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(Circulation. 2007;116:2325-2345.)
© 2007 American Heart Association, Inc.

Basic Science for Clinicians

Inherited Arrhythmias

A National Heart, Lung, and Blood Institute and Office of Rare Diseases Workshop Consensus Report About the Diagnosis, Phenotyping, Molecular Mechanisms, and Therapeutic Approaches for Primary Cardiomyopathies of Gene Mutations Affecting Ion Channel Function

Stephan E. Lehnart, MD, PhD; Michael J. Ackerman, MD, PhD; D. Woodrow Benson, Jr, MD, PhD; Ramon Brugada, MD; Colleen E. Clancy, PhD; J. Kevin Donahue, MD; Alfred L. George, Jr, MD; Augustus O. Grant, MD, PhD; Stephen C. Groft, PharmD; Craig T. January, MD, PhD; David A. Lathrop, PhD; W. Jonathan Lederer, MD, PhD; Jonathan C. Makielski, MD; Peter J. Mohler, PhD; Arthur Moss, MD; Jeanne M. Nerbonne, PhD; Timothy M. Olson, MD; Dennis A. Przywara, PhD; Jeffrey A. Towbin, MD; Lan-Hsiang Wang, PhD; Andrew R. Marks, MD

From the Departments of Physiology and Cellular Biophysics, Clyde and Helen Wu Center for Molecular Cardiology (S.E.L., A.R.M.), and Medicine (A.R.M.), College of Physicians and Surgeons of Columbia University, New York, NY; Molecular Pharmacology and Experimental Therapeutics (M.J.A.), Medicine/Division of Cardiovascular Diseases (M.J.A.), and Pediatric and Adolescent Medicine/Division of Pediatric Cardiology (M.J.A.), Mayo Clinic, Rochester, Minn; Divisions of Cardiology and Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio (D.W.B.); Montréal Heart Institute and University of Montreal Clinical Cardiovascular Genetics Center, Montréal, Québec, Canada (R.B.); Physiology and Biophysics, Institute for Computational Biomedicine, Weill Medical College of Cornell University, New York, NY (C.E.C.); Heart and Vascular Research Center, MetroHealth Hospital, Case Western Reserve University, Cleveland, Ohio (J.K.D.); Medicine, Division of Genetic Medicine, Vanderbilt University, Nashville, Tenn (A.L.G.); Medicine, Duke University, Durham, NC (A.O.G.); Office of Rare Diseases at the National Institutes of Health, Bethesda, Md (S.C.G.); Section of Cardiology, University of Wisconsin Hospital and Clinics, Madison (C.T.J.); Division of Cardiovascular Diseases, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md (D.A.L., D.A.P., L.W.); Medical Biotechnology Center, University of Maryland Biotechnology Institute, and Physiology, University of Maryland, Baltimore (W.J.L.); Medicine, Cardiovascular Medicine Section, University of Wisconsin, Madison (J.C.M.); Internal Medicine, Division of Cardiology, and Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City (P.J.M.); Medicine, Cardiology Division, University of Rochester Medical Center, Rochester, NY (A.M.); Center for Cardiovascular Research and Molecular Biology and Pharmacology, Washington University School of Medicine, St Louis, Mo (J.M.N.); Cardiovascular Genetics Laboratory, Division of Cardiovascular Diseases, Mayo Clinic, Rochester, Minn (T.M.O.); and Pediatrics (Cardiology), Baylor College of Medicine, Texas Children’s Hospital, Houston (J.A.T.).

Correspondence to Andrew R. Marks, MD, (e-mail arm42{at}columbia.edu) or Stephan E. Lehnart, MD, PhD (e-mail sel2004@columbia.edu), Department of Physiology and Cellular Biophysics, Clyde and Helen Wu Center for Molecular Cardiology, and Department of Medicine, College of Physicians and Surgeons of Columbia University, P&S 9-401 box 22, 630 W 168 St, New York, NY 10032.

	Abstract

Top Abstract Introduction Cardiac Na+ Channelopathies References

 
The National Heart, Lung, and Blood Institute and Office of Rare Diseases at the National Institutes of Health organized a workshop (September 14 to 15, 2006, in Bethesda, Md) to advise on new research directions needed for improved identification and treatment of rare inherited arrhythmias. These included the following: (1) Na⁺ channelopathies; (2) arrhythmias due to K⁺ channel mutations; and (3) arrhythmias due to other inherited arrhythmogenic mechanisms. Another major goal was to provide recommendations to support, enable, or facilitate research to improve future diagnosis and management of inherited arrhythmias. Classifications of electric heart diseases have proved to be exceedingly complex and in many respects contradictory. A new contemporary and rigorous classification of arrhythmogenic cardiomyopathies is proposed. This consensus report provides an important framework and overview to this increasingly heterogeneous group of primary cardiac membrane channel diseases. Of particular note, the present classification scheme recognizes the rapid evolution of molecular biology and novel therapeutic approaches in cardiology, as well as the introduction of many recently described diseases, and is unique in that it incorporates ion channelopathies as a primary cardiomyopathy in consensus with a recent American Heart Association Scientific Statement.

Key Words: arrhythmia • cardiomyopathies • death, sudden • electrophysiology • genetics • ion channels • long-QT syndrome

	Introduction

Top Abstract Introduction Cardiac Na+ Channelopathies References

 
The National Heart, Lung, and Blood Institute and Office of Rare Diseases at the National Institutes of Health organized a workshop to advise on new research directions needed for improved identification and treatment of rare inherited arrhythmias. During the workshop, current levels of understanding and gaps in knowledge of "rare inherited arrhythmias" were presented in 3 areas: (1) inherited channelopathies; (2) other inherited arrhythmias; and (3) implications for the future diagnosis and management of inherited arrhythmias. Elucidation of the basis of genetic diseases provides unique insights into the mechanisms responsible for more prevalent arrhythmias and sudden cardiac arrest (SCA) while also permitting the identification of therapeutic opportunities for treatment and prevention. From screening of larger patient cohorts, it has become clear that environmental and genetic modifiers play important roles for arrhythmia susceptibility and severity. Study of distinct forms of inherited arrhythmias requires evaluation in specific models to make accurate predictions of human pathophysiology. Appropriate in vivo arrhythmia models may vary from genetically engineered mouse or rabbit models to larger-animal models that more closely mimic human electrophysiological substrates. Workshop participants recommended multilevel translational approaches to identify and validate gene variations associated with arrhythmogenesis. Such approaches should span development of molecular and biophysical structure-function relationships and cellular mechanisms to examinations of organ and in vivo models. Integrated analysis by modeling approaches may aid in elucidation of arrhythmia mechanisms.

Opportunities to develop novel therapeutic approaches require comprehensive studies that characterize ion channel function and expression, regulation by intracellular signaling complexes, alterations in and cross talk of intracellular Ca²⁺ signals, and therapeutic interventions aimed at specific molecular defects. Future therapeutic and diagnostic approaches should include identification of novel surrogate clinical markers. It is also vital that training mechanisms be supported for young investigators in an integrated range of disciplines (including biology, medicine, computer modeling, bioengineering, information sciences) while providing scientific career opportunities.

Specific recommendations to the National Heart, Lung, and Blood Institute and Office of Rare Diseases to support, enable, or facilitate research in areas of key importance for the future understanding and treatment of inherited arrhythmic syndromes were as follows:

• Establish biological and computational models that appropriately reflect the molecular environment of the human heart and permit study of the functional effects of arrhythmogenic mutations. Biological models, including human stem cell–derived cardiomyocytes and genetically altered model organisms, should enhance the study of normal and mutant ion channel biosynthesis, assembly, macromolecular complexes, posttranslational regulation, trafficking, targeting (functional proteomics), and human arrhythmia mechanisms.
  
