported by evidence suggests myofilament Ca -sensitivity as a major structural evidence that CaM directly binds Nav1.5 and is a arrhythmic mechanism which is independent of gene mu- critical player in channel inactivation and permitting channel tation. As a molecular unit, the troponin complex and thin activation. However, a direct link between CaM mutations and filament proteins are responsible for sensing intracellular 2+ 171 VF has not been clearly demonstrated.105 Ultimately, subse- Ca fluctuations and triggering sarcomeric contraction. quent studies are needed to link CAM mutations to I current Although many myofilament proteins have been linked to Na 2+ and a reentry substrate in IVF. HCM arrhythmogenesis, alterations in Ca -sensitivity of the components of the thin filament have been most clearly Hypertrophic Cardiomyopathy linked with potentially fatal ventricular arrhythmias.172 This Hypertrophic cardiomyopathy (HCM) is an inherited cardiac is detailed below. disorder characterized by asymmetrical hypertrophy of the Although HCM carries an increased risk of lethal ventric- heart, with a prevalence of 1 in 500.147 This disease represents ular arrhythmias,173 AF is found commonly with a frequency the most common cause of arrhythmogenic SCD in the young, of 20% to 25% of all patients with HCM.174 The hemodynamic particularly in young athletes.148 HCM is associated with not mechanism of this may be related to atrial dilation second- only lethal ventricular arrhythmias but also AF.149,150 Since ary to elevated left ventricular filling pressures resulting in the association of the first gene mutation with HCM, that of left atrial dilation; however, the cellular mechanism of this is MYH7-encoded β-myosin heavy chain, multiple studies have unexplored among sarcomeric HCM. Furthermore, although determined that the majority of HCM cases are because of there have been some suggestions that the sarcomeric muta- mutations in genes encoding components of the cardiac sar- tions may predispose individuals for early and more severe comere.151–153 Although the genetic mutations in proteins en- AF,175,176 there have not been conclusive studies linking spe- coding cardiac myofilaments are the major cause of HCM, cific genotype to AF predisposition.150 1980 Circulation Research June 9, 2017

TNNT2-Encoded Cardiac TnT and TNNC1-Encoded Mutations in TNNC1, a rare cause of HCM, have been Cardiac TnC also linked with a predisposition to fatal arrhythmias. A TnC- are the Ca2+-sensing molecule of the myofilament. A31S mutation was identified in 3-year-old boy who had After CICR, free Ca2+ binds TnC, which increases its binding HCM and an out-of-hospital VF event. Despite being on β- affinity for TnI, pulling the TnI inhibitory domain away from blocker therapy, he had multiple breakthrough VF events with its binding site on actin through its interaction with the mo- appropriate ICD discharged. This mutation is located within lecular linker TnT.177 This permits the troponin–tropomyosin the inactive Ca2+-binding domain of TnC. When reconstituted complex to move further into the actin groove fully exposing in skinned porcine cardiac fibers, this resulted in increased the myosin binding sites on actin. Active actin–myosin cross- Ca2+-sensitivity of both TnC and the thin filament compared bridging then occurs and contraction begins.177 with wild-type.185 Should future studies confirm the presence Traditionally, mutations in TNNT2 were thought to be of increased cellular Ca2+ levels, this also raises the possibility more arrhythmogenic compared with other genetic subtypes of either a DAD-mediated trigger or formation of an arrhyth- of HCM.178 Although this belief has been called into question mogenic substrate for reentry. In addition, although rare, iden- recently,169 a significant body of evidence has linked HCM- tification and characterization of human mutations affecting associated TNNT2 mutations with the development of fatal other thin filament components will add additional mechanis- arrhythmias in the absence of other known predictors of ar- tic understanding to this process. rhythmia predisposition such as significant hypertrophy or fi- brosis. Many TnT mutations have been described that nearly JPH2-Encoded JPH2 Downloaded from universally increase Ca2+ sensitivity, and thus Ca2+-binding of A small subset of patients without mutations in sarcomeric TnT and the sarcomeric thin filament. It is thought that TnT genes host a genetic mutation in genes encoding Ca2+-handling serves as a molecular sink for dynamic Ca2+-buffering and that proteins, and some have been linked with a predisposition to increased Ca2+-sensitivity may lead to altered Ca2+-transient arrhythmia.186 JPH2-encoded