Calcium Revisited

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Basic Science for the Clinical Electrophysiologist Calcium Revisited New Insights Into the Molecular Basis of Long-QT Syndrome John R. Giudicessi, MD, PhD; Michael J. Ackerman, MD, PhD

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he first clinical description of long-QT syndrome (LQTS) occurred in 1957 when Drs Anton Jervell and Fred LangeNielsen postulated that syncopal/seizure episodes and high propensity for sudden cardiac death (SCD) observed in a subset of children with sensorineural deafness and otherwise unexplained heart rate–corrected QT interval prolongation on ECG stemmed from a novel congenital disorder.1 This was followed by independent descriptions of a syndrome characterized by a cardiac-only phenotype consisting of prolonged QT intervals and an increased risk for syncope, seizures, and SCD in the absence of sensorineural deafness by Drs Cesarino Romano and Owen Ward, in 1963 and 1964, respectively.2,3 During the past 5 decades, insights gleamed from a multitude of clinical, epidemiological, and molecular studies have demonstrated that LQTS is a collection of not only genetically and phenotypically diverse disorders of cardiac repolarization that encompasses the aforementioned predominantly autosomal-dominant, nonsyndromic Romano–Ward syndrome now simply referred to as LQTS4 and autosomal-recessive, multisystem, Jervell–Lange Nielsen syndrome but also exceedingly rare multisystem LQTS subtypes such as Timothy syndrome (TS) characterized by QT prolongation and an increased risk of SCD along with an array of extracardiac manifestations. Although mutations in calcium (Ca2+)-handling proteins, including the CACNA1C-encoded L-type Ca2+ channel (LTCC), RYR2-encoded ryanodine receptor-2 intracellular calcium release channel (RyR2), and many other auxiliary interacting proteins, are central to the pathogenesis of inherited cardiac arrhythmia syndromes, such as catecholaminergic ventricular tachycardia and Brugada syndrome, until recently, the contribution of dysfunctional Ca2+ handling to the pathogenesis of LQTS was limited to the extremely rare and highly lethal multisystem TS. However, the unexpected recent discoveries of multiple nonsyndromic LQTS-causative mutations in CACNA1C,5–7 syndromic LQTS-causative mutations in CALM1-3-encoded Calmodulin8,9 and TRDN-encoded Triadin,10 and common genetic variation at several novel genetic loci that modulate QT interval duration in health11 that encode or reside near known Ca2+-handling proteins suggest a larger role for Ca2+ cycling in cardiac repolarization and the pathogenesis of LQTS.

In this review, we examine existing paradigms and recent advances that shape our current understanding of the molecular basis of LQTS with a focus on the molecular insights provided by recent discoveries linking genetic variation in Ca2+-handling proteins to the pathogenesis of LQTS and modulation of cardiac repolarization in health.

Molecular Basis of Cardiac Ca2+ Cycling The carefully regulated flux of Ca2+ within and through cardiomyocytes governs both cardiac excitability and contractility. Not only does Ca2+ serve as the critical link between the electric stimuli generated by plasma membrane depolarization and mechanical contraction of the cardiomyocyte during excitation–contraction coupling (Figure 1)12 but also it generates a substantial inward/depolarizing current that modulates action potential (AP) duration and regulates several intracellular processes, including gene transcription. Although a detailed discussion of the major molecular players in cardiac excitation–contraction coupling/Ca2+ cycling is outside the scope of this review, a thorough review of the genetics, structure, and functionality of the Cav1.2 LTCC and RyR2 macromolecular complexes is contained within the Data Supplement to provide interested readers with sufficient background to appreciate how perturbations in these molecular constituents may contribute to the pathogenesis of LQTS.

Molecular Basis of LQTS During the early-to-mid 1990s, a series of linkage analysis and positional cloning studies identified KCNQ1 (LQT1), KCNH2 (LQT2), and SCN5A (LQT3) as genetic substrates for congenital LQTS.13–18 Subsequent epidemiological studies ascertained that disease-causative mutations in these 3 major genes account for an estimated 60% to 75% of genotype-positive LQTS cases (Table I in the Data Supplement).19,20 After the discovery of these 3 major LQTS-susceptibility genes, at least 14 additional minor LQTS-susceptibility genes have been described in the literature. The resulting LQTS subtypes can be further classified on the basis of whether they yield a nonsyndromic or multisystem clinical phenotype (Table I in the Data Supplement). Furthermore, because the

Received January 21, 2016; accepted May 27, 2016. From the Internal Medicine Residency and Clinician-Investigator Programs, Department of Medicine (J.R.G.) and Departments of Cardiovascular Diseases, Pediatrics (Division of Pediatric Cardiology), and Molecular Pharmacology & Experimental Therapeutics (M.J.A.), Windland Smith Rice Sudden Death Genomics Laboratory, Mayo Clinic, Rochester, MN. The Data Supplement is available at http://circep.ahajournals.org/lookup/suppl/doi:10.1161/CIRCEP.116.002480/-/DC1. Correspondence to Michael J. Ackerman, MD, PhD, Mayo Clinic Windland Smith Rice Sudden Death Genomics Laboratory, Mayo Clinic, Guggenheim 501, Rochester, MN 55905. E-mail [email protected] (Circ Arrhythm Electrophysiol. 2016;9:e002480. DOI: 10.1161/CIRCEP.116.002480.) © 2016 American Heart Association, Inc. Circ Arrhythm Electrophysiol is available at http://circep.ahajournals.org

