Mutations in Calmodulin Cause Ventricular Tachycardia and Sudden Cardiac Death

Abstract

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a devastating inherited disorder characterized by episodic syncope and/or sudden cardiac arrest during exercise or acute emotion in individuals without structural cardiac abnormalities. Although rare, CPVT is suspected to cause a substantial part of sudden cardiac deaths in young individuals. Mutations in RYR2, encoding the cardiac sarcoplasmic calcium channel, have been identified as causative in approximately half of all dominantly inherited CPVT cases. Applying a genome-wide linkage analysis in a large Swedish family with a severe dominantly inherited form of CPVT-like arrhythmias, we mapped the disease locus to chromosome 14q31-32. Sequencing CALM1 encoding calmodulin revealed a heterozygous missense mutation (c.161A>T [p.Asn53Ile]) segregating with the disease. A second, de novo, missense mutation (c.293A>G [p.Asn97Ser]) was subsequently identified in an individual of Iraqi origin; this individual was diagnosed with CPVT from a screening of 61 arrhythmia samples with no identified RYR2 mutations. Both CALM1 substitutions demonstrated compromised calcium binding, and p.Asn97Ser displayed an aberrant interaction with the RYR2 calmodulin-binding-domain peptide at low calcium concentrations. We conclude that calmodulin mutations can cause severe cardiac arrhythmia and that the calmodulin genes are candidates for genetic screening of individual cases and families with idiopathic ventricular tachycardia and unexplained sudden cardiac death. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a devastating inherited disorder characterized by episodic syncope and/or sudden cardiac arrest during exercise or acute emotion in individuals without structural cardiac abnormalities. Although rare, CPVT is suspected to cause a substantial part of sudden cardiac deaths in young individuals. Mutations in RYR2, encoding the cardiac sarcoplasmic calcium channel, have been identified as causative in approximately half of all dominantly inherited CPVT cases. Applying a genome-wide linkage analysis in a large Swedish family with a severe dominantly inherited form of CPVT-like arrhythmias, we mapped the disease locus to chromosome 14q31-32. Sequencing CALM1 encoding calmodulin revealed a heterozygous missense mutation (c.161A>T [p.Asn53Ile]) segregating with the disease. A second, de novo, missense mutation (c.293A>G [p.Asn97Ser]) was subsequently identified in an individual of Iraqi origin; this individual was diagnosed with CPVT from a screening of 61 arrhythmia samples with no identified RYR2 mutations. Both CALM1 substitutions demonstrated compromised calcium binding, and p.Asn97Ser displayed an aberrant interaction with the RYR2 calmodulin-binding-domain peptide at low calcium concentrations. We conclude that calmodulin mutations can cause severe cardiac arrhythmia and that the calmodulin genes are candidates for genetic screening of individual cases and families with idiopathic ventricular tachycardia and unexplained sudden cardiac death. Idiopathic ventricular tachycardia (VT) is a cardiac arrhythmia that is seen in individuals without structural heart disease. Depending on the ECG characteristics, it can be classified as monomorphic VT or polymorphic VT, the latter comprising a number of uncommon, often malignant familial disorders, including catecholaminergic polymorphic VT (CPVT [MIM 604772]).1Badhwar N. Scheinman M.M. Idiopathic ventricular tachycardia: Diagnosis and management.Curr. Probl. Cardiol. 2007; 32: 7-43Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar CPVT is characterized by episodic syncope and/or sudden cardiac arrest induced by exercise or acute emotion.2Coumel P. Fidelle J. Lucet V. Attuel P. Bouvrain Y. Catecholaminergic-induced severe ventricular arrhythmias with Adams-Stokes syndrome in children: Report of four cases.Br. Heart J. 1978; 40: 28-37Google Scholar, 3Napolitano C. Priori S.G. Diagnosis and treatment of catecholaminergic polymorphic ventricular tachycardia.Heart Rhythm. 2007; 4: 675-678Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar The ECG is usually within normal limits at rest and often displays prominent U waves, but ventricular arrhythmias might arise at times of adrenergic activation.4Aizawa Y. Komura S. Okada S. Chinushi M. Aizawa Y. Morita H. Ohe T. Distinct U wave changes in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT).Int. Heart J. 2006; 47: 381-389Crossref PubMed Scopus (38) Google Scholar, 5Viitasalo M. Oikarinen L. Väänänen H. Kontula K. Toivonen L. Swan H. U-waves and T-wave peak to T-wave end intervals in patients with catecholaminergic polymorphic ventricular tachycardia, effects of beta-blockers.Heart Rhythm. 