JPH2 is a member of the junc- dynamics. Overall, the degree of arrhythmia susceptibility tophilin family of proteins, which plays a critical role in main- http://circres.ahajournals.org/ seems to be directly correlated to the degree of increased Ca2+ taining the JMC in excitable cells, including striated muscle.6,7 sensitivity.179 JPH2 is the major family member found in the heart and spans For example, a mouse model of HCM (transgenic overex- the JMC, tethering the SR to the sarcolemma creating a fixed pression of I79N mutant TnT) exhibits increased cardiac con- cardiac dyad distance and serving a key role in negatively reg- tractility with reduced diastolic relaxation in the absence of ulating RyR2 opening (Figure 1B).7,187 Reflective of the critical significant fibrosis, as well as increased myofilament Ca2+ sen- role that this protein plays in maintain CICR and Ca2+ homeo- sitivity.180 This increased Ca2+-sensitivity was associated with stasis by RyR2 gating regulation, JPH2 plays a prominent role increased diastolic Ca2+ levels and intracellular Ca2+ overload in cardiomyopathy development, HF progression, and devel- 188–191 by guest on June 11, 2017 in isolated cardiomyocyte studies.181 Furthermore, TnT-I79N opment of EC coupling in the immature myocyte. These was associated with decreased Ca2+-transient amplitudes in diverse roles have been reviewed previously.192,193 An emerg- the face of elevated resting Ca2+ levels that caused ventricu- ing role of JPH2 is the development of Ca2+-mediated arrhyth- lar ectopy and VT.182 Although the precise mechanism has not mias, in particular congenital AF. Although the vast majority been clarified, the increased TnT Ca2+ sensitivity may lead of AF is acquired, reviewed in detail below, a specific mutation to DAD-mediated VT resulting from reduced myofilament (E169K) in JPH2 was linked with AF development in a small Ca2+ buffering or could cause reentrant arrhythmia through family with HCM.194 Expression of JPH2-E169K in mice a still undefined mechanism. The first option is supported in demonstrated a higher incidence of pacing-induced AF with other models of increased thin filament Ca2+ sensitivity. For increased SR Ca2+ leak and propensity of ectopic Ca2+ tran- example, the transgenic expression of fetal slow skeletal tro- sients after rapid pacing.194 This was associated with increased ponin I in place of TnI increased Ca2+ sensitivity in a manner RyR2-mediated SR Ca2+ leak because of loss of direct binding analogous to the electric remodeling found in pathological hy- between RyR2 and JPH2.189,194 This contributed to increased pertrophy.183 In this model, constitutive increase in Ca2+ sensi- diastolic Ca2+, increased NCX activity, and a predisposition tivity is associated with increased expression of NCX, which to DADs. Additional studies are needed to more thoroughly 2+ might result in increased INCX current to maintain Ca ho- dissect the molecular underpinnings of JPH2-mediated atrial meostasis during diastole when SERCA2a is also reduced.184 electric remodeling. Interestingly, this observation was noted in younger but not in older mice, reflecting the early age of onset of arrhythmias in CASQ2-Encoded CASQ2 and CALR3-Encoded TNNT2-positive subjects. The reentry hypothesis is supported CASQ3 by evidence that TNNI3 mutations can increase spatial dis- Rare mutations in other members of the JMC and RyR2 mac- persion of activation times across the myocardium, thereby romolecular complex have been linked to HCM. Genetic inter- promoting reentry. For example, TNNT2 mutations, including rogation of an Australian cohort of 252 unrelated individuals TnT-I19N, have been shown to associate with a short effective with HCM revealed a single mutation, D63E in CASQ2 and refractory period along with beat-to-beat variability in APD 2 mutations in the CALR3 gene (CALR3-R73Q and CASQ2- with increased spatial dispersion of conduction velocity.179 K82R) that were not identified in the ostensibly healthy Additional studies are required to directly prove 2 suggest- control population.195 To our knowledge, these are the only ed hypotheses and delineate the underlying arrhythmogenic mutations described in these genes among individuals with mechanisms associated with TNNT2 mutations. cardiomyopathy. The CASQ2-D63E was found in compound Landstrom et al Calcium Signaling and Cardiac Arrhythmias 1981