• Elucidate the molecular and physiological basis of arrhythmia triggers and substrates with the use of integrative animal, cell, and computational models.
  
• Identify new human arrhythmia-susceptibility genes, modifiers, and the mechanisms of their effects. Design and utilize more efficient (higher throughput) molecular, cellular, and integrative approaches to advance our understanding of Genotype–Phenotype interactions.
  
• Develop, test, and implement new therapeutic approaches to identify, treat, and prevent inherited arrhythmias based on genetic, molecular, and cellular mechanisms.
  
• Establish methods (including bioinformatics) to evaluate, integrate, and share structure-function genotype–phenotype relationships at the gene, protein, signaling complex, cell, organ, and in vivo levels to allow better prediction of the significance of specific genetic variants for arrhythmogenesis and to focus research efforts and therapy toward patient and family needs.
  

	Cardiac Na+ Channelopathies

Top Abstract Introduction Cardiac Na+ Channelopathies References

 
Mutations in SCN5A,the gene encoding the Na⁺ channel -subunit expressed in the human heart, cause inherited susceptibility to ventricular arrhythmias (congenital long-QT syndrome [LQTS] including prolongation of ventricular action potentials, dispersion of repolarization, QT-interval and T-wave abnormalities in surface ECG recordings [LQTS3*]; idiopathic ventricular fibrillation [VF]),^1–3 cardiac conduction disease (CoD),^4–6 and dilated cardiomyopathy (DCM) with atrial arrhythmia (Multigenetic arrhythmia syndromes are numbered sequentially to identify and discriminate specific genotype–phenotype relationships; refer to Table 1).^7,8 Mutations in SCN5A may also present with more complex phenotypes representing combinations of LQTS, CoD, and Brugada syndrome (BrS1). Examples of LQTS3 combined with either BrS1⁹ or congenital heart block,^10,11 cases of BrS1 with impaired conduction,¹² or combinations of all 3 phenotypes have been documented.¹³ Moreover, certain mutations may manifest different phenotypes in different individuals and families.

View larger version (15K): [in this window] [in a new window]   Table 1. Brugada and Related Arrhythmia Syndromes For a review of inherited conduction system abnormalities including mutations of genes not related to ion membrane transport, please see Wolf & Berul.183 Genes contributing to the same function and/or distinct phenotypes are marked similarly for ease of comparison within and between tables 1 through 5. indicates loss of function. *Sudden unexpected nocturnal death syndrome, estimated mortality rate 26–38/100,000 in young Thai men. $Idiopathic ventricular fibrillation without ECG signs of BrS1. #Lenègre-Lev disease (fibrofatty atrophy of His-Purkinje system). &Sudden Infant Death Syndrome: an estimated 10% to 15% of SIDS stems from LQTS-, BrS-, and CPVT-causing mutations. Approximately 50% of ion channel–related SIDS involves defects in SCN5A or other components of the Na+ channel macromolecular complex. +Glycerol-3-phosphate dehydrogenase 1–like gene. ¶Relative syndromic occurrence for a given genetic syndrome (in %); if not known (as indicated by ?) total numbers of published mutation carriers vary from 1 to 17.

Novel Clinical and Genetic Aspects of SCN5A Phenotypes
The voltage-gated Na⁺ channel (Na_v1.5 encoded by SCN5A), responsible for the initial upstroke of the action potential, represents an important drug target for antiarrhythmic class Ia blockers. SCN5A was mapped to chromosome 3p21, identifying it as a candidate gene for LQTS3.²⁷ Subsequently, a mutation in SCN5A was found in families with 3p21-linked LQTS3.²⁸ After linkage of SCN5A to LQTS3 and abnormal cardiac repolarization, distinct disease phenotypes, including conduction slowing, have been linked to SCN5A mutations. A subgroup of idiopathic VF patients with a distinctive ECG pattern characterized by apparent right bundle-branch block, ST-segment elevation, and sudden death was described as a new clinical entity referred to as Brugada syndrome (BrS1) (Table 1).¹⁴ Pharmacological Na⁺ channel block elicits or worsens the ECG features associated with BrS1.²⁷ Screening of families with BrS1 has revealed distinct mutations in the SCN5A gene.³ In a large Dutch family with a history of sudden death, mostly occurring at night, living members demonstrated ECG features compatible with BrS1 and LQTS3 corresponding to a novel mutation in the C-terminus of SCN5A (1795insD).¹⁹ Subsequently, a Y1795H mutation in a patient with BrS1 and a Y1795C mutation in a patient with LQTS3 were identified, providing evidence of the close interrelationship between BrS1 and LQTS3.²⁰ The effect of the 1795insD-SCN5A mutation during sinoatrial node pacemaking decreased the sinus rate by decreasing the diastolic depolarization rate, which may account for the bradycardia seen in LQTS3 patients, whereas sinoatrial node pauses or arrest may result from failure of sinoatrial nodal cells to repolarize.³⁰ In a large family with a Brugada-like phenotype, SCN5A could be excluded as a candidate gene indicating a distinct locus,¹⁵ and BrS2 was subsequently linked to GPD1L, the glycerol-3-phosphate dehydrogenase 1–like gene.²⁵ Mutations in GPD1L have also been implicated in sudden infant death syndrome (SIDS).²⁶ Sudden unexplained nocturnal death syndrome, described in southeast Asia, resembles BrS1 and has been linked to mutations in the SCN5A gene.³¹ It has also been suggested that BrS1 accounts for up to 60% of VF cases previously classified as idiopathic.³² This seems to be particularly prevalent in Southeast Asia and Japan, with mortality rates as high as 38 per 100 000,¹⁷ indicating that these arrhythmic syndromes may be much more common than previously thought.

Identification of SCN5A mutations in association with conduction system abnormalities has provided a major advance in our understanding of these disorders. Na⁺ channel mutations have also been identified in patients with atrioventricular block and sick sinus syndrome (SSS). In a kindred with progressive conduction system disease (Lenègre-Lev disease), heterozygous SCN5A mutations were identified,⁴ and additional SCN5A mutations have been associated with SSS (Table 2).^5,33

View larger version (19K): [in this window] [in a new window]   Table 2. Atrial Arrhythmia Syndromes Genes are highlighted by functional association and as given by tables 3 and 5. AT indicates atrial tachycardia; , gain of function; and , loss of function. ¶Relative syndromic occurrence has not been determined; total No. of mutation carriers published vary between 1 and 23 for any given syndrome.