DOI: 10.1161/CIRCEP.116.002480

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Figure 1. Cardiac excitation–contraction coupling. Depolarization of the transverse tubule (T-tubule) activates voltage-gated L-type Ca2+ channels (LTCC, designated in schema as Cav1.2) allowing calcium to enter the cytosol (1). The small amount of calcium influx through the LTCC triggers the large-scale release of calcium from stores in the sarcoplasmic reticulum through ryanodine receptors (RyR2) within calcium release units (CRU) producing a cytosolic calcium transient in a process deemed calcium-induced calcium release (2). Increased cytosolic calcium concentrations activate the cardiac myofilaments by binding to troponin C thereby inducing allosteric changes in myosin that results in cardiac muscle contraction during systole (3). Intracellular calcium is pumped back into the SR by the SR Ca2+ ATPase (SERCA2a) and expelled from the cell via the Na+/Ca2+ exchanger (NCX) (4) enabling the relaxation of the myofilaments during diastole (5).

majority of minor LQTS genes encode channel-interacting proteins that work in concert with the Nav1.5, Cav1.2, Kv11.1, and Kv7.1 pore-forming α-subunits, a current-centric model has been proposed as a means of summarizing the genetic and electrophysiological basis of the major and minor LQTSsusceptibility genes (Figure 2).21 Because the molecular basis of the major and minor LQTS-susceptibility genes linked to the function of INa, IKr, and IKs currents have been described recently in detail elsewhere,22 the following sections focus on examining the ≈5% to 10% of multisystem and nonsyndromic LQTS cases that arise from Ca2+-handling protein dysfunction.

TS: Aberrant Ca2+-Dependent and Ca -Independent Intracellular Processes 2+

TS is an extremely rare, largely sporadic form of multisystem LQTS characterized by severe QT prolongation on ECG (average heart rate–corrected QT interval ≈580 ms), high incidence of life-threatening cardiac arrhythmias/SCD, syndactyly, minor craniomaxillofacial abnormalities, and baldness at birth and variably expressed intermittent hypoglycemia,

immunodeficiency, congenital heart defects, and developmental delay/neurocognitive impairments along the autism spectrum.23–25 Initially, TS was thought to arise from a single recurrent mutation, G406R, in the alternatively spliced CACNA1C exon 8a that accounts for ≈20% of the Cav1.2 mRNA transcripts in the heart and brain and encodes the distal portion of the domain I transmembrane segment 6 (IS6) known to play a critical role in the voltage-dependent inactivation (VDI) of Cav1.2 (Figure 3).25–27 However, shortly after the elucidation of the molecular basis of typical TS now referred to as type I TS (TS1), 2 cases of atypical or type II TS (TS2) were reported in the literature.28 Interestingly, both cases were characterized by extreme QT prolongation (average ≈640 ms) complicated by multiple episodes of cardiac arrest in the absence of syndactyly and harbored de novo mutations in the CACNA1C exon 8–containing heart- and brain-predominant Cav1.2 splice variant.28 Interestingly, the exon 8 CACNA1C-G406R–positive TS2 proband displayed severe neurodevelopmental delay, craniofacial/odontic abnormalities, and possible nemaline skeletal myopathy during the first few months of life, whereas the exon 8 CACNA1C-G402S–positive TS2 proband seemed

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Figure 2. Current-centric classification of long-QT syndrome (LQTS) genotypes. The clinical phenotypes resulting from the abnormal ventricular cardiac action potential depolarization (purple) or repolarization (orange) are grouped according to the specific current perturbed by an underlying genetic defect. Blue circles represent mutations that confer a loss of function to the specified current, whereas green circles confer a gain of function. Solid lines indicate those disorders that are autosomal dominant, whereas dashed lines indicate those disorders that are autosomal recessive. Solid black outlines indicate nonsyndromic genotypes and solid orange outlines represent multisystem genotypes. ABS indicates ankyrin-B syndrome; COTS, cardiac-only Timothy syndrome; JLNS, Jervell and Lange-Nielson syndrome; ICa,L, L-type calcium current; IK1, inwardly rectifying potassium current; IKAch, G-protein–coupled inwardly rectifying potassium current; IKr, rapid component of the delayed rectifier potassium current; IKs, slow-component of the delayed rectifier potassium current; INa, cardiac sodium current; and TKO, triadin knockout syndrome. Adapted with permission from Giudicessi and Ackerman21. Authorization for this adaptation has been obtained both from the owner of the copyright in the original work and from the owner of copyright in the translation or adaptation.

developmentally normal until suffering an out-of-hospital cardiac arrest at age 4.28 It was postulated initially that the lack of syndactyly and the potentially more severe neurological/cardiac phenotypes anecdotally observed in TS2 versus TS1 were likely secondary to the differential tissue-specific expression of exon 8– and exon 8a–containing Cav1.2 splice variants.28 Although this is likely the case for exon 8 G406R–mediated TS2, the recent identification of exon 8 G402S in an otherwise developmentally normal adolescent woman with borderline QT prolongation who presented with an out-of-hospital cardiac arrest29 coupled with the normal development of the initial G402S-positive TS2 proband before his first cardiac arrest28 suggest that (1) CACNA1C-G402S results in a milder clinical phenotype more akin to nonsyndromic LQTS and (2) the neurodevelopmental sequelae observed in the initial CACNA1C-G402S–positive TS2 proband are more likely secondary to arrhythmia-induced anoxic brain injury than underlying CACNA1C-mediated developmental delay. Mechanistically, the IS6 and α-interaction domain–containing I-II loop linker of the Cav1.2 α-subunit form a continuous α-helix–rich region that allow Cavβ auxiliary subunits to bind and modulate Cav1.2 gating in a manner that supports

faster VDI.30,31 In addition, calmodulin (CaM)-mediated Ca2+-dependent inactivation (CDI) may also be dependent on Cavβ and the structural integrity of the Cav1.2 α-subunit IS6-α-interaction domain region suggesting that the Cav1.2 IS6-α-interaction domain/Cavβ complex serves as the common denominator by which VDI and CDI modulate Cav1.2 gating.32 Given the unique conformational flexibility imparted by glycine (G) residues and the common localization of TS-associated mutations to the distal IS6 region of Cav1.2, it is therefore not surprising that both TS1- and TS2-associated CACNA1C exon 8a (G406R) and exon 8 (G402S and G406R) mutations drastically reduce Cav1.2 VDI in vitro possibly via the alteration of Cavβ binding/function.25,28 Although subsequent studies, including those in TS-specific induced pluripotent stem cell (iPSC)–derived cardiomyocytes33 and cortical neurons,34 have reaffirmed the effect of CACNA1C-G406R on VDI, the effects of CACNA1CG406R on CDI reported to date are widely discordant.28,35,36 Furthermore, several alternatives to the IS6-α-interaction domain/Cavβ-mediated reduction in VDI hypothesis have been proposed, including formation of aberrant A kinase anchor protein–Cav1.2 complexes37 and facilitation by aberrant/