heterozygosity with 2 MYBPC3 mutations, which decrease mutation developed DCM and HF requiring cardiac trans- the likelihood of a truly causative biomarker. Conversely, the 2 plantation as adolescents.210 Interestingly, individuals hetero- CALR3 mutations were found in genotype-negative individu- zygous for this mutation tended to demonstrate HCM. This als. CALR is a Ca2+-binding chaperone in the sarcoplasmic/ raises the possibility of a dose-dependent effect with loss of endoplasmic reticulum, where it buffers Ca2+ and plays an PLN expression. In addition, multiple small genotyping stud- important role in the quality control of intracellular secretory ies identified a handful of heterozygous PLN mutations in in- pathway processes.196 CALR has 2 major isoforms, and little dividuals that were missense.211,212 Moreover, PLN mutations is known about the expression levels of CALR3 in myocardial have been identified in individuals with HCM exclusively. tissue. The functional implications of the CALR3 variants are These include a handful rare PLN promoter have been identi- presently unknown. In embryonic stem cell knockout model, fied in multiple independent cohorts.213–215 Overall, it seems CALR3 deficiency compromised the nuclear pore complex that mutations in PLN are a rare cause of both DCM and HCM and disrupted the nuclear import of the cardiac transcription accounting for <1% of all individuals with disease.213 factor MEF2C in a Ca2+-dependent manner.186,197 As discussed previously, myocyte relaxation during diastole is an active process mediated by ATP-expending pumping of Arrhythmogenic Cardiomyopathy cytosolic Ca2+ into the SR lumen via SERCA2a, which is nega- ARVC, also referred to as arrhythmogenic cardiomyopathy or tively regulated by PLN. Moreover, PLN inhibitory action can arrhythmogenic right ventricular dysplasia, is a relatively rare be reduced by PKA and CaMKII-mediate phosphorylation.216,217 type primary myocardial disease characterized by fibro-fatty Since the discovery of cardiomyopathy-associated mutations, Downloaded from replacement of myocardial tissue, cardiac arrhythmias, and an several mouse models have been made expressing putatively increased risk of SCD. This clinical entity has been recently pathogenic mutations. For example, transgenic overexpression reviewed.198 Traditionally, ARVC is considered a disease of of the PLN-R14del mutation, initially identified in a large fam- the cardiac desmosome, whereby mutations in components ily of DCM, leads to cardiac dilation, myocyte disarray, fibrosis, of this cell–cell adhesion structure are commonly identified 211 199,200 and early death in a mouse model. In vitro studies of PLN- http://circres.ahajournals.org/ in individuals with disease. There is some evidence that R14del expressed in HEK293 cells demonstrated superinhibi- RYR2 mutations may be a rare cause of ARVC or are present tion of Ca2+ affinity for SERCA2a that was not relieved by PKA with a prevalence that is significantly higher than rare RYR2 phosphorylation. Although the precise mechanism of PLN- variants in a control population.201,202 These clinical observa- mediated DCM remains unclear, it is possible that chronic sup- tions suggests that RYR2 variants play a role in the genetic pression of SERCA2a activity leads to increased cytosolic Ca2+ basis of traditional ARVC as either disease-causing mutations and a substrate for DAD arrhythmogenesis and, perhaps concur- or as a modifier susceptibility allele. In a mouse model of an rently, pathological myocardial remodeling resulting in HF. This ARVC-linked RYR2 variant, a reduced right ventricular end- possibility is supported by recent work exploring a PLN-R25C

by guest on June 11, 2017 diastolic volume was observed, but pathognomic fibrofatty mutation. Originally identified by WES of a DCM family with infiltration or structural abnormalities seen in patients with significant ventricular arrhythmias requiring ICD placement, ARVC were absent.59 Despite the possible link between RyR2 this mutation caused superinhibition of SERCA2a when virally and ARVC, currently there is insufficient evidence to impli- overexpressed in adult cardiomyocytes.218 This resulted in de- cate primary defects in Ca2+-signaling in the pathogenesis of creased SR Ca2+ content and reduced Ca2+ transient amplitude, this disorder.203 which is consistent with the loss of systolic function observed in Dilated Cardiomyopathy DCM. Furthermore, increased Ca2+ spark frequency and sponta- Familial dilated cardiomyopathy (DCM) is a genetic heart neous Ca2+ waves were seen, suggesting DAD-type arrhythmia muscle disease characterized by progressive dilation and susceptibility.218 Although the mechanism of increased SR Ca2+ dysfunction of the left or both ventricles. Mutations in >30 leak is unknown, it is possible that CaMKII activity is increased genes can cause congenital DCM, and most of these genes in the setting of elevated myocyte Ca2+ levels. Additional studies encode proteins that are part of the sarcomere or are structural specifically exploring the