TABLE 1. Mutations in the Cardiac Ryanodine Receptor Gene (RyR2) Associated With CPVT and ARVD2* Amino acid Nucleotide Disease Families Familial? Doma

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1 EDITORIAL Sudden Unexplained Death Caused by Cardiac Ryanodine Receptor (RyR2) Mutations About 10% to 20% of postmortem examinations of young people who were apparently healthy but died suddenly and unexpectedly reveal no morphologic abnormalities to explain their deaths. 1 In up to 50% of such cases, sudden unexplained death (SUD) is the first and only clinical manifestation. 2 Recent studies have suggested that primary arrhythmogenic disorders such as congenital long QT syndrome, Brugada syndrome, idiopathic ventricular fibrillation, and catecholaminergic polymorphic ventricular tachycardia (CPVT) may underlie a substantial proportion of SUD cases. 2-6 In all these arrhythmogenic cardiac disorders, there is no evidence of pathological abnormalities of the heart, and death would be classified as being of unknown etiology based on traditional postmortem examination. However, recent linkage of these syndromes to causative disease genes (mostly cardiac ion channels) has provided the opportunity to elucidate a pathogenic basis for SUD. 2,3 In the current issue of the Mayo Clinic Proceedings, Tester et al 7 elegantly demonstrate, using molecular genetic analysis, that mutations in the cardiac ryanodine receptor gene (RyR2) may be associated with about 14% of SUD cases. Mutations in RyR2 have previously been linked to CPVT 5,8 and arrhythmogenic right ventricular dysplasia type 2 (ARVD2) 9 (Table 1). Moreover, mutations in the calsequestrin gene (CASQ2) have been linked to an autosomal recessive form of CPVT. 15 Unlike ARVD2, which is associated with progressive degeneration of the right ventricular myocardium, CPVT is not characterized by structural heart disease. Although CPVT is characterized by normal electrocardiographic patterns at rest, it is arguably one of the most malignant types of inherited cardiac arrhythmia syndromes. Exercise or sympathetic nervous system activation induces ventricular arrhythmias, syncope, or sudden cardiac death in most patients with CPVT, 16,17 leading to mortality rates of 30% to 50% at age 35 years. 8,17,18 The findings by Tester et al 7 also suggest the involvement of catecholamines in the onset of lethal arrhythmias of 7 patients, 3 died during exertion, and 1 died during potential emotional stress. Clinical electrophysiologic studies of patients with RyR2 Address reprint requests and correspondence to Andrew R. Marks, MD, Department of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons, 630 W 168th St, P&S 9-401, Box 65, New York, NY ( Mayo Foundation for Medical Education and Research mutations have revealed that CPVT can be induced by exercise or catecholamine infusion, but not by programmed electrical stimulation. 11,16,17,19 Taken together, these findings strongly suggest that catecholamine-sensitive automaticity functions as a cellular mechanism of CPVT. MOLECULAR PATHOGENESIS OF CPVT Catecholaminergic polymorphic ventricular tachycardia linked RyR2 mutant channels show features consistent with the clinical phenotype. 20 The cardiac ryanodine receptor (RyR2) is the major calcium (Ca 2+ ) release channel on the sarcoplasmic reticulum in cardiomyocytes. 21 During excitation-contraction coupling, intracellular Ca 2+ stored in the sarcoplasmic reticulum is released via RyR2 to initiate contraction of the myofilaments (Figure 1, A). Exercise- or stress-induced activation of the sympathetic nervous system results in protein kinase A (PKA) phosphorylation of RyR2, which dissociates the inhibitory subunit See also page 1380 calstabin2 (also known as FKBP12.6) from the channel complex and increases Ca 2+ -induced activation of the RyR2 channel (Figure 1, B). 