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Figure 3. Electrophysiological and clinical manifestations of putative long-QT syndrome (LQTS)–causative gain-of-function CACNA1C mutations. A, The location of Timothy syndrome (TS; maroon circles)-, cardiac-only Timothy syndrome (COTS; orange circles)-, and LQTS (CACNA1C-LQTS; green circles)-causative CACNA1C mutations are depicted on the Cav1.2 linear protein topology. Biogenic (small dash), kinetic (solid), and mixed (large dash) generalized electrophysiological manifestations of individual CACNA1C mutations are indicated by contrasting circle outlines. B, Summary of the electrophysiological and clinical manifestations of TS, COTS, and LQTS. EP indicates electrophysiological; HCM, hypertrophic cardiomyopathy; and SCD, sudden cardiac death.

excess calmodulin kinase II activity.38,39As such, the precise mechanisms by which TS1- and TS2-causative mutations reduce Cav1.2 VDI and the potential contribution of impaired Cav1.2 CDI to the generation of the multisystem TS phenotype remain incompletely understood. In the heart, the reduction of Cav1.2 VDI conferred by TS1- and TS2-causative CACNA1C mutations is predicted in silico to accentuate the inward depolarizing current and prolong AP repolarization/duration.25,28 Although subsequent studies in both iPSC cardiomyocytes33 and a TS mouse model40 concluded that increased Ca2+ influx through mutant Cav1.2-TS channels during the plateau phase prolongs cardiac AP duration, both studies also demonstrated that TS ventricular myocytes are in a constant state of Ca2+ overload and thus prone to spontaneous ectopic Ca2+ release from sarcoplasmic reticulum (SR) and the generation of potentially arrhythmogenic delayed after-depolarizations (DADs). As such, the proarrhythmic state observed in TS likely arises from the generation of early after-depolarizations secondary to increased myocyte refractoriness and DADs secondary to spontaneous

SR Ca2+ release during phase 2/3 and phase 4 of the cardiac AP, respectively. Interestingly, roscovitine (Seliciclib), a cyclin-dependent kinase inhibitor previously shown to enhance Cav1.2 VDI in heterologous expression systems via direct extracellular binding,41,42 rescued the electrophysiological perturbations observed in TS iPSC-derived cardiomyocytes,33 suggesting that agents with similar mechanisms of action may prove beneficial in the treatment of TS. Although the contribution of increased Ca2+ influx to the proarrhythmic TS cardiac phenotype is relatively well understood, precisely how TS-causative CACNA1C mutations generate a myriad of variably expressed extracardiac phenotypes, particularly in nonexcitable tissues, remains unclear. At present, there is evidence to suggest that Ca2+-dependent processes likely underlie the intermittent hypoglycemia (excessive Ca2+-mediated insulin release from pancreatic β cells)43 and craniofacial dysmorphism (mandibular chondrocyte/osteoblast hypertrophy via increased Ca2+-dependent calcineurin/ nuclear factor of activated T-cell transcription factors signaling)44 aspects of the multisystem TS phenotype. However, the

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5  Giudicessi and Ackerman  Calcium and LQTS apparent lack of neurodevelopmental delay and other extracardiac manifestations in the 2 CACNA1C-G402S–positive TS2 cases,28,29 despite the fact that CACNA1C-G402S and CACNA1C-G406R result in similar increases in Ca2+ influx in vitro, suggests that perturbation of Ca2+-independent processes may also contribute to multiorgan dysfunction in TS. Consistent with this theory, Krey et al45 demonstrated that TS1 iPSC-derived neurons displayed marked impairment of dendrite formation, a common feature of neurodevelopmental/autism spectrum disorders, independent of Ca2+ influx through mutant TS1-Cav1.2 channels. Based on an elegant series of experiments, Krey et al45 propose that conformational changes in TS1-Cav1.2 channels, unrelated to their Ca2+-permeating properties, lead to decreased binding/recruitment of Gem, a guanosine triphosphate-binding protein, to the Cav1.2 macromolecular complex which then precipitates the loss of RhoA GTPase inhibition, aberrant cytoskeletal remodeling, and ultimately dendritic retraction in TS1 iPSC-derived neurons. As such, there is ample evidence to suggest that electrophysiological perturbations in L-type calcium current (ICaL)33,40 underlie the proarrhythmic component of the TS cardiac phenotype, whereas disruption of downstream Ca2+-dependent (eg, calcineurin/nuclear factor of activated T cells)44 and Ca2+-independent (eg, Gem/ RhoA/ROCK)45 signaling cascades may underlie many of the extracardiac manifestations. Lastly, our current understanding of the molecular basis of TS is further complicated by the recent identification of 3 additional CACNA1C mutations (CACNA1C-G402R, -I1166T, and -A1473G), including 2 mutations that reside outside of exon 8/8a, in patients with TS-like phenotypes (Figure 3).46–48 Interestingly, all putative TS-causative mutations described to date localize to the hydrophobic residuerich boundaries between the last transmembrane segment (S6) and interdomain linkers of their respective Cav1.2 domains/ repeats (Figure 3), suggesting that these regions may contribute to a key tertiary/quaternary structure(s) within the Cav1.2 channel that regulate gating kinetics and function as a scaffold for the interaction/binding of downstream signaling proteins. At present, it remains to be seen if the novel phenotypes (eg, hypotonia, joint hyperflexibility/contractures, clinodactyly, etc)46,47 associated with these recently described TS-like cases represent true genotype–phenotype correlations secondary to unique electrophysiological perturbations (eg, increased ICaL window current with CACNA1C-I1166T)47 or are simply the result of variable expressivity. Regardless, these findings suggest that expanded CACNA1C sequencing should be considered for any patient with a TS-like phenotype and previous negative exon 8/8a targeted screening.