interaction between SERCA2a func- proteins needed to conduct mechanical force in the cardio- tion and RyR2 gating are needed to clarify this relationship. myocyte.204 The remaining genes encode proteins that play HRC-Encoded HRC various roles within cardiomyocytes to ensure proper contrac- Candidate gene-based genetic interrogation of a cohort of pa- tile function. Various studies suggest that mutations in sarco- tients with DCM for HRC-encoded HRC identified an S96A meric genes205–207 and nonsarcomeric genes208,209 can alter Ca2+ polymorphism that was statistically associated with the de- homeostasis, although the affected proteins are not directly velopment of ventricular arrhythmias. The presence of the involved in Ca2+ handling. On the contrary, there is a clear minor allele variant conferred a hazard ratio of 4.2 for VT role of defective Ca2+ handling in DCM pathogenesis among or VF among individuals with DCM.219 HRC is part of the patients with inherited mutations in phospholamban (PLN) SERCA2a macromolecular complex and serves as a regula- and HRC. tor of SR Ca2+ reuptake (Figure 4). It has been shown to bind PLN-Encoded PLN the SR luminal side of SERCA2a and interact with TRDN.32,33 Rare mutations and polymorphisms localizing to PLN have Subsequent studies utilizing adenoviral overexpression in rat been linked to patients with inherited cardiomyopathy. In a ventricular myocytes demonstrated reduced SR store Ca2+ large family with multiple generations of cardiomyopathy, reuptake and increased Ca2+ sparks with HRC-S96A expres- kindred homozygous for a PLN-L39X nonsense (early stop) sion compared with wild-type.220 Interestingly, this phenotype 1982 Circulation Research June 9, 2017

was exacerbated after myocardial ischemia and resulted in the development of AF. The primary arrhythmia mechanisms spontaneous Ca2+ waves.220 This suggested an arrhythmia- contributing to AF are focal ectopic firing and reentrant activ- susceptibility allele that may alter RyR2 gating function in the ity (Figure 7). setting of ischemic stress. Although in vivo studies are needed Ca2+-Dependent Triggered Activity in AF to validate these observations, the findings together suggest a Experimental studies in animal AF models and atrial samples relatively common variant that may be clinically silent until an from patients with AF revealed that abnormal atrial Ca2+- acquired myocardial stress or injury. Furthermore, these find- signaling likely plays a role in AF pathophysiology by con- ings suggest a molecular mechanism of cross talk between SR tributing to afterdepolarization-mediated triggered activity, reuptake via SERCA2a and SR release via RyR2. Whether conduction block, and Ca2+-driven subcellular alternans.36,235 this association is direct through common binding partners, Cellular DAD-mediated triggered activity was demonstrat- indirect through signal transduction molecules, or a combina- ed in atrial myocytes from patients with paroxysmal AF tion of both, remains unknown. (Figure 7A).236 These patients were in sinus rhythm at the time Calcium-Dependent Acquired Arrhythmias of tissue collection for weeks, thus excluding confounding effects of high atrial rate-induced atrial remodeling. Several Although many arrhythmias can be caused by heritable muta- factors contribute to the increased incidence of spontaneous tions in cardiac ion channels and channel-interacting proteins, SR Ca2+ release events, including increased SR Ca2+ load and Ca2+-mediated arrhythmias can also develop in the setting of enhanced RyR2-mediated SR Ca2+ release. The SR Ca2+ stores

Downloaded from acquired diseases of the myocardium. These common types are overloaded because of increased SERCA2a activity sec- of arrhythmias include AF and ventricular tachyarrhythmias ondary to PLN phosphorylation resulting in inactivation of encountered in patients with structural heart disease.75,221 In the inhibitory protein.236 Increased SR Ca2+ leak was caused this review, we will not discuss arrhythmias that occur in con- by increased RyR2 protein levels and RyR2 activity levels, junction with structural heart disease. whereas RyR2 phosphorylation levels were unaltered.194,237 http://circres.ahajournals.org/ Heart Failure Enhanced RyR2 protein expression during paroxysmal AF HF is a clinical diagnosis, which is defined as any abnormal- seems to be caused, in part, by a reduced expression of the ity in cardiac structure or function that results in failure of the micro-RNA cluster miR-106b-25, which enhances post-tran- heart to meet the metabolic demands of the body. Affecting scriptional regulation of RyR2.238 Consistent with these data millions of people worldwide, an estimated 1 in 5 people will is the finding that miR-106b-25–deficient mice are more sus- develop HF during their lifetime, making it one of the most ceptible to pacing-induced AF, atrial ectopy, and increased SR 2+ 238 deadly, morbid, and expensive diseases known.222 Although Ca release events. Recent transcriptomic analyses suggest gains have been made in reducing mortality, there is recent that there may be additional alterations in miRNA and mRNA