2008; 5: 1382-1388Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar The arrhythmias, typically bidirectional and/or polymorphic VT, can develop into ventricular fibrillation and sudden death, leading to a high mortality rate (30%–50% by the age of 30) for this disorder.6Fisher J.D. Krikler D. Hallidie-Smith K.A. Familial polymorphic ventricular arrhythmias: A quarter century of successful medical treatment based on serial exercise-pharmacologic testing.J. Am. Coll. Cardiol. 1999; 34: 2015-2022Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar CPVT often manifests in childhood, and a family history of juvenile sudden death and stress-induced syncope is present in approximately one-third of the cases. It can present as sudden death in children without any prior signs or warning,6Fisher J.D. Krikler D. Hallidie-Smith K.A. Familial polymorphic ventricular arrhythmias: A quarter century of successful medical treatment based on serial exercise-pharmacologic testing.J. Am. Coll. Cardiol. 1999; 34: 2015-2022Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar and it has been estimated to cause up to 15% of unexplained sudden cardiac deaths in young people.7Liu N. Ruan Y. Priori S.G. Catecholaminergic polymorphic ventricular tachycardia.Prog. Cardiovasc. Dis. 2008; 51: 23-30Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar Previously, familial assessment revealed little evidence of the condition in many victims of unexplained sudden death.8Behr E.R. Dalageorgou C. Christiansen M. Syrris P. Hughes S. Tome Esteban M.T. Rowland E. Jeffery S. McKenna W.J. Sudden arrhythmic death syndrome: Familial evaluation identifies inheritable heart disease in the majority of families.Eur. Heart J. 2008; 29: 1670-1680Crossref PubMed Scopus (333) Google Scholar, 9Tester D.J. Spoon D.B. Valdivia H.H. Makielski J.C. Ackerman M.J. 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: 1380-1384Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar Mutations in the ryanodine receptor 2 gene (RYR2 [MIM 180902]) are known to cause dominantly inherited CPVT10Priori S.G. Napolitano C. Tiso N. Memmi M. Vignati G. Bloise R. Sorrentino V. Danieli G.A. Mutations in the cardiac ryanodine receptor gene (hRyR2) underlie catecholaminergic polymorphic ventricular tachycardia.Circulation. 2001; 103: 196-200Crossref PubMed Scopus (1145) Google Scholar (CPVT1 [MIM 604772], and more than 70 different mutations are currently known. A less common autosomal-recessive form of the disorder (CPVT2 [MIM 611938]) is caused by mutations in the calsequestrin-2 gene11Lahat H. Pras E. Olender T. Avidan N. Ben-Asher E. Man O. Levy-Nissenbaum E. Khoury A. Lorber A. Goldman B. et al.A missense mutation in a highly conserved region of CASQ2 is associated with autosomal recessive catecholamine-induced polymorphic ventricular tachycardia in Bedouin families from Israel.Am. J. Hum. Genet. 2001; 69: 1378-1384Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar (CASQ2 [MIM 114251]). Mutations in these genes together explain a little more than half of all familial CPVT cases. Mutations in KCNJ2 [MIM 600681] and ANK2 [MIM 106410], normally causing Andersen-Tawil Syndrome (ATS [MIM 170390]) and type 4 long-QT syndrome (LQT4 [MIM 600919]), respectively, have in addition been found in a low number of individuals diagnosed with CPVT.12Mohler P.J. Splawski I. Napolitano C. Bottelli G. Sharpe L. Timothy K. Priori S.G. Keating M.T. Bennett V. A cardiac arrhythmia syndrome caused by loss of ankyrin-B function.Proc. Natl. Acad. Sci. USA. 2004; 101: 9137-9142Crossref PubMed Scopus (276) Google Scholar, 13Tester D.J. Arya P. Will M. Haglund C.M. Farley A.L. Makielski J.C. Ackerman M.J. Genotypic heterogeneity and phenotypic mimicry among unrelated patients referred for catecholaminergic polymorphic ventricular tachycardia genetic testing.Heart Rhythm. 2006; 3: 800-805Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar Finally, a locus for a severe form of CPVT (CPVT3 [MIM 614021]) was in 2007 localized to chromosome 7 without the identification of the disease gene.14Bhuiyan Z.A. Hamdan M.A. Shamsi E.T. Postma A.V. Mannens M.M. Wilde A.A. Al-Gazali L. A novel early onset lethal form of catecholaminergic polymorphic ventricular tachycardia maps to chromosome 7p14-p22.J. Cardiovasc. Electrophysiol. 