Nonsyndromic Long-QT Syndrome: Evolving Role of the L-Type Ca2+ Channel In 2013, a combination of whole-exome sequencing, gene prioritization, and candidate-based screening, unearthed 4 novel putative disease-causative missense mutations in CACNA1C in LQTS patients with isolated QT prolongation devoid of any congenital heart defects or extracardiac manifestations that define TS clinically.5 Interestingly, 3 out of 4 LQTS-causative CACNA1C mutations (K834E, P857L, and P857R) localized

to the PEST (proline [P]-, glutamic acid [E]-, serine [S]-, and threonine [T]-rich) domain of Cav1.2’s II-III linker (Figure IA in the Data Supplement) that is believed to serve as a proteolytic signal peptide that when unmasked marks the LTCC for rapid degradation via calpain-mediated and ubiquitin-proteasome–mediated mechanisms.5,49–51 Given the previous observation that deletion of the Cav1.2 LTCC’s 2 PEST domains (PEST1 in the I-II linker and the aforementioned PEST3 in the II-III linker) individually increased the stability and current density of heterologously expressed Cav1.2 channels,50 it is not entirely surprising that in vitro functional characterization of P857R-CACNA1C revealed a significant increase in both Cav1.2 cell surface expression and peak current density without significantly altering Cav1.2 channel kinetics.5 Collectively, these findings suggest that rare CACNA1C mutations localizing to the Cav1.2 PEST3 domain may disrupt normal PEST3-mediated Cav1.2 degradation and impart a biogenic ICaL gain of function that manifests clinically as an isolated LQTS phenotype without any other cardiac or extracardiac abnormalities (CACNA1C-LQTS). After the initial whole-exome sequencing–aided discovery by Boczek et al,5 screening of CACNA1C in 2 independent LQTS cohorts identified 8 additional LQTS-causative mutations shown to impart an ICaL gain of function in vitro through a variety of electrophysiological mechanisms (Figure 3).6,7 In addition, subsequent screening of genotypenegative LQTS individuals with documented echocardiographic evidence of hypertrophic cardiomyopathy and a family history of hypertrophic cardiomyopathy–like phenotypes led to the identification of a novel genetic hotspot in the distal portion of the Cav1.2 I-II linker (CACNA1C exon 12) responsible for a newly described clinical entity, cardiac-only TS (COTS), characterized by the concomitant but variably expressed phenotypes of LQTS, hypertrophic cardiomyopathy, congenital heart defects, and SCD in the absence of any extracardiac symptoms (Figure 3).52 With these discoveries, the initial hypothesis that TS and CACNA1C-LQTS may be differentiated by distinct electrophysiological mechanisms was turned on its head as several of the newly discovered CACNA1C-LQTS/COTS mutations imparted an ICaL gain of function via the slowing of VDI analogous to the effect observed for the TS1- and TS2-causative CACNA1C mutations.6,7 Furthermore, in silico action potential modeling predicted that many CACNA1C-LQTS mutations result in a more pronounced ICaL gain of function (AP duration prolongation and increased cytosolic Ca2+ concentration) than the canonical TS exon 8a G406R-CACNA1C mutation when the estimated percentage of affected Cav1.2 transcripts are accounted for.7 These findings suggest that the stark phenotypic differences between TS, COTS, and CACNA1CLQTS cannot be explained by distinct electrophysiological mechanisms readily appreciated in heterologous expression systems, the degree of increased cytosolic Ca2+ influx, or mutation localization to specific Cav1.2 domains such as the S6/interdomain linker boundaries as summarized in Figure 3. As such, the extracardiac manifestations of TS syndrome and the cardiac hypertrophy/congenital heart defects observed in TS and COTS may be (1) independent of the Ca2+-permeating

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6  Giudicessi and Ackerman  Calcium and LQTS properties of mutant Cav1.2 channels (eg, perturbation of aforementioned Ca2+-dependent (calcineurin/nuclear factor of activated T cells) or Ca2+-independent (GEM/RhoA/ ROCK) signaling cascades that culminate in aberrant gene expression) and (2) modulated by as of yet unrecognized genetic/epigenetic mechanisms (eg, interindividual variation in splicing patterns, auxiliary subunit expression/function, or regulatory noncoding RNAs).53