by guest on June 11, 2017 evidence that these gains have plateaued and that global bur- that have not been fully explored in patients with paroxysmal 239 den of HF remains high.223,224 Given this, there have been rapid AF. Taken all of these studies together, it is clear that ad- advances in the pharmacological management of HF, as well ditional investigation is needed to assess the potential effects 2+ as guidelines shifts for recommended therapies, which target of intracellular Ca modulation on the pathogenesis and pro- a growing number of molecular mechanisms.225 Pathological gression of AF. alterations in cardiomyocyte Ca2+ cycling have emerged as a In patients with persistent AF, an increased prevalence 2+ prominent component of the molecular dysfunction that oc- of spontaneous SR Ca release events and DADs have also 235,240,241 curs in HF. Understanding these mechanisms has been cen- been reported. The activity of single RyR2 channels 242,243 tral to the development of recent novel therapies. These topics was found to be increased in patients with cAF. Increased have been heavily reviewed.226–228 Furthermore, a substantial levels of PKA and CaMKII-mediated phosphorylation of body of evidence exists linking these pathological altera- RyR2 have been reported in patients and large animal mod- 242,244,245 tions in Ca2+ cycling to arrhythmic predisposition during HF els of cAF. Functionally, however, it seems that mainly remodeling. These arrhythmias are the cause of a significant CaMKII phosphorylation of RyR2 promotes excessive chan- 2+ 243 proportion of SCD that occurs during HF.229,230 Just as HF is a nel activation and SR Ca leak. In addition, reduced inter- complex and varied disease, the arrhythmic substrate from ab- actions of RyR2 with channel-stabilizing subunits such as errant Ca2+ signaling is a broad subject and has been the topic FKBP12.6 and JPH2 may contribute to increased diastolic SR 2+ 194,246 2+ of multiple comprehensive reviews.15,231,232 Ca leak and triggered activity. The enhanced SR Ca leak is more likely to lead to triggered activity because of up- Atrial Fibrillation regulation of NCX in patients with cAF.243 AF represents the most common type of cardiac arrhythmia Finally, AF has been reported in patients with CPVT, which observed in the general population.233 This disease often pro- is not surprising because mutant RyR2 channels cause SR Ca2+ gresses from a more intermittent form (paroxysmal AF) to per- leak in both the atria and ventricles.247,248 Studies in mouse sistent (chronic) AF (cAF) that lasts for >7 days at a time.36,221 models of CPVT caused by an RyR2 mutant confirmed en- Numerous factors can promote the occurrence of AF, includ- hanced SR Ca2+ leak in atrial myocytes, consistent with DADs ing genetic determinants (Figure 6), extracardiac factors (ie, and triggered activity.249,250 In addition, atrial conduction slow- sleep apnea, obesity, hypertension, autonomic imbalance), ing has been reported in an RyR2 knockin mouse model of as well as remodeling of the cardiac tissue.36,234 In this sec- CVPT, which may be caused by acute Ca2+-dependent inhibi- tion, we will focus on the potential involvement of Ca2+ in tion of Na+-channels and a chronic downregulation of Nav1.5 Landstrom et al Calcium Signaling and Cardiac Arrhythmias 1983 Downloaded from http://circres.ahajournals.org/

Figure 7. Calcium-dependent arrhythmia mechanisms in atrial fibrillation (AF). Schematic diagram delineating which changes in by guest on June 11, 2017 intracellular Ca2+-handling promote arrhythmia mechanisms leading to AF. Enhanced ryanodine receptor type-2 (RyR2)–mediated Ca2+ release leads to activation of Na+/Ca2+-exchanger (NCX), which in turn can cause a delayed afterdepolarization (DAD)–mediated triggered 2+ action potential (AP). Shortening of the AP duration (APD) because of reduction of ICa,L (L-type Ca current) and membrane hyperpolarization + because of upregulation of IK1 (inward rectifer K current) promote reentry. See main text for further details. CaM indicates calmodulin; CaMKII, Ca2+/calmodulin-dependent protein kinase II; Cav1.2, L-type Ca2+ channels; Cn, calcineurin; FKBP12.6, FK506-binding protein 12.6; miR-26, micro–RNA-26; NFAT, nuclear factor of activated T cells; and SERCA2a, sarco/endoplasmic reticulum ATPase type-2a.