Newly Recognized Phenotypes Associated With SCN5A Mutations
Genome-wide linkage analyses led to identification of an SCN5A missense mutation (D1275N) that cosegregated among 22 family members with a "novel" phenotype of DCM and atrial fibrillation (AF) (Table 1).^7,8,23,53 Variably expressed phenotypic traits included defects in impulse generation (SSS) and conduction (atrioventricular node and bundle-branch block), previously linked to loss-of-function mutations in SCN5A. Electric dysfunction (sinus bradycardia and SSS) typically preceded clinically apparent myocardial disease, and relatively slow ventricular rates in mutation carriers with AF ruled out tachycardia-induced cardiomyopathy. Subsequent mutation scanning in 156 probands with idiopathic DCM identified additional heterozygous missense (T220I, R814W, D1595H) and truncation (2550 to 2551insTG) SCN5A mutations, segregating with cardiac disease or arising de novo in 2.2% of the DCM cohort.⁷ Among probands and their relatives with an SCN5A mutation, 27% had early features of DCM (mean age at diagnosis, 20.3 years), 38% had DCM (47.9 years), and 43% had AF (27.8 years). The same T220I and D1275N substitutions were previously reported as recessive loss-of-function alleles in SSS and familial atrial standstill, respectively.^5,33 The link between cardiac Na⁺ channel loss of function and structural heart disease is supported by fibrosis and cardiac myocyte degeneration occurring both in aging heterozygous SCN5A^+/– mice and in patients with BrS1.^54,55 These observations implicate SCN5A in the pathogenesis of both electric and myopathic heart disease and support a recent American Heart Association Scientific Statement that classifies ion channelopathies as a primary cardiomyopathy.⁵⁶

Unexplained Aspects: Variable Expressivity and Penetrance
Although mutations in Na⁺ channels may lead to DCM and arrhythmias, many unanswered questions remain. For instance, is SCN5A also a disease gene for isolated AF or DCM? Earlier studies in European BrS1 patients have excluded mutation carriers with cardiomyopathy. Histology analysis of cardiac tissue from human mutation carriers or heterozygous SCN5A^+/– knockout mice has demonstrated age-dependent fibrosis in conjunction with CoD.^54,57 What are the mechanisms for development of DCM and myocardial fibrosis, and do other proteins that interact with the Na⁺ channel complex modulate phenotypic expression? Factors that underlie the chamber-specific manifestation of Na⁺ channel mutations may include interacting proteins. For instance, the dystrophin complex has been implicated in Na⁺ channel interaction and plays a key role in preserving integrity of the myocellular membrane.⁵⁸ Moreover, common polymorphisms like H558R can (1) modify the phenotype of an existing SCN5A mutation and (2) produce a decrease in depolarization reserve through the differential function seen in the 2 ubiquitous alternatively spliced transcripts present in all humans. Thus, although not sufficient to cause BrS1 by itself, a quantitatively smaller loss-of-function defect associated with positive H558R status has been shown to confer susceptibility for AF.^59,60,61 Future studies will need to characterize the relationship between the SCN5A genotype, polymorphisms, and the cardiac phenotype.

Biophysical Mechanisms of SCN5A Arrhythmias
Mechanisms contributing to arrhythmias include loss of function by synthesis of nonfunctional Na⁺ channels and mutations in functionally expressed Na⁺ channels that either increase or decrease Na⁺ current. One existing challenge is to understand the structure-function relationship of Na⁺ channels under physiological conditions and in appropriate disease models. An important property of Na⁺ channels is intrinsic modal gating, defined by the probability of a single channel residing in and switching between multiple and distinct modes. Cardiac Na⁺ channels regularly exhibit 2 modes of gating, distinguished by short and long channel open times. Whereas fast modes account for 99% of Na⁺ channel gating, heart rate–dependent slow gating components are important in pathological states.⁶² Models predict that maintained depolarization results in the passage of the channel into a series of inactivated states. In 1 in every 500 to 1000 depolarizations, inactivation fails, and channels open and shut by deactivation for several seconds. These opening bursts can contribute substantial inward current during the action potential plateau. Na⁺ channel mutations may result in incomplete inactivation during maintained depolarization,^28,63 a decrease in the level of channel expression, or acceleration of inactivation.⁶³ The resulting clinical phenotypes include LQTS3, BrS1, and heart block. LQTS3-associated Na⁺ channel mutations enhance slow gating components and increase late persistent inward current as the basis for early afterdepolarizations as triggers of ventricular tachycardia (VT)/VF, especially at slow heart rates. A delK1500 mutation in the Na⁺ channel III-IV interdomain linker identified in a family with LQTS3, BrS1, and CoD resulted in reduction of inactivation and an increased late current component.²¹ Deletion of amino acid residues 1505 to 1507 (KPQ) in the cardiac Na⁺ channel cause autosomal dominant LQTS3 and excessive prolongation of the action potential at low heart rates that predispose to development of fatal arrhythmias, typically at rest or during sleep. Mice heterozygous for a knockin KPQ deletion resemble the LQTS3 phenotype and show an increased late Na⁺ current during the plateau phase of the action potential.⁶⁴ SSS was identified as the first recessive disorder of SCN5A, and biophysical characterization of the Na⁺ channel mutants demonstrated loss of function or significant impairments in channel gating (inactivation) that predict reduced myocardial excitability.⁵

Complex Biophysical Phenotypes
Examples of LQTS3 combined with either BrS1¹⁹ or congenital heart block^65,66 and cases of BrS1 with impaired conduction have been documented.⁶⁷ In 1 unique family, all 3 clinical phenotypes occur together.²¹ The increase in the late Na⁺ current component shifts the current-voltage relation during repolarization such that reactivation of Ca_v1.2 and early afterdepolarizations occur. The subgroup of patients with BrS1 Na⁺ channel mutations has a conduction disturbance: Slow conduction delays endocardial-to-epicardial activation, resulting in paradoxical endocardial-to-epicardial repolarization (normal repolarization occurs in the epicardial-to-endocardial direction) and ST-segment and T-wave changes. This subgroup of patients also has HV interval prolongation during electrophysiological study, confirming the presence of conduction slowing. In 2007, we do not fully understand the reasons for interfamily and interindividual clinical variability association with SCN5A mutations. For some of the mixed arrhythmia phenotypes, the pattern of Na⁺ channel dysfunction suggests plausible mechanisms responsible for multiple manifestations. However, for most of these mixed disorders, we cannot exclude a role for other genetic or environmental factors in determining the type of clinical disease associated with particular mutations.

Predicting Effects of Specific Na⁺ Channel Kinetic Perturbations on Myocardial Dynamics
Common single-scale approaches often fail to reveal the most sought after information: how disruptions in proteins due to mutations and consequently through complex interactions (behaviors of cells) lead to triggers that result in recurrent disorganized cardiac excitation, an arrhythmia hallmark. Computational approaches permit utilization and integration of experimentally and clinically obtained information gathered at individual system scales to understand arrhythmogenesis. Simulations can be undertaken to relate the integrated electrophysiological behavior of the cell to state-specific single-channel events. Such approaches may allow prediction of the effect of a mutation that alters a single voltage-dependent transition or, even more complex, multiple discrete transitions on the whole-cell behavior due to multiple nonlinear interactions within the cellular environment and cell-to-cell coupling in cardiac tissue.

An absolute requirement for using computational methods to make connections between Na⁺ channel defects and disease is the development of sufficiently detailed models that accurately recapitulate all the basic features of Na⁺ channel gating. Numerous experimental studies have shown that arrhythmia-linked mutations tend to affect single or specific multiple discrete transitions. Virtual transgenic cardiomyocytes have been used to demonstrate how specific defects disrupt channel-gating kinetics and underlie likely cellular arrhythmogenic mechanisms. Several investigations showed how modal gating of the Na⁺ channel contributes to LQTS3.^68,69 Modeling approaches have also been used to investigate dynamics of channel gating under conditions of changing voltage, so-called nonequilibrium gating, to identify the mechanism of rate dependence of slow-gating, noninactivating current and to reveal a possible mechanism of conduction slowing.^70–72 Modeling studies have helped to explain the propensity to drug-induced arrhythmia in blacks who carry a common SCN5A-S1103Y polymorphism.¹²

Unanswered Questions and Opportunities for Future Research
In addition to its role in impulse transmission in the specialized conduction system, atria, and ventricles, Na⁺ channel function contributes to maintenance of the action potential plateau and excitation-contraction coupling. As a result of the complex physiological roles, Na⁺ channelopathies may result in multiple distinct or overlapping phenotypes, including mixed arrhythmic phenotypes, primary CoD, and DCM. SCN5A mutations in CoD are characterized by depolarization/repolarization abnormalities and the extent and site of conduction block. The most profound phenotype is atrial standstill, with loss of atrial excitability and prolonged ventricular depolarization. Heterozygous mutation carriers exhibit mild conduction abnormalities and no evidence of sudden death or Brugada-like symptoms. In congenital SSS, compound heterozygous pairing of SCN5A mutations and biophysical characterization showed defects consistent with impaired inactivation and slowed recovery from inactivation but no persistent late currents. The combination of SCN5A-D1275N with a Cx40 promoter polymorphism was associated with late-onset SSS (Table 2). The challenge of low-penetrance alleles is to identify silent carriers with loss-of-function SCN5A mutations that develop a phenotype later in life and to explain mechanisms that rescue heart rhythm in early childhood. It is important that future studies investigate associated fibrosis mechanisms in patient tissues and develop a rationale for SSS treatment in the elderly.