23 Recent work from our laboratory and others has revealed that under baseline (resting) conditions, CPVT-associated mutant RyR2 channels exhibit normal single channel properties (with low activity at diastolic intracellular Ca 2+ concentrations), 20,24 which is not surprising because patients with CPVT do not have arrhythmias at rest. 5,16 However, after PKA phosphorylation, mutant RyR2 channels display significantly increased activities, suggesting that these channels are more active than wild-type channels during exercise because they show increased Ca 2+ -dependent activation at low intracellular Ca 2+ concentrations (Figure 1, C). 20,24 Mutant RyR2 channels found in patients with CPVT have decreased binding affinities for calstabin2, which is believed to cause increased RyR2 activity after PKA phosphorylation. 17,20 Interestingly, calstabin2-deficient mice consistently show exercise-induced ventricular arrhythmias and sudden cardiac death. 20 RyR2 channels isolated from calstabin2-deficient mice under resting conditions display a minor increase in channel activity compared with those from wild-type mice. However, RyR2 single channel activity isolated from calstabin2-deficient mice after exercise was greatly increased compared with that from wild-type mice, suggesting that these calstabin2-deficient channels might become leaky during diastole when the mice are exercised. 20 Similarly, CPVT-mutant RyR2 channels that have decreased calstabin2-binding affinity might become Mayo Clin Proc. November 2004;79(11):

2 TABLE 1. Mutations in the Cardiac Ryanodine Receptor Gene (RyR2) Associated With CPVT and ARVD2* Amino acid Nucleotide Disease Families Familial? Domain Reference R176Q 527G>A ARVD2 1 1 N-term 9 R420W 1258C>T ARVD N-term 7, 10 L433P 1298T>C ARVD2 1 1 N-term 9 S2246L 6737C>T CPVT Mid-RyR2 5, 7, 11 V2306I 6916G>A CPVT 1 1 Mid-RyR2 12 R2311D NK CPVT 1 0 Mid-RyR2 11 P2328S 6982C>T CPVT 1 1 Mid-RyR2 8 N2386I 7157A>T ARVD Mid-RyR2 9 A2387P 7159G>C CPVT 1 1 Mid-RyR2 13 Y2392C 7175A>G ARVD2 1 1 Mid-RyR2 10 R2474S 7422G>C CPVT 1 0 Mid-RyR2 5 T2504M 7511C>T ARVD2 1 1 Mid-RyR2 9 L2534V NK CPVT 1 0 Mid-RyR2 14 L3778F 11332C>T CPVT 1 1 C-term 11 G3946S 11836G>A CPVT C-term 11 N4097S 12290A>G CPVT 1 1 C-term 7 N4104K 12312C>G CPVT 1 0 C-term 5 E4146K 12436G>A CPVT 1 0 C-term 7 T4158P 12472A>C CPVT 1 0 C-term 7 Q4201R 12601C>A CPVT 1 1 C-term 8 R4497C 13489C>T CPVT C-term 5, 7 N4504I 13512G>A CPVT 1 1 C-term 13 A4608P 13819G>C CPVT 1? C-term 13 V4653F 13957A>G CPVT 1 1 C-term 8 V4771I 14311G>A CPVT 1 0 C-term 11 A4860G 14579C>G CPVT 1 1 C-term 11 I4867M 14601T>G CPVT 1 1 C-term 11 V4880A 14639T>C CPVT 1? C-term 13 N4895D NK CPVT 1 0 C-term 11 P4902L 14705C>T CPVT 1 1 C-term 12 E4950K 14848G>A CPVT 1 0 C-term 11 R4959Q 14876G>A CPVT 1 0 C-term 12 *Highlighted rows represent mutations identified in the study by Tester et al. 7 ARVD2 = arrhythmogenic right ventricular dysplasia type 2; CPVT = catecholaminergic polymorphic ventricular tachycardia; C-term = carboxyterminus; NK = not known; N-term = aminoterminus. In the column Familial?, 0 represents no, and 1 represents yes. Confirmed in one family. Phenotype not well described. leaky in patients during exercise, thereby initiating ventricular arrhythmias or sudden cardiac death. 5,16 The RyR2 mutations linked to CPVT and ARVD2 cluster in 3 regions of the channel, corresponding to similar domains in RyR1 linked to malignant hyperthermia and central core disease. 13 The 3 novel RyR2 mutations discovered by Tester et al 7 also fall within these 3 well-defined regions in RyR2. Mutations linked to malignant hyperthermia and central core disease cause skeletal muscle disease by altering the Ca 2+ -dependent regulation of RyR1 in ways similar to the mutations in RyR2. 25 For example, single channel studies of mutant RyR1 channels isolated from malignant hyperthermic pigs containing an R615C missense mutation revealed an increased sensitivity to activation by Ca 2+ and decreased sensitivity to inhibition by magnesium (Mg 2+ ). 