expression.251,252 This study suggests a possible mechanistic of triggered activity, including cytosolic Ca2+ alternans (see link between abnormal SR Ca2+ release and reduced conduc- below), which play a critical role in the initiation of AF in tion velocity and a slower AP upstroke, which might contrib- humans.256 ute to reentry. Overall, abnormal Ca2+ signaling and enhanced diastolic SR Ca2+ leak along with cellular DAD-mediated trig- Ca2+-Dependent Reentry in AF gered activity may support AF induction by producing DADs Reentry requires a suitable vulnerable substrate, as well as a and could promote AF persistence by increasing heterogeneity trigger that acts on the substrate to initiate reentry (discussed (dispersion) of excitability, thereby causing conduction block above). Atrial remodeling is induced by atrial arrhythmias and that increases the susceptibility to AF-maintaining reentry. has the potential to increase the likelihood of ectopic activ- In addition to DADs, late phase 3 EADs have been ob- ity and reentry through multiple mechanisms. The persistence 2+ 2+ served in dogs after rapid atrial pacing (which causes Ca2+ of abnormal Ca signaling and enhanced diastolic SR Ca 2+ loading of the cells; Figure 7B).239 This is somewhat surpris- leak can activate ion channels and trigger Ca -dependent signaling pathways, thereby promoting the evolution of atri- ing because EADs typically occur in the setting of APD pro- al remodeling and the progression of AF to more persistent longation, whereas the atrial APD is usually abbreviated in forms.257 For example, small-conductance Ca2+-dependent K+- most models of AF. Several changes favor the development of channels (SK channels) govern the risk of human AF likely 2+ EADs in cAF, including SR Ca leak via RyR2 can promote by decreasing APD and promoting reentry,258 and the associa- ICa,L reactivation, the upregulation of INa,L, and enhancement tion between SK channels and RyR2 as the potential internal 243,253–255 of INCX. Nevertheless, the potential role of late phase source of SK channel activation, position SK channels as an 3 EADs in the development of AF requires further investiga- important Ca2+-dependent link between triggered activity with 259,260 tion. Other mechanisms may also contribute to the formation reentry. Furthermore, reduced ICa,L in AF causes APD 1984 Circulation Research June 9, 2017

shortening and promotes reentry.235 Its reduction is complex pacing of normal Langendorff-perfused whole rabbit hearts, and involves downregulation of the Cav1.2 subunit expression RyR2 refractoriness initiates SR Ca2+-release alternans in by the calcineurin-NFAT system and Cav1.2 breakdown by the ventricle without concomitant changes in diastolic SR Ca2+-dependent calpain proteases.235 Reduced Ca1.3 might Ca2+ alternans, which points to a potential role of RyR2 dys- 261 2+ 271 also contribute. Reentry-promoting increased IK1 may result function in Ca alternans during pacing. However, in this from a Ca2+-dependent enhancement in expression of Kir2.1- model, RyR2-related Ca2+ alternans did not play a major role subunits because of a calcineurin-NFAT–mediated decrease in for the transition of spatially concordant to spatially discor- micro–RNA-26.235 dant alternans and the transition of alternans to VF, which In atria, the primary mechanism leading to alternans rather depended on APD and CV restitution.271 These find- results from abnormalities in Ca2+ signaling (Ca2+-driven ings can be interpreted to suggest that although RyR2-related alternans), with APD alternans being secondary to Ca2+- Ca2+ alternans is involved in the initiation of arrhythmias, the alternans.262 Despite some controversies about whether Ca2+- maintenance of VF might be less dependent on intracellular alternans results from fluctuations in SR Ca2+ content or from SR Ca2+-release oscillations. Of note, studies using optical changes in RyR2 refractoriness, Ca2+-alternans can be ob- mapping of voltage and Ca2+ were not yet performed in the served in both animal models of AF and in humans with AF. diseased ventricle or atrium, thus the consequences of dys- For instance, SR Ca2+ leak increases the susceptibility to Ca2+- functional RyR2 (and other ECC components) for arrhythmia alternans and atrial arrhythmias in mice with CPVT mutations maintenance, particularly in the diseased atrium, remain un- in RyR2,263 and in rabbits with chronic myocardial infarction known and require thorough addressing in subsequent studies. Downloaded from or hypertension-induced atrial remodeling Ca2+-alternans is a Simultaneous high-resolution optical mapping of voltage and prominent feature of the arrhythmogenic substrate.264,265 Atrial Ca2+ in perfused intact human atria of sinus rhythm and pa- cardiomyocytes from patients with cAF are also more prone to tients with AF should be performed to obtain first hints