Factors Accounting for Chamber-Specific Phenotype Expression
Na⁺ channels interact with multiple proteins, are targeted to specific membrane locations, and are regulated by a multitude of mechanisms. Therefore, genetic, functional, and modulatory mechanisms may all contribute to chamber-specific phenotype expression.^43,73 Mutations that affect any of these associated mechanisms may cause alterations in Na⁺ channel expression within the atria, ventricles, or conduction system. An important future research question will be to identify factors that underlie chamber-specific manifestation of Na⁺ channel mutations and their interaction with modulatory molecules.

An additional challenge is to understand the variable phenotype expression associated with the same or similar SCN5A mutations, the identification of Na⁺ channel interactions with other molecular and cellular proteins, and genetic modifiers. For instance, the functional consequences of phosphorylation by protein kinase A and protein kinase C are controversial. The controls of trafficking and channel expression localization are unresolved questions. Few high-resolution data are available to support structure-function correlations. Nuclear magnetic resonance and circular dichroism studies have been performed on small segments of the protein. The high structural resolution afforded by x-ray crystallography has not been applied to the Na⁺ channel, and structural data from K⁺ channels suggest that the motifs inferred from sequence data are simplistic.

The understanding of channel modulation by age-related factors and by sex hormones is limited, which may explain why some arrhythmia triggers are gender mediated. Therefore, it seems important to develop models of age- and environment-dependent factors to understand arrhythmia syndromes. Although Na⁺ channel behavior appears to be consistent throughout species, the common mouse models show important phenotypic differences from human disease. Ideally, animal or computational models need to replicate all electrophysiological abnormalities seen in the human heart.

Understanding how SCN5A mutations confer arrhythmia susceptibility and other phenotypes should proceed to appropriate animal models. Such studies will be guided by predictions made with the use of computational modeling approaches and in vitro studies with recombinant Na⁺ channels expressed in their native environment. Studies in transgenic models provide additional insights not readily predictable from heterologous expression. The LQTS3-associated Na⁺ channel mutations enhance the slow components of gating observed in the normal phenotype. The resulting increase in the late current shifts the current-voltage trajectory in the inward direction and causes membrane oscillations that are the basis for early afterdepolarizations. The challenge will reside in developing novel therapeutic approaches that are tailored to precise genotypes or are capable of correcting specific Na⁺ channel dysfunctions.

Cardiac K⁺ Channelopathies
Normal Cardiac K⁺ Channel Function
Multiple K⁺ channels, with distinct physiological roles, have been identified in the heart. Myocardial K⁺ channels function, for example, to control resting membrane potentials, action potential plateau potentials, and both the initiation and the duration of membrane repolarization. Available evidence suggests that cardiac K⁺ channels reflect the homomeric or heteromeric assembly of 4 voltage-gated (K_v) or inward rectifier (Kir) pore-forming -subunits, together with accessory subunits and regulatory proteins. Heterologous expression of KCNQ1 (KvLQT1) alone yields rapidly activating K_v currents, whereas coexpression with KCNE1 (minK) produces slowly activating currents that resemble the slow component of cardiac delayed rectification, I_Ks, suggesting that cardiac I_Ks channels reflect the coassembly of the KCNQ1 and minK proteins. Although expression of KCNH2 (HERG) reveals currents that are similar to the rapid cardiac delayed rectifier, I_Kr, both KCNE1 (minK) and KCNE2 (MiRP1) have been suggested to function in the generation of cardiac I_Kr channels (Table 3).

View larger version (19K): [in this window] [in a new window]   Table 3. Long QT and Related Arrhythmia Syndromes Genes contributing to the same membrane currents and/or distinct phenotypes are marked similarly for ease of comparison within and between Tables 1 through 5. indicates gain of function; , loss of function. *LQTS indicates Romano-Ward (RW) syndrome resulting from autosomal-dominant heterozygous mutations, nomenclature is considered historical because of low average QTc penetrance of 60%;148 mechanism-based classification by protein dysfunction is preferable. £LQTS- and CPVT1-causing mutations probably account for 10% to 15% of SIDS.10, 184, 185 $Ankyrin-B syndrome (ABS) including sinus bradycardia, paroxysmal AF, VF, polyphasic T waves; see also Tables 2 and 5. #Andersen Tawil syndrome (ATS): 50% to 60% are KCNJ2 mutation carriers including periodic muscle paralysis and developmental abnormalities. &See also Table 5. +Jervelle and Lange-Nielsen syndrome type 1 (JLNS1) with autosomal-recessive inheritance resulting in homozygous loss-of-function mutations, congenital deafness, QTc prolongation, and ventricular tachyarrhythmias. ++Jervelle and Lange-Nielsen syndrome type 2 (JLNS2) with compound heterozygous mutations; asymmetric T waves with rapid terminal configuration. ¶Relative syndromic occurrence for a given genetic syndrome (in %).

Dysfunction in K⁺ Channel–Based Cellular Pathways and LQTS
LQTS is associated with increased risk of syncope and sudden death from ventricular tachyarrhythmias, including torsades des pointes and VF. Several LQTS genes have been identified, and most encode the subunits of repolarizing voltage-gated K_v channels. LQTS-linked mutations in the K⁺ channel pore-forming -subunit genes KCNQ1 (LQTS1) and KCNH2 (LQTS2) account for the majority of inherited cases (multigenetic arrhythmia syndromes are numbered sequentially to identify and discriminate specific genotype–phenotype relationships; refer to Table 3). Mutations in K⁺ channel accessory subunits KCNE1 (LQTS5) and KCNE2 (LQTS6) occur in <1%. Mutations in KCNJ2, encoding the inward rectifier K⁺ channel -subunit Kir2.1, a component of cardiac I_K1, have been linked to LQTS7 or Andersen Tawil syndrome. In 25% of LQTS families, the genetic link remains to be established.

Heterozygous mutations in KCNQ1 and KCNH2 were first linked to the autosomal dominant inherited LQTS (Romano-Ward). Numerous KCNQ1 (LQTS1) and KCNH2 (LQTS2) mutations have been identified throughout the (KvLQT1 and HERG) protein sequences, and expression studies have suggested that all are loss-of-function mutations due to haploinsufficiency or to the generation of dominant negative subunits, which result in reduction in functional cell surface expression of I_Ks or I_Kr channels. In addition, many KCNH2 mutations affect the processing of the channel proteins and the trafficking of assembled K⁺ channels to the cell surface. LQTS7 mutations in KCNJ2 are also loss-of-function mutations that result in reduced I_K1 density. Computer simulations confirm that reduced I_Ks, I_Kr, or I_K1 densities will result in prolongation of ventricular action potentials. Given the intrinsic heterogeneities in channel densities and action potential waveforms in the ventricular myocardium, KCNQ1, KCNH2, KCNE1, KCNE2, and KCNJ2 mutations result in heterogeneous dispersion of repolarization. Patients with autosomal recessive (Jervelle and Lange-Nielsen) LQTS syndrome are homozygous for loss-of-function mutations in either KCNQ1 or KCNE1 (Table 3). These patients have a more severe cardiac phenotype and complete loss of I_Ks in the hair cells and endolymph of the inner ear, resulting in congenital deafness.