26 This suggests that Ca 2+ leaking from the sarcoplasmic reticulum may play an important role in the pathogenesis of both skeletal muscle and cardiac diseases linked to mutations in RyR2 genes. THERAPEUTIC APPROACHES FOR CPVT Clinical studies support the concept that systemic β- blockers can prevent cardiac events in patients with CPVT, consistent with the notion that prevention of sympathetic nervous system activation and PKA phosphorylation of mutant RyR2 channels may prevent Ca 2+ leaking from the sarcoplasmic reticulum during diastole, which triggers arrhythmias. 20 Although results are promising based on follow-up reports of small groups of patients with CPVT, sudden cardiac death has been reported while patients were receiving β-blocker treatment. 16,18,19,27 It should also be noted that, although β-blockers seem to prevent most car Mayo Clin Proc. November 2004;79(11):

3 FIGURE 1. Physiological regulation of the ryanodine receptor (RyR2) macromolecular complex. A, The RyR2 macromolecular complex includes 4 identical RyR2 subunits (orange, numbers 1 through 4 indicate the 4 monomers). Each RyR2 subunit binds one calstabin2 (FKBP12.6, yellow triangle), as well as the protein kinase A (PKA) catalytic and regulatory subunits (RII) (purple), muscle A-kinase anchoring protein (makap) (purple), protein phosphatase 2A (PP2A) and its targeting protein PR130 (gray), and protein phosphatase 1 (PP1) and its targeting protein sphinophilin (green). (For clarity reasons, accessory molecules are shown only for 1 of the 4 RyR2 subunits, except for calstabin2, which is shown for all 4 RyR2 subunits.) B, In normal hearts, exercise activates the β-adrenergic signaling pathway, leading to activation of PKA, increased calcium (Ca 2+ ) release through RyR2, and improved cardiac contractility. Protein kinase A phosphorylation of RyR2 leads to phosphorylation of 1 to 2 (of 4) serine 2809 (Ser 2809 ) amino acids, which causes dissociation of 1 to 2 (of 4) calstabin2 molecules from the RyR2 complex. During exercise, the RyR2 complex remains closed during diastole. C, In catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited RyR2 mutation reduces the calstabin2-binding affinity for RyR2. After exercise, 2 to 4 calstabin2 molecules are released from the channel complex, leading to increased Ca 2+ -dependent activation and release of Ca 2+ from the sarcoplasmic reticulum (SR) during diastole (Leak) that can activate depolarizations and trigger fatal ventricular arrhythmias. Modified from Trends Biochem Sci, 22 with permission from Elsevier Health Sciences. Mayo Clin Proc. November 2004;79(11):

4 diac events, they do not usually abolish the arrhythmias, which can still be provoked at higher heart rates. 19 In such patients, an implantable cardioverter-defibrillator should be considered. Therapies are currently being developed to treat the molecular defect underlying CPVT more specifically and to limit adverse effects associated with β-adrenergic receptor blockade. 21 In proof-of-principle experiments, we recently showed that a genetically altered calstabin2 protein is capable of binding to PKA-phosphorylated CPVT-mutant RyR2, indicating that stabilizing the RyR2 channel complex might be a promising therapeutic strategy for CPVT. 20 The 1,4-benzothiazepine derivative JTV519 also effectively enhances calstabin2 binding to PKA-phosphorylated wild-type and CPVT-mutant RyR2 channels. 17,28,29 By inducing a conformational change in RyR2 that allows calstabin2 to rebind to the PKA-phosphorylated channel, this experimental drug inhibited Ca 2+ leak from the sarcoplasmic reticulum during diastole and prevented cardiac arrhythmias in calstabin2-deficient mice. 29 These findings indicate that JTV519 and its derivatives may constitute a novel class of drugs that could lead to the development of a gene-defect specific treatment of arrhythmias in patients with CPVT. 21,30 IMPLICATIONS FOR GENETIC COUNSELING Like most arrhythmogenic cardiac disorders that cause sudden death, CPVT is inherited as an autosomal dominant trait, with 50% of offspring from an affected individual being at risk of developing the same disease. 