about Ca2+-alternans, an effect that seems to involve an increased ac- the putative role of intracellular Ca2+ oscillations for the fibril- 272 tivation of adenosine A2A receptors with subsequent enhance- latory process during pacing-induced AF. http://circres.ahajournals.org/ ment of RyR2-mediated SR Ca2+ leak.266 Overall, computer modeling clearly suggests that elevated Ca2+-driven APD al- Therapeutic Approaches to Correcting ternans leads to increased arrhythmia vulnerability, complex- Calcium Mishandling ity and persistence because of increased heterogeneity of Ca2+-handling within cardiomyocytes has been recognized repolarization in atria.267 as a potential target for the treatment of cardiac disease for There is evidence that abnormal intracellular Ca2+-handling a long time. One class of drugs known as Ca2+ channel an- promotes atrial remodeling. Mice with cardiac-restricted over- tagonists targets the voltage-gated sarcolemmal Ca2+ channels expression of a repressor form of the cAMP-response element and is currently being used clinically to treat hypertension, by guest on June 11, 2017 modulator (CREM-Tg mice) develop atrial dilatation, abnor- angina pectoris, cardiomyopathy, and cardiac arrhythmias. mal cardiomyocyte growth, atrial fibrosis along with conduc- Fleckenstein273 described the first Ca2+ channel blockers as tion disturbance leading to spontaneous AF.268 By crossing the new drugs for the treatment of coronary disease ≈50 years CREM-Tg mice with RyR2-S2814A mice, in which RyR2 ago. During decades of subsequent studies, the role of Ca2+ phosphorylation by CaMKII is inhibited, the development of a channels in cardiac muscle contraction was elucidated (for substrate for spontaneous AF was prevented.269 These studies review, see274). Moreover, the biophysical and genetic iden- suggest that RyR2-mediated SR Ca2+ leak is involved in atrial tities of various voltage-gated Ca2+ channels were subse- remodeling, potentially by activation of calcineurin-NFAT– quently described.275,276 Several classes of antagonists have mediated changes in gene transcription.269 been described (ie, benzothiazepines, phenylalkylamines, In addition, there is emerging evidence that intracellular and dihydropyridines), and are now part of the formulary for Ca2+ signaling regulates the proliferation and the transition the treatment of cardiac diseases including arrhythmias. Ca2+ of fibroblasts to collagen-secreting myofibroblasts, thereby channel blockers are able to decrease the automaticity of ecto- promoting reentry.235 Transient-receptor potential canonical-3 pic foci in the heart and have emerging uses in many arrhyth- channels are key mediators of the fibroblast-to myofibro- mias. For example, T-type Ca2+ channel blockers and LTCC blast transition and their increase in AF involves the NFAT- blockers have efficacy in reducing AF arrhythmia burden and micro–RNA-26 pathway.270 Overall, these findings indicate can prevent electric remodeling.277–279 Moreover, although that abnormal RyR2-mediated SR Ca2+ leak and the related mainstay for treatment of CPVT is β-blockade, there has been 2+ Ca -dependent signaling may drive AF progression via these early evidence that also blocking ICa,L with the LTCC blocker and possibly other unrecognized remodeling pathways, lead- verapamil prevented ventricular arrhythmias.279 Overall, it is 2+ ing to the evolution of AF-maintaining substrate for reentry thought that reduced ICa,L results in less Ca overload of the (Figure 7). myocyte, reducing predisposition to ectopy that can trigger In summary, despite some controversies about the precise arrhythmias. role of RyR2 in AF, there is good evidence for contribution of During the past 15 years or so, several groups includ- abnormal Ca2+ signaling to the formation of the trigger and the ing our own have tried to develop pharmaceutical agents that substrate for reentry in both animal models and humans with target the intracellular Ca2+ release channel. To our knowl- AF.221 However, it is unknown whether intracellular Ca2+ os- edge, the first example of an experimental small molecule cillations are required and sufficient to sustain high-frequen- is K201 (also referred to as JTV519), a 1,4-benzothiazepine cy foci once the arrhythmia persists. During high-frequency shown to normalize RyR2 gating in a canine model with Landstrom et al Calcium Signaling and Cardiac Arrhythmias 1985