Dysfunction in K⁺ Channel–Based Cellular Pathways and Short-QT Syndrome
Short-QT syndrome (SQTS) is a more recently described syndrome characterized by ECG shortening of the QT interval and episodes of syncope, paroxysmal AF, and life-threatening cardiac arrhythmias. SQTS usually affects young and otherwise healthy individuals with no structural heart disease; it may be expressed as sporadic cases as well as in families. SQTS was originally identified in a family with short QT and AF, including a member who suffered SCA.⁸⁹ Families with a high incidence of SCA⁹⁰ and missense mutations in KCNH2 were linked to SQTS1 (Table 4).^{46,91,92–94}

View larger version (7K): [in this window] [in a new window]   Table 4. Short-QT Syndromes indicates gain of function. ¶Relative syndromic occurrence for a given syndrome has not been determined; total No. of mutation carriers published vary between 1 and 9 (incidence not known).

The biophysical analysis showed a gain-of-function defect in KCNH2 (HERG). Shortly thereafter, a mutation in KCNQ1 responsible for SQTS2 was identified in a 70-year-old individual who suffered VF and had a QTc interval of 302 ms after resuscitation.⁹³ Similar to SQTS1, biophysical analysis showed a gain-of-function defect in KCNQ1 (KvLQT1). A second gain-of-function mutation in KCNQ1 has been associated with SQTS2 and AF in a newborn patient.³⁵ An additional form, SQTS3, has been linked to mutations in KCNJ2 underlying I_K1.⁹⁴ When expressed in CHO cells, the mutant Kir2.1 subunits generated I_K1-like channels that did not rectify as much as the wild-type channels, resulting in larger outward K⁺ currents in the physiological range of membrane potentials. Gain-of-function defects in 3 different K⁺ channel genes have therefore now been identified in SQTS; there will likely be more. Clinical and biophysical research efforts have enabled the investigation of possible therapeutic interventions, such as quinidine, which was found to improve (prolong) the QT interval in mutation carriers and which is presently recommended in patients who cannot receive a defibrillator or who have received multiple implantable cardioverter-defibrillator (ICD) shocks.^90,95

Metabolism-Sensing ATP-Sensitive K⁺ Channel Variants
Heart failure (HF) is a disease with a high prevalence of SCA and arrhythmias. The significance of the sarcolemmal ATP-sensitive K⁺ (K_ATP) channel for cardiac protection from HF is indicated by mutations that produce abnormal channel phenotypes with compromised metabolic signaling in patients with inherited cardiomyopathy.⁹⁶ A causal relationship between K_ATP channel dysfunction and the development of HF has been shown in models of experimental hypertension in which knockout of the KCNJ11 gene, encoding the Kir6.2 pore-forming subunit of the K_ATP channel, predisposed to HF and sudden death.⁹⁷ In the heart, K_ATP channel activity has been linked to homeostatic shortening of the action potential under stress and a deficit in repolarization reserve, as demonstrated in Kir6.2-knockout hearts with an increased risk for triggered activity and ventricular arrhythmia.⁹⁸ Defects in hypertension-induced metabolic distress signals via the K_ATP channel may cause pathological Ca²⁺ overload, aggravated cardiac remodeling, and arrhythmias that could potentially be targeted by novel pharmacological approaches.

The significance of K_ATP channels in human disease is further underscored by an ABCC9 missense mutation (T1547I) in the SUR2A nucleotide sensing channel subunit conferring risk for adrenergic-mediated AF originating from the vein of Marshall, a recognized source for adrenergic AF (Table 2).⁴⁷ Targeted knockout of the K_ATP channel verified the pathogenic link between channel dysfunction and predisposition to adrenergic AF. ABCC9 mutation–induced AF susceptibility was cured by radiofrequency ablation disrupting the substrate for arrhythmia conferred by K_ATP channelopathy.⁴⁷

Current Challenges and Important Issues for Future Studies
Since identification of the LQTS-causing genes KCNQ1 and KCNH2, considerable progress has been made in the characterization of specific gene mutations in affected families, the discovery of additional LQTS genes, and linkage of novel phenotypes such as SIDS (Table 3). Refined clinical phenotyping of LQTS families has led to improved, disease-specific, therapeutic paradigms. Nevertheless, important problems remain such as the identification of the genetic and environmental factors that affect the well-documented individual variations in disease susceptibility, induction (onset), presentation, and severity. Studies focused on the identification and characterization of gene modifiers and proteins will provide new insights into cellular and systemic disease mechanisms while facilitating the development of improved paradigms for risk assessment and therapeutic intervention. Although AF is the most common arrhythmia, genetic defects responsible for AF and their molecular mechanisms remain incompletely understood (Table 2).

Phenotypic characterization of SQTS is in its early stages (Table 4), although it is now well accepted that a QT interval <320 ms indicates increased SCA risk. It also appears that a gradient of phenotypic severity likely exists according to QT interval length similar to LQTS.⁹⁹ From a therapeutic point of view, the first line of therapy in individuals recovered from SCA or with a history of syncope is the implantation of an ICD.⁹⁰ Although some of the class III antiarrhythmic agents appear suitable for SQTS1,^91,95,100 no specific pharmacological treatment for SQTS2 and SQTS3 is yet known. Similar to LQTS, SQTS is a polygenetic disease highlighting the need to characterize each individual form to specifically direct therapeutic approaches.

Apart from the identification of novel LQTS genes, mutations, modifier genes, and proteins, delineation of protein trafficking mechanisms and regulatory pathways has emerged as an important area. Proteomics of ion channel macromolecular complexes is increasingly recognized as important. Genetically engineered large-animal models of LQTS will be necessary to help to translate basic sciences into the human context. An improved transitioning process into clinical medicine will be necessary to translate complex genotype–phenotype relations from the basic sciences. Renaming and realignment of disease entities based on improved genotype–phenotype characterization according to channel and modifier dysfunction are important to facilitate comprehensive characterization beyond preliminary or early phenotype description. Current recommendations suggest that "inclusive" phenotype classification systems such as "cardiomyopathy" are preferable to more exclusive ones such as "electric heart disease," which tend to underestimate phenotype expression.⁵⁶ Finally, continuous improvement of risk assessment and stratification in mutation carriers together with development of targeted, gene, or mutation-specific therapies is necessary to provide more effective and specific therapeutic options.