Calmodulinopathic LQTS: Impaired LTCC Ca2+-Dependent Inactivation Over the past several years, independent whole-exome sequencing studies by Crotti et al8, Makita et al54, and Reed et al9 linked heterozygous sporadic/de novo mutations in the identical CALM1-, CALM2-, and CALM3encoded CaM, a ubiquitous Ca2+ sensor essential for the Ca2+-dependent regulation of an array of intracellular processes, to what was described initially as a rare multisystem disorder characterized by marked QT prolongation and recurrent cardiac arrest (often in infancy) accompanied by variably expressed congenital heart defects, seizures, and neurodevelopmental delays. However, the recent description of CALM2-positive cases with a milder phenotype consisting of later onset cardiac events (>1 year of life) in the absence of discernible neurodevelopmental delay,54 raises the possibility that the phenotype initially observed in calmodulinopathic LQTS8 may (1) depend on the severity of the Ca2+ binding impairment/reduction in Ca2+ affinity conferred by mutant CaMs, (2) occur secondary to the poorly understood effects of the differential temporospatial/tissue-specific expression of the 3 CALM genes (in the human heart CALM1, CALM2, and CALM3 is expressed at ≈1:2:5 ratio),8 and (3) represent neurological sequelae of recurrent cardiac arrests early in life rather than an intrinsic manifestation of CaM mutations. Interestingly, all LQTS-causative CALM1-3 mutations described to date localize at or immediately proximal to Ca2+coordinating residues of the C-terminal lobe (C-lobe) of CaM (Figure 4A) and impart a marked reduction in Ca2+-binding affinity.8,9,54 Despite the fact that CaM regulates a number of cardiac ion channels, the predominant effect of LQTScausative CALM1-3 mutations seems to be the loss of CDI resulting in unrestrained Ca2+ influx as the effects on RyR2 (reduced CaM binding) and Nav1.5 (increased INa late current) were mild and widely variable.55–58 In contrast, CaM mutations that produce a predominantly catecholaminergic ventricular tachycardia phenotype localize to both the N-terminal-lobe (N-lobe; N54I) and C-lobe (N98S) and increase RyR2-binding affinity/single channel open probability and the incidence of spontaneous Ca2+ release from the SR with little to no effect on Ca2+ binding affinity (Figure 4A).57,58 The fact that all LQTS-causative CaM mutations, including those that yield a LQTS/catecholaminergic ventricular tachycardia overlap phenotype (N98S, D132E, and Q136V), localize to the CaM C-lobe raises the possibility of a functional bipartition whereby the CaM N- and C-lobes are responsible for different sets of cellular functions. One example of partitioned CaM function potentially pertinent to the pathogenesis of calmodulinopathic LQTS is the presence of a dual-phase

CDI unique to LTCCs (Figure 4B). In comparison to other voltage-gated calcium channel classes where the lower Ca2+ affinity N-lobe mediates CDI and the higher Ca2+ affinity C-lobe either potentiates Ca2+ entry during Ca2+-dependent facilitation or plays no role in Ca2+-dependent regulation,59,60 the LTCCs’ N-lobe and C-lobe underlie distinct slow and rapid components of CDI, respectively (Figure 4B).61 As such, the localization and function of LQTS-causative CALM mutations as well as modest effects on RyR2 or Nav1.5 suggest that calmodulinopathic LQTS primarily arises from the impairment of C-lobe–mediated rapid CDI (Figure 4C). However, given the ubiquitous nature of CaM, significant functional redundancy, and vast number of intracellular CaM targets, without more in-depth studies, it remains to be seen if more pervasive defects in ion channel regulation (eg, K+ channels, other voltage-gated Ca2+ channels, etc) and CaMdependent signaling cascades contribute to the molecular basis of calmodulinopathic LQTS.

Triadin Knockout Syndrome: Remodeling of the Calcium Release Unit Molecular Architecture Most recently, Altmann et al10 described a rare autosomalrecessive form of LQTS characterized by transient/consistent QT prolongation with extensive precordial (V1 through V4) T-wave inversions, severe and often β-blocker- and left cardiac sympathetic denervation-refractory exercise-induced cardiac events (eg, syncope, sudden cardiac arrest, and SCD) in early childhood, and mild-to-moderate proximal skeletal muscle weakness that arises secondary to either homozygous or compound heterozygous frameshift/null mutations in TRDN-encoded triadin, a key structural component of the cardiac release unit (Figure 5A). Although no functional studies were undertaken with the initial clinical and genetic description of Triadin knockout (TKO) syndrome, insights gleamed from ventricular arrhythmia-prone TRDN-null mice suggest that complete ablation of triadin, as would be expected in TKO syndrome patients, significantly (1) disrupts the approximation of the T-tubule and the junctional SR within the cardiac dyad, (2) reduces the expression of key junctional SR proteins including RyR2, calsequestrin 2, and junctin, and (3) reduces the colocalization/juxtaposition of Cav1.2/RyR2 and RyR2/Calsequestrin2 in the cardiac release unit (Figure 5B).62 This radical remodeling of the cardiac release unit molecular architecture reduces SR Ca2+ release leading to impaired Cav1.2 LTCC CDI, unrestrained cytosolic Ca2+ influx via ICaL, and subsequent SR Ca2+ overload (Figure 5B).62–64 Interestingly, in TRDN-null myocytes, Cav1.2 activation and inactivation remain slower even when Ba2+, which does not trigger SR Ca2+ release or Cav1.2 CDI, is used as the charge carrier, suggesting that the loss of Triadin intrinsically alters Cav1.2 gating by as of yet undefined mechanisms independent of SR Ca2+-release–triggered Cav1.2 CDI.62,65 Collectively, the misappropriation of calsequestrin 2 in the SR,62,66 SR Ca2+ overload,62 and reduction in junctin-mediated RyR2 inhibition at high SR luminal Ca2+ concentrations67 that arise secondary to remodeling of cardiac release unit molecular architecture in TRDN-null

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Figure 4. Molecular basis of calmodulinopathic long-QT syndrome (LQTS). A, The location of LQTS (maroon solid circles), catecholaminergic ventricular tachycardia (CPVT; green solid circles), and LQTS/CPVT overlap (green/maroon dashed circles)-causative CALM1, CALM2, and CALM3 mutations in the identical calmodulin linear protein topology. B, Schematic model for dual-phase L-type calcium channel (LTCC) calcium-dependent inactivation (CDI). The amino (N)- and carboxy (C)-terminal lobes of calcium-free calmodulin (apoCaM) preassociate with the LTCC carboxy (C)-terminal IQ motif–containing calcium inactivation region. After activation of the LTCC, binding of calcium to calmodulin (Ca2+/CaM) induces a conformational shift that allows the N-lobe and C-lobe to bind the N-terminal spatial Ca2+-transforming element (NSCaTE) and C-terminal IQ motif, respectively, leading to CDI. C, Schematic model for perturbed LTCC dual-phase calciumdependent inactivation in calmodulinopathic LQTS. LQTS-causative CALM mutations result in loss of calcium-binding affinity to the C-lobe of calmodulin and reduce/abolish the rapid component of CDI leading to intracellular calcium overload.