tachycardia-induced HF.280 Subsequently, K201 was shown including dantrolene,295 carvedilol analogues,296 and tetra- to prevent lethal ventricular tachyarrhythmias in a mouse caine derivatives.297 In addition to LTCC and RyR2, other Ca2+ model of CPVT by stabilizing RyR2 channels.281 Studies channels, Ca2+ transporters, and Ca2+-dependent signaling using recombinantly expressed RyR2 channels with CPVT- molecules (such as CaM, CaMKII) are potential therapeutic linked missense mutations showed that K201 can normalize targets. mutant channel activity.282 In addition, K201 was shown to exert antiarrhythmic effects against AF in an experimental Concluding Remarks guinea pig model.283 Although K201 normalizes defective The past 3 decades have seen a remarkable expansion in iden- RyR2 channels, this compound also inhibits various other tifying the genetic and molecular causes of both congenital + targets including annexin V and K channels, raising con- and acquired cardiac arrhythmias. Although the specific mo- cerns about potential off-target and proarrhythmic side-ef- lecular mechanisms of arrhythmic remodeling of the heart are 284,285 fects. The proposed mechanism of RyR2 stabilization, as diverse as the many ways in which arrhythmia can pres- through normalization of the binding stoichiometry of RyR2 ent, Ca2+ is a critical and central player in many. Progress into and FKBP12.6, remains controversial. For example, the identifying the role of Ca2+ in arrhythmias has led to novel un- 2+ role of FKBP12.6 in reducing RyR2-mediated Ca leak has derstanding of the physiological and pathological regulation 77,286 been debated and this topic has been robustly reviewed. of the cardiomyocyte. When coupled to rapid advancement in Furthermore, dissociating FKBP12.6 from RyR2 by FK506 genetic sequencing platforms, and recent breakthroughs in the did not affect suppression of spontaneous Ca2+ release events Downloaded from development of both in vitro and in vivo models of disease, in rat ventricular myocytes questioning the role of FKBP12.6 these advances offer the possibility of revolutionizing the di- 287 binding in the mechanism of FK506. Although debate ex- agnosis and treatment of these common and potentially life- ists in the field, there is a preponderance of datam, which threatening conditions. suggests that RyR2 stabilization can be achieved by small molecules. Since discovery of this first molecule, newer gen- http://circres.ahajournals.org/ Sources of Funding erations of RyR2 stabilizing molecules have been developed. Dr Landstrom is supported by the National Institutes of Health (NIH) For example, a 1,4-benzothiazepine named S107—a more L40-HL129273 and K08-HL136839, Baylor College of Medicine specific RyR2-blocker—was shown to prevent ventricular Department of Pediatrics Pilot Grant Award, and the Pediatric and arrhythmias in a CPVT mouse model heterozygous for mu- Congenital Electrophysiology Society Paul C. Gillette Award. Dr tation R2474S.80 Moreover, S107 has been shown to inhibit Dobrev is supported by DZHK (German Center for Cardiovascular the RyR2-mediated diastolic SR Ca2+ leak in atrial myocytes Research), the German Research Foundation DFG (Do 769/4-1), and a NIH grant (R01-HL131517). Dr Wehrens is supported by NIH in many RYR2 mutation knockin models and decreased the grants R01-HL089598, R01-HL091947, R01-HL117641, and R41- 250 incidence of burst pacing-induced AF. Although thought HL129570, and American Heart Association grant 13EIA14560061. by guest on June 11, 2017 to have less off-target effects on a host of other receptors than K201, there have yet to be comprehensive studies on Disclosures the major ion channels responsible for EC-coupling and Ca2+ Dr Wehrens is a founding partner of Elex Biotech, a start-up company 80 homeostasis. that developed drug molecules that target ryanodine receptors for the Although flecainide is a class IC antiarrhythmic drug with treatment of cardiac arrhythmia disorders. The other authors report Na+ channel–blocking properties, this drug has also been no conflicts. shown to inhibit RyR2 and exert therapeutic effects in mouse models of, and patients with, CPVT.288 This is most salient in References patients for whom β-blockade is less effective. For example, 1. Cheng H, Lederer WJ. Calcium sparks. Physiol Rev. 2008;88:1491–1545. flecainide has been shown to inhibit aberrant RyR2 activity and doi: 10.1152/physrev.00030.2007. reduce spontaneous Ca2+ waves in both patients with CPVT 2. Ringer, S. A further contribution regarding the influence of the differ- ent constituents of the blood on the contraction of the heart. J Physiol. -/- refractory to β-blocker therapy and in Casq2 mouse mod- 1883;4:29–42. els of CPVT in numerous independent studies.289–291 Although 3. Bers DM. Cardiac excitation-contraction coupling. Nature. 2002;415:198– the suppression of aberrant SR Ca2+ release seems a consis- 205. doi: 10.1038/415198a. 4. Heijman J, Voigt N, Wehrens XH, Dobrev D. Calcium dysregulation in tent effect of flecainide, other investigators have questioned atrial fibrillation: the role of CaMKII. Front Pharmacol. 2014;5:30. doi: whether RyR2 is the primary molecular target. For example, 10.3389/fphar.2014.00030. increasing the threshold for triggered activity by action on the 5. 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