Arrhythmias Linked to Ca²⁺ Transport Mechanisms
Dynamic Roles of Ca²⁺ in Arrhythmogenesis
Changes in intracellular Ca²⁺ cycling within macroscopic cardiac regions contribute to an unstable conduction substrate and the development of ectopic electric activity.^101,102 Underlying this change in electric activity are characteristic changes in intracellular Ca²⁺ signals and Ca²⁺ storage organelle (sarcoplasmic reticulum [SR]) content occurring in subcellular compartments in a tissue-specific manner.^103–105 The primary feature of Ca²⁺-dependent arrhythmias is an unstable state of SR Ca²⁺ storage, which may be rate dependent and sensitive to hormonal and pharmacological modulation. Thus, cellular and molecular studies have linked inherited arrhythmias (eg, LQTS4 or CPVT1) to arrhythmogenic mechanisms that are similar to those seen in more common disease forms such as HF.^43,105–109 Changes in the subcellular spatial organization of heart cells may further contribute to abnormal Ca²⁺ signaling.^109–111 Additionally, changes in the Ca²⁺ sensitivity of excitation-contraction coupling and Ca²⁺ cycling proteins can directly contribute to arrhythmogenesis.^107,112,113

Under resting, diastolic conditions, spontaneous elementary Ca²⁺ release events (sparks) occur at a rate of 100 s^–1 per cardiomyocyte, each representing the activity of a single functional SR Ca²⁺ release complex containing tens to hundreds of cardiac RyR2 channels. Each Ca²⁺ spark produces a local SR Ca²⁺ depletion within the SR lumen visualized as Ca²⁺ "blinks."¹¹⁴ SR Ca²⁺ overload produces higher spark rates that may form abnormal intracellular Ca²⁺ waves that alter the electric properties of cardiomyocytes. In HF, T-tubule remodeling results in signaling changes affecting SR Ca²⁺ release structures. The consequence of T-tubule remodeling is dyssynchronous SR Ca²⁺ release that may disrupt Ca_V1.2-RyR2 signaling and contribute to arrhythmogenesis.

Timothy Syndrome and Ca_V1.2 Mutations
Timothy syndrome (TS) is a multisystem disorder characterized by congenital heart disease including LQTS8 (Tables 3 and 5) and cardiac arrhythmias including bradycardia, atrioventricular block, torsades de pointes, VT, and VF contributing to early mortality.⁸²

View larger version (23K): [in this window] [in a new window]   Table 5. Ca2+-Dependent Arrhythmia Syndromes AT indicates atrial tachycardia; LVH, left ventricular hypertrophy; black boxes, genes directly involved in Ca2+ transport function; , gain of function; ARVC2, Arrhythmogenic Right Ventricular Cardiomyopathy type 2 (atypical form of ARVC); and ATS, Andersen Tawil syndrome. *Timothy syndrome (TS): multisystem disorder including congenital heart disease, AF, VT, autism, syndactyly in 100%, musculoskeletal disease, immune dysfunction; CACNA1C have been associated with more severe arrhythmia risk, absence of syndactyly, and nemaline rod myopathy. #QTc intervals of select groups of RYR2 mutation carriers were reported as slightly but significantly longer (for comparison to LTQS, see Tester et al186). ¶Estimated occurrence within a given syndrome. £LQTS- and CPVT1-causing mutations may account for 10% to 15% of SIDS.

Common features include syndactyly, dysmorphic facial features, myopia, immunodeficiency with recurrent infections, intermittent hypoglycemia, and hypothermia. Children with TS show developmental delay, autism, and generalized cognitive impairment. TS results from a de novo, gain-of-function missense mutation in splice exon 8A of CACNA1C that encodes the pore-forming -subunit (Ca_V1.2) of the cardiac L-type Ca²⁺ channel, a key protein in excitation-contraction coupling in the heart that is also expressed in the brain, smooth muscle, immune system, teeth, and testes.¹²⁵ Heterologous expression of mutant and wild-type channels demonstrated that the causative mutation, G406R, results in loss of voltage-dependent channel inactivation likely to induce intracellular Ca²⁺ overload in multiple cell types. In an atypical case of TS with severe QT interval prolongation, skeletal nemaline myopathy, but no syndactyly, a de novo missense mutation was found that was affecting the dominant cardiac splice exon 8. One analogous TS-associated mutation, G406R, and another, G402S, both cause loss of voltage-dependent channel inactivation, leading to maintained inward Ca²⁺ currents possibly by affecting a gating hinge mechanism.⁸³ CACNA1C mutations in TS include cardiac and autism spectrum phenotypes, indicating the importance of including the central nervous system in the disease characterization (Table 5).

Catecholaminergic Polymorphic VT Resulting From RYR2 and CASQ2 Mutations
Catecholaminergic polymorphic VT (CPVT) is a familial cardiomyopathy characterized by stress-induced ventricular arrhythmias that result in syncope and sudden death in children or young adults. Two major genetic variants have been identified: The majority of cases have been linked to missense mutations in the RYR2 gene encoding the ryanodine receptor, the principal intracellular Ca²⁺ release channel of the heart (CPVT1)^115,116; additionally, unrelated cases have been linked to a recessive form caused by homozygous mutations in the CASQ2 gene encoding calsequestrin2 (CPVT2), a low-affinity, high-capacity Ca²⁺ buffering protein of the SR Ca²⁺ storage organelle.^120,126 Recently, mutations in RYR2 associated with a CPVT1-like phenotype were discovered as an uncommon cause of SIDS.¹¹⁷

Different RYR2 missense mutations resulted in a gain-of-function defect characterized by high channel open probability during sympathetic stimulation consistent with a "leaky" channel phenotype.^107,112 The inability of CPVT-mutant RyR2 to achieve stable channel closure is amplified by protein kinase A phosphorylation of RyR2-Ser2808 and was associated with abnormally decreased calstabin2 (FKBP12.6) binding compared with wild-type RyR2 channels.^107,112 Accordingly, RyR2-S2808A knockin prevented VT and sudden death, indicating that protein kinase A phosphorylation of Ser2808 is a key mediator of RyR2 dysfunction during catecholaminergic VT.¹²⁷ The mechanism of stress-induced arrhythmias in the calstabin2-deficient background appears to be triggered activity, as evidenced by intracellular Ca²⁺ leak causing a transient inward current (I_ti) and delayed afterdepolarizations.^107,108 Intracellular Ca²⁺ leak and delayed afterdepolarizations have been confirmed in HL-1 cells expressing mutant RyR2 and in a CPVT1 knockin mouse model.^128,129 Neutralizing a charge in a mutant calstabin2-D37S increases binding to and rescues mutant RyR2 channel function and prevents SR Ca²⁺ leak in vitro and in vivo.^107,130 JTV519, a 1,4-benzothiazepine, normalized mutant RyR2 channel function.¹¹² Because all CPVT mutant RyR2 channels showed a reduced calstabin2 binding affinity, the arrhythmogenic consequences of calstabin2 deficiency were investigated in a knockout mouse. Stress testing by treadmill exercise followed by epinephrine injection resulted in polymorphic sustained VT in calstabin2^–/–-deficient mice, which could not be prevented by JTV519 treatment.^107,131 However, JTV519 prevented sustained VT in haploinsufficient calstabin2^+/– mice, consistent with in vivo rebinding of calstabin2 to RyR2 and normalization of single-channel function.¹³¹

Phospholamban Mutations
Heart muscle relaxation occurs from SR Ca²⁺ reuptake mediated by SERCA2a Ca²⁺ pumps. Phospholamban (PLN), a 52–amino acid transmembrane SR protein, inhibits SERCA2a in its dephosphorylated state. SERCA2a activity is decreased in human HF, potentially contributing to intracellular Ca²⁺ overload and arrhythmias. A PLN-R9C mutation in the cytosolic PLN domain occurs in inherited, rapidly progressive DCM (Table 5), and transgenic mice expressing the mutant PLN^R9C protein resemble the human phenotype.¹³² PLN^R9C traps protein kinase A, preventing PLN phosphorylation and resulting in chronic SERCA2a inhibition. A homozygous PLN mutation (L39stop) results in PLN deficiency and progressive DCM, indicating that lack of PLN expression is detrimental to humans.¹²⁴ The PLN-Arg14del mutation in the human PLN gene results in superinhibition of SERCA2a activity, indicating that a gain of inhibitory PLN function predisposes to DCM and potentially early death from arrhythmias.¹³³ Whether PLN mutations and SERCA2a dysregulation directly cause cardiac arrhythmias needs to be addressed by future research.