2007; 18: 1060-1066Crossref PubMed Scopus (61) Google Scholar Tightly controlled cycling of the intracellular calcium concentration is the basis for cardiac muscle contraction and determination of the heart rhythm. RYR2 is the sarcoplasmic reticulum (SR) calcium release channel. Upon a small increase in local intracellular Ca2+ concentration (from approximately 100 nM to a few micromolar), this channel switches from a closed to an open conformation, resulting in a large influx of Ca2+ from the SR storage and ultimately causing muscle contraction. The current molecular understanding of RYR2-associated VT is that RYR2 mutations render the tetrameric RYR2 complex “leaky,” thereby leading to increased local Ca2+ concentrations (Ca2+ sparks), untimely activation of nearby RYR2 clusters through calcium-induced calcium release (CICR), and eventual arrhythmia.15Cerrone M. Napolitano C. Priori S.G. Catecholaminergic polymorphic ventricular tachycardia: A paradigm to understand mechanisms of arrhythmias associated to impaired Ca(2+) regulation.Heart Rhythm. 2009; 6: 1652-1659Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 16Kontula K. Laitinen P.J. Lehtonen A. Toivonen L. Viitasalo M. Swan H. Catecholaminergic polymorphic ventricular tachycardia: Recent mechanistic insights.Cardiovasc. Res. 2005; 67: 379-387Crossref PubMed Scopus (84) Google Scholar The precise molecular mechanism is, however, still unclear, and it has been put forward that an abnormal interaction with one or more of the associated proteins or ions, such as FKBP12.6 or CASQ2, or abnormal activation by extraluminal or intraluminal Ca2+ ions also might play a role, as reviewed by Kontula et al.16Kontula K. Laitinen P.J. Lehtonen A. Toivonen L. Viitasalo M. Swan H. Catecholaminergic polymorphic ventricular tachycardia: Recent mechanistic insights.Cardiovasc. Res. 2005; 67: 379-387Crossref PubMed Scopus (84) Google Scholar and Mohler et al.17Mohler P.J. Wehrens X.H. Mechanisms of human arrhythmia syndromes: Abnormal cardiac macromolecular interactions.Physiology (Bethesda). 2007; 22: 342-350Crossref PubMed Scopus (27) Google Scholar Here we report a Swedish multiplex family presenting with a history of ventricular arrhythmias, syncopes and sudden death, predominantly in association with physical exercise or stress (Figure 1A). The index case (II:6), a now 42-year old man of Swedish ethnic origin, presented with syncope while playing football at the age of 12. The ECG at admission (Figure 1B, upper panel) showed sinus bradycardia (HR = 45/min) and prominent U-wave in V2 and V3, but no evidence of QT prolongation (QTc = 0.42 s). He had a history of loss of consciousness on at least four occasions during physical activity and once in connection with a fire alarm. A 24 hr ECG registration revealed ventricular extrasystoles (VES), bigemini, and paired VES (Figure 1B, lower panel) during football training, but no symptoms were reported. He was started on β1-adrenergic-receptor blocker treatment. At follow up, a 24 hr ECG and exercise test still showed some VES and bigemini under conditions of increasing load and heart rate. A genomic DNA sample from the index case was later screened for mutations in a panel of arrhythmia-associated genes, including RYR2 and CASQ2, where no mutations were identified. An older brother (II:4), aged 23, had a history of repeated syncope during exercise. During an exercise test, he displayed polymorphic VT (an ECG from that time is not available). After treatment with β1-adrenergic-receptor blocker, a follow-up exercise test showed VES at high loads. The family history also included a brother (II:3) who drowned during a swimming competition at age 15 after having had prior episodes of syncope and an older sister (II:2) who from or After a later of ventricular fibrillation was by treatment with β1-adrenergic-receptor blocker and A sister presented at the age of 7 with a history of repeated syncope during physical family with syncope from age at age having been with β1-adrenergic-receptor blocker for family started having syncope at age was under treatment with β1-adrenergic-receptor blocker, and cardiac arrest at age After and and had an An older sister presented with syncope from age and under treatment with β1-adrenergic-receptor A sister presented with syncope at age and were put on β1-adrenergic-receptor blocker after having syncope and of to the cases of syncope in and was on at the phenotypic of the family is characterized by CPVT-like with symptoms including syncope and cases of sudden death or cardiac The family no were in with the of the and on human After from all the we genomic DNA from samples from all the to linkage and by an in were with the The were all older than at the time of were by the and to the for with were with analysis of genetic maps gene Genet. PubMed Scopus Google Scholar and from the of monomorphic and a of and an of was was with S. B. K. L. M.A. D. J. P. de M.J. A for association and linkage J. Hum. Genet. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar and in a of in linkage with was for the identification of resulting in the of from the A linkage analysis with and a disease gene of was with with from the and genetic from the were identified and with a of (Figure in the A follow-up analysis with and in these was and included an from the family and and and individuals with the were were from the The analysis the locus on chromosome and mapped the disease locus to chromosome (Figure A analysis the of the chromosome disease in all and of the individuals (Figure of the mutation in this The peak on chromosome approximately from to a region that 70 genes (Figure and of the of calmodulin in calcium and heart CALM1 was for the and the and of CALM1 were with S. H. on the for and for PubMed Google Scholar The were and on at Sequencing one family index and one family revealed a heterozygous missense mutation in mutation in a from to without the to the in the index case (Figure A for for the was by with that the was present in all individuals and in all of the individuals in the of individuals medical from and showed that the mutation was among these sample an to a with a of and mutations in J. Hum. Genet. Full Text Full Text PDF PubMed Scopus (113) Google Scholar that the mutation is highly to be a normal but for mutations in we a for of the on the basis of of were with and referred for RYR2 mutation analysis at the in genetic to any RYR2 mutation in 61 of these were in samples with aberrant as as samples with normal were are in this screening a heterozygous CALM1 missense mutation was identified in an individual of Iraqi The mutation was in and in an (Figure DNA revealed that this mutation was in the and the of showed signs of heart arrhythmias, the of a de mutation in this A analysis the The individual with the de CALM1 mutation was a who presented at age 4 with a cardiac arrest to while was a and was by treatment with β1-adrenergic-receptor An ECG and were within normal and was no evidence of QT prolongation of family was An exercise ECG and on and ventricular and were within normal An of idiopathic was at the demonstrated prominent U in the but no evidence of the long-QT or (Figure upper was including cardiac and and were An exercise ECG while was β1-adrenergic-receptor blocker demonstrated ventricular with and of into (Figure lower to be bidirectional at this a of CPVT was but genetic of RYR2, CASQ2, and KCNJ2 to any mutations in these a follow-up genetic test of arrhythmia-associated genes, including and no mutations were more in and at age 15 a cardiac After an was and but was diagnosed with [MIM aged was heart to from treatment. not syncope or An ECG demonstrated and aged had syncope in with ECG was and exercise for had induced changes without An was as and Both CALM1 mutations to an and highly that to the severe end of the of CPVT-like with an to with β1-adrenergic-receptor blocker was with symptoms in the Iraqi case and in individuals and in family The that p.Asn97Ser is a de is with the of the associated disease as a highly malignant form with early and is with the RYR2-associated is caused by de S.G. Napolitano C. Memmi M. B. M. L. Bloise R. R. et and molecular of patients with catecholaminergic polymorphic ventricular tachycardia.Circulation. PubMed Scopus Google Scholar CALM1 missense mutations in the a of all CALM1 were in individuals. missense mutations were identified among these were identified were present among the missense mutations in this A Iraqi was not the mutation was de novo, and the mutation rate is to be the in all missense mutations in CALM1 were found from the through the release an of and from and of A also to present evidence for any identified calmodulin mutations. The of identified missense mutations is not that calmodulin is known as one of the conserved displays an in all and has the from (Figure The of genes in the human [MIM and all encoding calmodulin the any changes in this is a four that one calcium It is as a and is of a of small intracellular changes to a of intracellular in