mice is hypothesized to increase the frequency of spontaneous ectopic Ca2+ release from the SR leading to proarrhythmic DADs, particularly in the setting of β-adrenergic stimulation.62,65 In addition, the slowed Cav1.2 inactivation observed in TRDN-null mice theoretically could prolong action potential duration and create an electric substrate favorable for the generation of early after-depolarization– triggered ventricular arrhythmias, the predominant arrhythmogenic mechanism in most LQTS subtypes. Thus, in TKO syndrome, calmodulinopathic LQTS, and to a lesser extent in non-Ca2+-mediated LQTS subtypes (eg, INamediated LQT3) the perturbation/modulation of a myriad of Ca2+-dependent events, including disrupted Cav1.2 kinetics/enhanced ICa,L current, increased spontaneous SR Ca2+ release via RyR2, and promotion of SR Ca2+ loading via NCX, likely contribute to an underlying proarrhythmic electrophysiological substrate capable of triggering DAD- and

early after-depolarization–mediated arrhythmias.55,56,62,68 Although it is not clear whether a DAD- or early afterdepolarization–mediated mechanism is predominantly responsible for the ventricular arrhythmias observed in TRDN-null mice, treatment of TRDN-null myocytes with the DHP Ca2+ channel blocker nifedipine abolished SR Ca2+ overload and spontaneous SR Ca2+ release, suggesting that DHPs and other class IV antiarrhythmics may one day play a role in the treatment of patients with TKO syndrome.10,62 Ultimately, these observations in TRDN-null mice function to substantiate the hypothesized molecular basis of TKO syndrome in humans and provide a reasonable launching point for future inquiries aimed at elucidating the precise arrhythmogenic mechanism(s) that underlie the severe medically refractory adrenergically mediated cardiac events that plague TKO syndrome patients with the hope that these insights will catalyze novel therapeutic strategies.

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Figure 5. Molecular basis of triadin knockout (TKO) syndrome. A, Artistic rendering of the molecular architecture of a normally functioning cardiac dyad composed of closely juxtaposed Cav1.2 L-type calcium channels (LTCC) and ryanodine receptor-2 (RyR2) intracellular calcium release channels and appropriate sequestration of calcium in the sarcoplasmic reticulum (SR) as a result of the appropriate expression/function of triadin, junctin, junctophilin-2, and calsequestrin in calcium release units (CRUs). B, Artistic rendering of the remodeled cardiac dyad molecular architecture in TKO syndrome and the resulting Ca2+ overload that occurs secondary to the loss of Cav1.2 LTCC and RyR2 juxtaposition, normal triadin, junctin, and junctophilin expression/function, and proper calcium/calsequestrin localization in the SR based on observations in triadin-null mice.

Modulation of Cardiac Repolarization by Ca2+ Handling Proteins in Health In recent years, the association of QT prolongation/shortening with an increased risk of SCD in the general population69–71 and the barrier that drug-induced QT prolongation has presented to new drug development has served as an impetus for QT interval genome-wide association studies in several large population-based cohorts such as the Framingham Heart Study and the Cardiovascular Heart Study. Beginning in 2009, 3 large genome-wide association study meta-analyses, including the mammoth QT-IGC consortium expanded genomewide association study meta-analysis of 76  061 individuals of European descent,11 built on existing single cohort studies by collectively demonstrating that common variants in 6 established (NOS1AP, KCNQ1, KCNE1, KCNH2, SCN5A, and KCNJ2) and 29 novel genetic loci collectively explain ≈8% to 10% of heritable QT interval variability.11,72,73 Interestingly, a surprising number of these novel genetic loci localize within or near genes that encode intracellular Ca2+-handling proteins, including ATP2A2-encoded SR ATPase 2a calcium pump, SERCA-regulator PLN-encoded phospholamban, and the SLC8A1-encoded NCX1 Na+/Ca2+ exchanger whose roles in excitation–contraction coupling were discussed previously and are summarized in Figure 1.11 Furthermore, mutational analysis of the 6 genes (ATPA2A2, CAV1, CAV2, SLC8A1, SRL, and TRPM7), in closest proximity

to the novel genetic loci with the strongest statistical significance and highest biological plausibility in an international cohort of 298 unrelated LQT1-LQT3 negative LQTS probands, identified potentially pathogenic frameshift mutations in ATP2A2-encoded SR ATPase 2a and the TRPM7-encoded transient receptor channel melastatin 7 Mg2+/Ca2+ channel known to mediate Ca2+ influx and regulate expression of other Ca2+-handling proteins.11 Although much work remains to fully understand the physiological ramifications of common and rare genetic variation in these newly identified genetic loci, the above studies highlight a larger than anticipated role for Ca2+ flux in cardiac repolarization/modulation of QT interval in health and hint at a potentially even greater role for Ca2+-handling proteins in the pathogenesis of congenital and acquired LQTS.