Arrhythmias Linked to Accessory, Regulatory, and Scaffolding Proteins
Dysfunction in Ankyrin-B
How do defects in proteins not comprising membrane ion transporters result in arrhythmias? Ion channels and transporters in higher vertebrates function within specialized cellular microdomains (compartments) and depend on local regulation by protein complexes. Ankyrins are membrane-adaptor proteins that link structurally unrelated ion channels, transporters, and cell adhesion molecules with the spectrin- and actin-based cytoskeleton¹³⁴ in many cell types.¹³⁵ Two unique ankyrin gene products, ankyrin-B (ANK2) and ankyrin-G (ANK3), target ion channels and transporters to distinct cardiomyocyte membranes. Human ANK2 variants resulting in ankyrin-B loss of function cause the "ankyrin-B syndrome" (LQTS4) originally identified in E1425G carriers (prolonged QTc not common in all variant carriers), including bradycardia, AF, CoD, and increased risk for catecholaminergic sudden death (Tables 3 and 5).^43,122 Heterozygous mice with reduced ankyrin-B expression resemble the LQTS4 phenotype and implicate aberrant intracellular Ca²⁺ homeostasis in the generation of stress-induced arrhythmias.⁴³ Loss-of-function ankyrin-B mutations result in loss of cellular targeting and expression of the ankyrin-binding proteins Na⁺/K⁺ ATPase, IP₃ receptor, and Na⁺/Ca²⁺ exchanger to their appropriate membrane microdomains.^43,136 With the use of a combined neonatal cardiomyocyte overexpression and rescue strategy, distinct functional classes of ANK2 loss-of-function variants have been identified that correspond with the severity of arrhythmia expression in the respective mutation carriers.⁴⁵ Importantly, variants with less severe in vitro phenotypes (eg, L1622I) appear to be present in a small percentage of the population.^44,45 The principal voltage-gated Na⁺ channel in the heart, Na_v1.5, is directly associated with ankyrin-G (encoded by ANK3) required for correct Na_v1.5 targeting and expression.¹³⁷ Human variants in SCN5A that disrupt interaction between ankyrin-G and Na_v1.5 result in decreased Na_v1.5 expression at the cardiomyocyte intercalated disc and cause BrS1.¹³⁷ Dysfunction in ankyrin-based pathways in human arrhythmias clearly demonstrates the importance of precise ion channel and transporter targeting and localization pathways.

Caveolin-3 Mutations Cause LQTS9
Caveolin-3 (Cav-3) is a muscle-specific isoform and principal protein component of caveolae, 50 to 100 nm invaginations that represent subcompartments of the plasma membrane. Cav-3 contains a 20–amino acid scaffolding domain (residues 54 to 73) that is critical for interaction with associated signaling molecules. Cav-3 copurifies with dystrophin and dysferlin, and mutations in these 3 proteins have been linked to inherited muscular dystrophies. In Japanese brothers with hypertrophic cardiomyopathy whose father with hypertrophic cardiomyopathy had died suddenly, a T63S substitution in CAV3 was identified.¹³⁸ Four novel mutations in CAV3 were found recently that result in increased late Na⁺ current and LQTS9 arrhythmias⁸⁴ similar to LQTS3-associated SCN5A mutations (Table 3). Cav-3 and Na_V1.5 colocalize to caveolae in human myocardium. More recently, missense mutations in Cav-3 were discovered in a small subset of black infants dying from apparent SIDS. Similar to the patients with LQTS, the SIDS-associated Cav-3 mutations accentuated the late Na⁺ current attributed to the otherwise intact Na⁺ channel -subunit.⁸⁵ Future studies are needed to address the molecular mechanisms that result in the gain-of-function defects associated with these mutations.

SCN4B Mutations Cause LQTS10
Akin to the K⁺ channel ß-subunits responsible for LQTS5 and LQTS6, the Na⁺ channel ß4 subunit encoded by SCN4B has been established as a novel, albeit rare, LQTS-susceptibility gene (LQTS10; Table 3). Identified in a multigenerational Mexican-mestizo family, the missense mutation conferred a secondary gain of function on the Na⁺ channel such that the accentuated late Na⁺ current mimicked that of classic LQTS3-associated mutations in SCN5A.⁸⁶

Intracellular Targeting and Signaling Defects Indicate Common Mechanisms
Overlap between genetic defects responsible for cardiomyopathies and arrhythmias is important in the understanding of complex disease phenotypes.⁵⁶ Interactions between ion channels and structural proteins of the cardiomyocyte cytoskeleton and sarcomere, as well as nuclear proteins, need to be evaluated. For instance, DCM may occur because of disruption of the link between the sarcolemma and sarcomere, and young male dystrophin mutation carriers and their mothers develop both cardiomyopathy and arrhythmias. Interactions between cytoskeletal proteins, such as dystrophin, with Na⁺ (and other ion) channels may provide an explanation for how structural defects translate into arrhythmic mechanisms. Interactions with the dystrophin-sarcoglycan complex indicate that a monogenetic disruption may cause a multitude of distinct defects.¹³⁹ In arrhythmogenic right ventricular cardiomyopathy, desmosome function is disturbed, leading to disruption of intercalated discs and fibrofatty replacement of the myocardium. Arrhythmias may result after disruption of membrane-bound channels from the desmosome apparatus. At least 5 of the LQTS-susceptibility genes (ANK2, KCNE1, KCNE2, CAV3, and SCN4B) do not encode pore-forming ion channel -subunits, and these findings strongly support the notion that defective protein-protein interactions or targeting mechanisms contribute to arrhythmogenesis (Table 3). Specific animal models are required along with cellular studies to elucidate mechanisms and dissect protein-protein interactions and their specific regulation.

Summary and Future Directions
Arrhythmia mechanisms caused by defective targeting and expression of ion transporters (eg, ankyrin mutations) may depend critically on the native cardiac cellular environment, which differs from heterologous expression systems because of proteome- and cell type–specific expression characteristics. Moreover, specific genetic defects in ion transporters may cause structural pathology (eg, fibrosis), implicating an important role of fibroblasts or other cell types that may directly contribute to the arrhythmogenic substrate. Important unresolved questions include the following: How does spatial remodeling influence Ca²⁺-dependent arrhythmogenesis? How does microdomain signaling influence cellwide function? How do ion channels and transporters interact with cytoskeletal, regulatory, and other effector proteins? How do changes in the spatial organization of heart cells influence the ability of the conductive medium to generate and support altered signal propagation? In addition, how does this cellular architecture differ among the various types of electrically excitable cell types, as well as with the nonexcitable cells of the heart?

Arrhythmias are a common cause of death and morbidity in cardiomyopathies associated with HF and abnormalities of the conduction system. Intracellular Ca²⁺ leak from protein kinase A hyperphosphorylated RyR2 channels may represent an important defect contributing to arrhythmias and disease progression.^140,141 Current evidence supports stress-induced intracellular Ca²⁺ leak as the trigger mechanism of CPVT.^107,108,142 In CPVT1, mortality in RYR2 mutation carriers at 35 years of age reaches 35% to 50%,^112,115 indicating that stress-induced RyR2 Ca²⁺ leak represents a very aggressive arrhythmia mechanism. Molecular determinants of the RyR2 channel closed state instability include RyR2 protein kinase A phosphorylation, calstabin2 depletion, and decreased sensitivity to Mg²⁺ inhibition.^107,112 Future studies are needed to characterize the mechanisms of RyR2 channel leak and cytoplasmic regulation at the structural level. Intracellular Ca²⁺ leak has been recognized as a pathogenic mechanism that activates a Ca²⁺-dependent transient inward current (I_ti) and delayed afterdepolarizations, leading to triggered activity.^108,143,144 Overlap between intracellular Ca²⁺ changes and altered plasma membrane ion currents occurs also in multigenetic forms of arrhythmias. Thus, characterization of Ca²⁺-dependent mechanisms contributing to arrhythmogenic membrane instabilities will aid in conceptualizing arrhythmia triggers versus substrates, as well as in developing rational therapeutic interventions.