the and for of local and Ca2+ by calmodulin in complex with a Ca2+ 2008; Full Text Full Text PDF PubMed Scopus Google Scholar The the and and the the and The identified are in the is on the of the of in the and the is one of the of in the calmodulin (Figure to and the activity of a large number of intracellular but in of the calmodulin are the in with any or by the peptide complex in the we a to the CALM1 are in from a from M.T. C. Christiansen M. L. J. of and the of in human and 1999; PubMed Scopus Google Scholar and we this into a a mutations were with are in The calmodulin were in and were with and and and all was in and that Ca2+ be The Ca2+ concentration of the was to be and with the calcium and and an proteins were by and concentrations were by at The and of was by the of and calmodulin was calcium to a large in calmodulin and the it is to Ca2+ to the by the changes in M.A. to calmodulin having effects on the of 2006; PubMed Scopus Google Scholar were with while the at was on a 4 with a at The Ca2+ demonstrated a in the Ca2+ for the p.Asn97Ser (Figure as by an concentration of to Ca2+ ions calmodulin to Ca2+ for calmodulin Ca2+ The on the demonstrated a but Ca2+ and an half of Ca2+ the is in the of we that this Ca2+ a Ca2+ and to increased of Ca2+ for the to at lower Ca2+ A less is that it an increased the and we that to calcium of calmodulin and that the different effects on the of Ca2+ the and of the interaction the calmodulin and a peptide from RYR2 the calmodulin was the of calmodulin to the peptide from the of calmodulin the of the peptide to RYR2 to calmodulin to an in on the is by a large increase in of the RYR2 calmodulin not any The at of RYR2 was with and without addition of of calmodulin under four different no Ca2+ including conditions conditions of calmodulin by and effects on Ca2+ and for Sci. 6: PubMed Scopus Google Scholar and Ca2+ conditions all in 100 The concentrations for and conditions were controlled with an as C. S. D. in to in 2004; PubMed Scopus Google Scholar A concentration of RYR2 peptide was with no calmodulin (RYR2 calmodulin and 100 nM or calmodulin and of calmodulin were at low calcium concentrations nM a in the was demonstrated for p.Asn97Ser as to and The p.Asn97Ser induced a and no increase in (Figure to the seen calmodulin to the ryanodine receptor J. to calmodulin to an in on the ryanodine 2001; PubMed Scopus Google Scholar peptide in the of little or no in the was and calmodulin at or calcium concentrations and that for the p.Asn97Ser the interaction is at low intracellular Ca2+ concentrations and is at to high Ca2+ concentrations. It is that calmodulin to RYR2, and it has been demonstrated that calmodulin the that RYR2 has an open at concentrations to 100 N. L. 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PubMed Scopus (61) Google Scholar are the human to the molecular disease in an aberrant interaction is causative of CPVT-like The not a compromised RYR2 interaction in the of is that we the interaction by a small peptide of the large RYR2 molecular it is at present the the of RYR2 in the of was different from the and p.Asn97Ser and it is that a different molecular mechanism is in play in family be of the mechanism for mutations in CPVT that calmodulin and is for a number of and in contraction. such and the calcium channel, and the P.J. Splawski I. Napolitano C. Bottelli G. Sharpe L. Timothy K. Priori S.G. Keating M.T. Bennett V. A cardiac arrhythmia syndrome caused by loss of ankyrin-B function.Proc. Natl. Acad. Sci. USA. 2004; 101: 9137-9142Crossref PubMed Scopus (276) Google Scholar the screened included individuals who had arrhythmias or sudden cardiac death and had been referred for RYR2 CALM1 mutations were the family index case and the Iraqi case were included in this the of CALM1 mutations is low from this individuals were found to be for RYR2 the of CALM1 and RYR2 mutations were in this characterized and sample the and phenotypic of CALM1 mutations in CPVT and screening of large this is we that CALM1 is a to we have identified CALM1 missense mutations that to of CPVT or CPVT-like genetic in families in with this severe disorder CALM1 the of treatment for children and young individuals with disease mutations. a of the calmodulin genes are candidates for genetic screening of individuals with CPVT-like arrhythmia and unexplained sudden cardiac death. We all and families for We and K. for was by the the the the and the with and and The for presented are as Sequencing in Man The for the in this are for for for and for