Concluding Remarks The discovery and subsequent investigation of rare and common genetic variation in Ca2+-handling proteins has illuminated a previously underappreciated and rapidly expanding role for Ca2+ signaling in cardiac repolarization and the pathogenesis of nonsyndromic and multisystem forms of congenital LQTS. Undoubtedly, these discoveries will provide a rich foundation for future inquiries, aided by the continued use of next-generation sequencing technologies and patient-specific iPSC-derived cardiomyocytes, aimed at (1) determining the

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9  Giudicessi and Ackerman  Calcium and LQTS genetic substrates that underlie the roughly 15% to 20% of LQTS cases that remain genetically elusive, (2) elucidating the novel genetic/epigenetic determinants that underlie the variable phenotypic expressivity observed in most LQTS subtypes and the dramatic phenotypic divergences observed between CACNA1C-mediated TS, CACNA1C-mediated COTS, and CACNA1C-LQTS, (3) developing a deeper understanding of the pathophysiological mechanisms of LQTS-causative mutations in Ca2+-handling proteins in hopes that such insights will lead to the development of individualized, genotype-guided therapeutic strategies with improved efficacy and safety profiles, and (4) further investigating the potential contribution of an increased burden of common genetic variation in Ca2+handling proteins to the pathogenesis of drug-induced LQTS. Because we continue to revisit the role of Ca2+ in cardiac repolarization and the pathogenesis of nonsyndromic and multisystem forms of LQTS, there is renewed hope that insights gleamed from these efforts will lead to the development of novel approaches to the diagnosis, risk stratification, and treatment of LQTS patients, particularly those with high-risk LQTS subtypes such as TS, calmodulinopathic LQTS, and TKO syndrome who are among those at greatest risk of succumbing to potentially lethal ventricular arrhythmias.

Sources of Funding This work was supported by the Windland Smith Rice Sudden Comprehensive Sudden Cardiac Death Program (to Dr Ackerman). Dr Giudicessi thanks the Mayo Clinic Internal Medicine Residency and Clinician Investigator Training Programs for fostering an outstanding environment for physician-scientist training.

Disclosures Dr Ackerman is a consultant for Boston Scientific, Gilead Sciences, Medtronic, and St. Jude Medical. Dr Ackerman and Mayo Clinic receive sales-based royalties from Transgenomic for their FAMILIONLQTS and FAMILION-CPVT genetic tests. However, none of these entities participated in this study. The other authors reports no conflicts.

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Calcium Revisited: New Insights Into the Molecular Basis of Long-QT Syndrome John R. Giudicessi and Michael J. Ackerman Circ Arrhythm Electrophysiol. 2016;9: doi: 10.1161/CIRCEP.116.002480 Circulation: Arrhythmia and Electrophysiology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2016 American Heart Association, Inc. All rights reserved. Print ISSN: 1941-3149. Online ISSN: 1941-3084

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SUPPLEMENTAL MATERIAL Genetics and Structure of the L-Type Ca2+ Channel Macromolecular Complex Although three types of voltage-gated calcium channels, including the CACNA1G-encoded nodal T-type Cav 3.1 and the CACNA1A-encoded neuronal P/Q-type Cav 2.1 pore-forming α1-subunits, are expressed in the human heart, the CACNA1C-encoded Cav 1.2 α1-subunit, which conducts the “long lasting” L-type Ca2+ current (ICaL), represents the most prevalent and only class of voltagegated calcium channels currently linked to the pathogenesis of LQTS or any other heritable arrhythmia syndrome.1,

2

Human CACNA1C spans >500 kb of chromosome 12 and is comprised of 55 exons, of which 19 are subject to alternative splicing, providing the theoretical substrate for the generation of a staggering number of Cav 1.2 splice variants each with the potential to impart their own unique functional consequences.3 Interestingly, those Cav 1.2 splice variants studied to date display significant differences in channel kinetics3-6 , dihydropyridine affinity7, 8 , and regulatory properties5,

9

that explain many of the functional characteristics that differentiate smooth muscle-

and cardiac-specific Cav 1.2 α1-subunit isoforms (Supplemental Table 2). Although the adult cardiac Cav 1.2 reference isoform is defined traditionally as a combination of the presence of mutually exclusive exons 1a, 8, and 32 along with the absence of 75 nucleotides following exon 910,

11

, quantitative analysis of mRNA expression in human hearts demonstrated substantial inter-

individual variation in overall CACNA1C expression levels and splicing patterns across the majority of the 12 identified splicing loci (Supplemental Table 2).12 Collectively, these findings suggest that variable splicing patterns can influence profoundly Cav 1.2 function and that the electrophysiological and clinical phenotypes associated with CACNA1C mutations may depend

on the Cav 1.2 splice variant(s) in which they reside as well as the degree to which particular Cav 1.2 splice variants are expressed in a given host. Structurally, the ~220kDa Cav 1.2 α1-subunit contains ~2100 amino acids divided into four structural repeats (domains I-IV) each composed of six helical transmembrane domains (S1S6) linked by three intracellular loops that collectively form a pseudo-tetrameric structure akin to voltage-gated sodium channels (Supplemental Figure 1a).10 In addition, several intracellular Cav 1.2 structural domains play a critical role in optimizing channel activity. First, the I-II loop linker α-interaction domain (AID) facilitates β2 subunit binding that enhances Cav 1.2 trafficking and modulates voltage-dependent inactivation (VDI).13 Secondly, the amino (N)-terminal spatial Ca2+-transforming element14 and the carboxy (C)-terminal “IQ”-motif15,