Implications for Diagnosis and Management of Inherited Arrhythmias
More than 400 different mutations have been reported among the 10 LQTS-susceptibility genes, yet a small amount of patient-related data exists on the relationship of specific ion channel mutations, their coding characteristics, and the influence of the biophysical dysfunction on patients’ clinical course.¹⁴⁵

State of Genetic Testing for Cardiac Channelopathies
Arrhythmia Susceptibility Genes
LQTS is a potentially lethal, heritable arrhythmia syndrome affecting 1 in 2500 people. To date, 10 LQTS-susceptibility genes have been identified (Table 3); 5 of these genes encode critical ion channel pore-forming -subunits, and 3 of them encode ß-subunits. The other 2 genes encode the adapter proteins ankyrin-B and caveolin-3. Mutations in the genes responsible for LQTS1–3 account for 75%, and the rest of the known susceptibility genes collectively account for <3% of LQTS. The concept that inherited arrhythmia syndromes like LQTS may originate from defects in scaffolding or adaptor proteins points to a new class of candidate genes possibly responsible for the 20% to 25% of unexplained LQTS cases. Tables 1 to 5 summarize genetic arrhythmia syndromes, susceptibility genes, and their relative frequency within a syndrome and provide an overview of concealed versus overlapping or complex phenotypes.

Identification of mutations in the KCNQ1-, KCNH2-, and SCN5A-encoded K⁺ and Na⁺ channel -subunits as the cause of LQTS1–3, respectively (Table 3), in concert with a growing body of evidence implicating ion channels in episodic syndromes of excitable tissues, defined a new class of diseases, channelopathies.¹⁴⁶ Genetic testing has indicated that LQTS is a common risk factor in arrhythmogenesis. Over the past decade, LQTS genetic testing was performed in research laboratories while genotype–phenotype relationships were being determined.¹⁴⁷ Today, genetic testing is commercially available, and clinical diagnostic screening consists of comprehensive open reading frame sequence analysis (60 translated exons) of 5 LQTS-susceptibility genes: KCNQ1 (LQT1), KCNH2 (LQT2), SCN5A (LQT3), KCNE1 (LQT5), and KCNE2 (LQT6).¹⁴⁸ Hundreds of mutations have been reported within these 5 genes, with the vast majority yielding LQTS1–3 (Table 3).^149,150 Among patients with definitive clinical evidence of LQTS, the yield of genetic testing is 75%: LQTS1 (30% to 35%), LQTS2 (25% to 30%), LQTS3 (5% to 10%), LQTS5 (1%), and LQTS6 (1%).^151,152 The majority of known mutations are missense mutations, and approximately half are unique, novel mutations. Thus, conversion from a sequencing-based genetic test to a chip-based genetic test containing prespecified mutations is not yet possible. Besides these 5 LQTS genes, there are now 5 additional susceptibility genes for LQTS-related syndromes, including the following: ANK2 (LQTS4, <1%), KCNJ2 (LQTS7, <1% and 50% to 65% of Andersen Tawil syndrome), CACNA1C (LQTS8 or TS1), CAV3 (LQTS9, <1%), and SCN4B (LQTS10, <1%). Clinical genetic testing for the rare subtypes of LQTS is not yet available. Still, the pathogenesis for 20% to 25% of LQTS remains unexplained. Possible reasons for this include (1) missed regions and false-negative results within the currently explored gene regions of interest, (2) disease-causing mutations in noncoding sequence (eg, promoters, introns) of the known LQTS-susceptibility genes, and (3) novel or unknown LQTS genes. "Allelic dropout" is a mechanism responsible for some previous false-negative genetic test results,¹⁵³ and the new discovery of CAV3-LQTS9 and SCN4B-LQTS10 indicates that all 3 reasons are likely.

Genetic testing has to be combined with clinical evaluation and management of patients and is now most available for LQTS. In contrast to LQTS loss-of-function K⁺ channel mutations, gain-of-function mutations in KCNH2, KCNQ1, and KCNJ2 confer susceptibility for SQTS (Table 4). It is unclear what percentage of SQTS is explained by mutations in these 3 genes because a specific genetic test is not currently available. In contrast to gain-of-function, LQTS3-causing Na⁺ channel mutations, loss-of-function mutations in SCN5A cause BrS1 (Table 1). A SCN5A-only gene test is available clinically.¹⁵⁴ However, mutations in SCN5A account for only 20% to 30% of BrS1. Recently, a mutation in GPD1L-encoded glycerol-3-phosphate dehydrogenase 1-like gene has been implicated as a novel cause of BrS2 and SIDS (Table 1).^25,26 Finally, mutations in RYR2 and CASQ2 cause 50% to 60% of CPVT (Table 5). A targeted examination of 38 of the 105 RYR2 translated exons has been released recently as a commercially available clinical genetic test for CPVT.

Genotype–Phenotype Relationships
Understanding the pathogenetic link between genotype and clinical phenotype is vital to the diagnosis and treatment of inherited arrhythmias. This includes the biophysical phenotype of abnormal protein function (eg, effects on currents), the cellular phenotype caused by this abnormal function (eg, effects on action potential, Ca²⁺ loading), and the tissue and organ phenotype (eg, ECG change, type of arrhythmia) that characterizes the clinical phenotype (eg, syncope, sudden death) (Figure).¹⁵⁵ At each phenotype level, environmental factors (eg, acidosis, autonomic nerve activity, ion concentrations) and other genetic factors (genetic background and modifier genes) may affect the phenotypes. Better understanding of these complex interrelations is especially important for inherited arrhythmia syndromes and has immediate implications for the discovery of new mutations and for cost-effective screening. Most importantly, insights into these relationships may point the way to improved treatment.

View larger version (80K): [in this window] [in a new window]   Figure. Integrative study of arrhythmic cardiomyopathies (refer to Implications for the Diagnosis and Management of Inherited Arrhythmias for conceptual discussion).

In inherited arrhythmia, the relationship between a genotype and a clinical phenotype is not necessarily linear. As an example, mutations in the cardiac Na⁺ channel gene SCN5A may lead to different arrhythmia syndromes, including LQTS3 and/or BrS1 (Tables 1 and 3). Even if one restricts consideration to loss-of-function mutations for SCN5A, multiple clinical phenotypes such as BrS1, idiopathic VF without BrS1, AF, SSS, and CoD can result (Table 2). Finally, even single SCN5A mutations can exhibit multiple phenotypes.^21,5 Incomplete penetrance and variability of the clinical phenotype present additional challenges, as seen commonly with Andersen Tawil syndrome mutations in KCNJ2.¹⁵⁶ Conversely, an apparently single clinical phenotype can result from >1 genotype. For example, CPVT may result from mutations in the different Ca²⁺-handling genes RYR2,^116,157,158 CASQ2,¹²⁰ ANK2,⁴³ and perhaps KCNJ2.^80,123

Several approaches are used to study the link between genotype and clinical phenotype (Figure). The first is the careful definition of the clinical syndrome and classic genetic linkage study. This makes the connection but does not fill in the pathogenetic gaps of the biophysical phenotype, cellular phenotype, and tissue