19 The study by Tester et al 7 suggests that the presence of CPVT should be excluded in all cases of SUD in which the autopsy is negative for anatomical and histopathological findings. Another disorder responsible for 10% to 15% of SUD cases is the congenital long QT syndrome, which may manifest with similar signs and symptoms. 3,31 This disease is inherited as an autosomal dominant or recessive trait, but it can also be caused by de novo mutations. 6 The diagnosis of a congenital arrhythmogenic disorder at autopsy will enable appropriate clinical screening of surviving family members. Family screening and counseling are particularly important with the advent of preventive therapies for sudden cardiac death (eg, β-blockers and implantable cardioverterdefibrillators). The findings by Tester et al clearly underscore the importance of a thorough review of the autopsy report by the physicians caring for family members of patients with SUD. Although some cases may indeed remain unexplained, others will have a plausible explanation (ie, hypertrophic cardiomyopathy or an anomalous coronary artery). Once it is verified that the sudden death is truly unexplained, an accurate exploration of the family history is warranted. Screening methods for family members may include a 12-lead electrocardiogram, Holter recordings, exercise stress tests, and echocardiograms. 31 Unfortunately, mutational analysis to unveil a congenital arrhythmogenic disorder is currently performed only in specialized research laboratories. The screening of CPVT genes can be performed on DNA extracted from paraffin-embedded tissues. At present, genetic analysis identifies disease-causing mutations in about two thirds of patients with long QT syndrome, 32 10% to 20% of patients with Brugada syndrome, and about half of patients with CPVT. 11 This investigation is best undertaken in centers with expertise in the molecular diagnosis and management of inherited cardiac diseases that cause premature sudden death and underscores the need for new technology that will allow rapid genetic diagnosis at low cost. In the future, the results from a molecular forensic autopsy may also be used as a tool of preventive medicine. Xander H. T. Wehrens, MD, PhD Andrew R. Marks, MD Department of Physiology and Cellular Biophysics Center for Molecular Cardiology Columbia University College of Physicians and Surgeons New York, NY 1. Anderson RE, Hill RB, Broudy DW, Key CR, Pathak D. A populationbased autopsy study of sudden, unexpected deaths from natural causes among persons 5 to 39 years old during a 12-year period. Hum Pathol. 1994;25: Chugh SS, Senashova O, Watts A, et al. Postmortem molecular screening in unexplained sudden death. J Am Coll Cardiol. 2004;43: Ackerman MJ, Tester DJ, Porter CJ, Edwards WD. Molecular diagnosis of the inherited long-qt syndrome in a woman who died after near-drowning. N Engl J Med. 1999;341: Virmani R, Burke AP, Farb A. Sudden cardiac death. Cardiovasc Pathol. 2001;10: Priori SG, Napolitano C, Tiso N, et al. Mutations in the cardiac ryanodine receptor gene (hryr2) underlie catecholaminergic polymorphic ventricular tachycardia. Circulation. 2001;103: Wehrens XH, Vos MA, Doevendans PA, Wellens HJ. Novel insights in the congenital long QT syndrome. Ann Intern Med. 2002;137: Tester DJ, Spoon DB, Valdivia HH, Makielski JC, Ackerman MJ. Targeted mutational analysis of the RyR2-encoded cardiac ryanodine receptor in sudden unexplained death: a molecular autopsy of 49 medical examiner/ coroner s cases. Mayo Clin Proc. 2004;79: Laitinen PJ, Brown KM, Piippo K, et al. Mutations of the cardiac ryanodine receptor (RyR2) gene in familial polymorphic ventricular tachycardia. Circulation. 2001;103: Tiso N, Stephan DA, Nava A, et al. Identification of mutations in the cardiac ryanodine receptor gene in families affected with arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2). Hum Mol Genet. 2001;10: Bauce B, Rampazzo A, Basso C, et al. 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