16

containing Ca2+-

inactivation region bind calmodulin (CaM) and mediate calcium-dependent inactivation (CDI), a crucial negative feedback mechanism for the prevention of intracellular Ca 2+ overload. Thirdly, the C-terminus also facilitates β-adrenergic modulation via the binding of the A kinase anchor protein 15 (AKAP15)/protein kinase A (PKA) complex to atypical leucine zipper motifs 17 referred to as the AKAP binding domain (ABD). Lastly, the far distal portion of C-terminus is cleaved proteolytically in vivo to generate an auto-inhibitory peptide containing prominent PKA phosphorylation sites and the ABD, which significantly reduces channel activity/voltagedependent activation by non-covalently binding to the truncated Cav 1.2 channels (Supplemental Figure 1a). Like most cardiac voltage-gated ion channels, the optimum function and regulation of Cav 1.2 depends on a macromolecular complex composed of multiple auxiliary proteins (Supplemental Figure 1b). At present, the Cav 1.2 α1-subunit, CACNA2D1-encoded α2 δsubunit, and CACNB2-encoded β2 -subunit, assembled in a 1:1:1 ratio, is necessary and sufficient

to recapitulate the ICaL current in vitro (Supplemental Figure 1b).18 Functionally, the α2 δsubunit, which is comprised of two disulfide-linked proteins (α2 and δ) derived from a single post-translationally cleaved

transcript/precursor protein19 ,

activation and inactivation20,

21

increases the speed of Cav 1.2

, whereas the aforementioned β2 -subunit predominantly promotes

trafficking of Cav 1.2 to the cell surface13 . As such, both the α2 δ and β2 subunits function to increase ICaL current density, albeit by distinct mechanisms. The role of a third class of auxiliary proteins, the CACNG4, 6, 7, and 8-encoded transmembrane-spanning glycoprotein γ subunits, in the heart remains unclear. However, the recent discovery of γ subunits in cardiac tissue coupled with in vitro evidence that γ subunits modulate Cav 2.1 voltage-dependence in a β-subunit dependent fashion suggest that contrary to popular belief, γ subunits may contribute to the function of the cardiac Cav 2.1 macromolecular complex in vivo.22 Genetics and Structure of RyR2 Intracellular Ca 2+ Release Channel Macromolecular Complex Currently, three mammalian ryanodine receptor isoforms (RyR1-3) have been identified, but only RyR2 is expressed at appreciable levels in the human heart.23 Although direct perturbation of RyR2 function is not linked currently to LQTS pathogenesis, defective RyR2 function predominantly results in catecholaminergic polymorphic ventricular tachycardia (CPVT), but is also

anecdotally linked

cardiomyopathy,

to

a trio of cardiomyopathies [arrhythmogenic right ventricular

hypertrophic

cardiomyopathy

cardiomyopathy] reviewed in detail elsewhere. 1,

(HCM),

and

nonischemic

dilated

24, 25

Structurally, the RYR2 gene encodes a single 560kDa monomer comprised of a vast cytosolic N-terminal domain connected to a smaller C-terminal domain by six SR-spanning transmembrane domains that assemble to form the massive homo-tetrameric RyR2 channel

(Supplemental Figure 1c). Similar to the LTCC, the RyR2 channel functions as a macromolecular complex dependent on an array of cytoplasmic and luminal SR auxiliary interacting proteins for proper function. The cytoplasmic N-terminal domain serves as a binding site-containing scaffold for a multitude of auxiliary interacting proteins that modulate Ca2+release from the C-terminal pore region including the channel-stabilizing/tetramer-coupling FK506-binding protein 12.6 (FKBP12.6)/calstabin 1 26 , Ca2+-dependent Ca2+ release inhibiting CaM27 , and Ca2+-release inhibiting sorcin28 (Supplemental Figure 1d). Furthermore, the Nterminal domain also contains three highly conserved leucine/isoleucine zipper (LIZ) motifs that provide binding sites for phosphatases [protein phosphatases 1 (PP1) and 2A (PP2A)] 29 and PKA30 as well as Ca2+/CaM-dependent protein kinase II (CaMKII) phosphorylation sites31 that augment the probability the RyR2 channel assumes the open conformation thereby increasing (PKA and CAMKII) or decreasing (PP1 and PP2A) channel sensitivity to Ca2+-dependent activation (Supplemental Figure 1d). Within the SR lumen, RyR2 activity is modulated in response to luminal Ca2+ concentrations by a complex consisting of the low affinity Ca 2+ storage protein, calsequestrin2, which is encoded by CASQ2 and three structural SR-spanning transmembrane proteins, TRDNencoded triadin, JCN-encoded junctin, and JPH2-encoded junctophilin-2, which maintain the structural and functional integrity of the LTCC-RyR2 calcium release unit [CRU,(Supplemental Figure 1d)].32,

33

When SR luminal Ca2+ concentrations are high, calsequestrin2 oligomerizes to

function as a Ca2+ buffer. However, as RyR2 opens and SR luminal Ca 2+ concentrations decline, calsequestrin2 reverts to its monomeric form and via an interaction with triadin inhibits RyR2 activity contributing to the termination of Ca2+ release that occurs with each heartbeat. 34 A summary of the cytoplasmic and luminal RyR2 auxiliary interacting proteins is depicted visually

in Supplemental Figure S1 and the role of PKA, CaMKII, and other regulatory pathways in the regulation of RyR2 and Cav 1.2 during EC coupling are reviewed in more complete detail elsewhere.1

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SUPPLEMENTAL TABLES Supplemental Table 1 | Genetic basis of non-syndromic and multisystem long QT syndrome subtypes. Gene (Genotype)

Acronym

OMIM

Protein

Functional Effect

Mode of Inheritance

Frequency

Ref.

LQT1 LQT2 LQT3

192500 613688 603830

Kv7.1 Kv11.1 Nav1.5

Reduced IKs Reduced IKr Increased INa

AD; AR AD AD

~30-35% ~25-30% ~5-10%

35, 36

LQT11 LQT9 LQT5 LQT6 LQT13 LQT10 LQT12 LQT8

611820 611818 613695 613693 613485 611819 612955 N/A

Yotiao Caveolin 3 M inK M iRP1 Kir3.4 Nav1.5 4-subunit Syntrophin-1 Cav1.2

Reduced IKs Increased INa Reduced IKs Reduced IKr Reduced IK,Ach Increased INa Increased INa Increased ICa,L

AD AD AD AD AD AD AD AD