Reduced Na + Current in Native Cardiomyocytes of a Brugada Syndrome Patient Associated With β-2-Syntrophin Mutation
2018; Wolters Kluwer; Volume: 11; Issue: 11 Linguagem: Inglês
10.1161/circgen.118.002263
ISSN2574-8300
AutoresConstanze Schmidt, Felix Wiedmann, Ibrahim El‐Battrawy, Markus Fritz, Antonius Ratte, Carsten J. Beller, Siegfried Lang, Boris Rudic, Rainer Schimpf, İbrahim Akın, Matthias Karck, Martin Borggrefe, Hugo A. Katus, Xiao-Bo Zhou, Dierk Thomas,
Tópico(s)Neuroscience and Neural Engineering
ResumoHomeCirculation: Genomic and Precision MedicineVol. 11, No. 11Reduced Na+ Current in Native Cardiomyocytes of a Brugada Syndrome Patient Associated With β-2-Syntrophin Mutation Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBReduced Na+ Current in Native Cardiomyocytes of a Brugada Syndrome Patient Associated With β-2-Syntrophin Mutation Constanze Schmidt, MD, Felix Wiedmann, MD, Ibrahim El-Battrawy, MD, Markus Fritz, MD, Antonius Ratte, , MD, Carsten J. Beller, MD, Siegfried Lang, PhD, Boris Rudic, MD, Rainer Schimpf, MD, Ibrahim Akin, MD, Matthias Karck, MD, Martin Borggrefe, MD, Hugo A. Katus, MD, PhD, Xiao-Bo Zhou, MD and Dierk Thomas, MD Constanze SchmidtConstanze Schmidt Department of Cardiology, University Hospital Heidelberg (C.S., F.W., A.R., H.A.K., D.T.) DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, University Hospital Heidelberg (C.S., F.W., I.E.-B., S.L., B.R., R.S., I.A., M.B., H.A.K., X.-B.Z., D.T.) HCR (Heidelberg Center for Heart Rhythm Disorders), University Hospital Heidelberg (C.S., F.W., A.R. H.A.K., D.T.) , Felix WiedmannFelix Wiedmann Department of Cardiology, University Hospital Heidelberg (C.S., F.W., A.R., H.A.K., D.T.) DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, University Hospital Heidelberg (C.S., F.W., I.E.-B., S.L., B.R., R.S., I.A., M.B., H.A.K., X.-B.Z., D.T.) HCR (Heidelberg Center for Heart Rhythm Disorders), University Hospital Heidelberg (C.S., F.W., A.R. H.A.K., D.T.) , Ibrahim El-BattrawyIbrahim El-Battrawy HCR (Heidelberg Center for Heart Rhythm Disorders), University Hospital Heidelberg (C.S., F.W., A.R. H.A.K., D.T.) First Department of Medicine, University Medical Center Mannheim, Germany (I.E.-B., S.L., B.R., R.S., I.A., M.B., X.-B.Z.). , Markus FritzMarkus Fritz Department of Cardiac Surgery, University Hospital Heidelberg (M.F., C.J.B., M.K.) , Antonius RatteAntonius Ratte Department of Cardiology, University Hospital Heidelberg (C.S., F.W., A.R., H.A.K., D.T.) HCR (Heidelberg Center for Heart Rhythm Disorders), University Hospital Heidelberg (C.S., F.W., A.R. H.A.K., D.T.) , Carsten J. BellerCarsten J. Beller Department of Cardiac Surgery, University Hospital Heidelberg (M.F., C.J.B., M.K.) , Siegfried LangSiegfried Lang HCR (Heidelberg Center for Heart Rhythm Disorders), University Hospital Heidelberg (C.S., F.W., A.R. H.A.K., D.T.) First Department of Medicine, University Medical Center Mannheim, Germany (I.E.-B., S.L., B.R., R.S., I.A., M.B., X.-B.Z.). , Boris RudicBoris Rudic HCR (Heidelberg Center for Heart Rhythm Disorders), University Hospital Heidelberg (C.S., F.W., A.R. H.A.K., D.T.) First Department of Medicine, University Medical Center Mannheim, Germany (I.E.-B., S.L., B.R., R.S., I.A., M.B., X.-B.Z.). , Rainer SchimpfRainer Schimpf HCR (Heidelberg Center for Heart Rhythm Disorders), University Hospital Heidelberg (C.S., F.W., A.R. H.A.K., D.T.) First Department of Medicine, University Medical Center Mannheim, Germany (I.E.-B., S.L., B.R., R.S., I.A., M.B., X.-B.Z.). , Ibrahim AkinIbrahim Akin HCR (Heidelberg Center for Heart Rhythm Disorders), University Hospital Heidelberg (C.S., F.W., A.R. H.A.K., D.T.) First Department of Medicine, University Medical Center Mannheim, Germany (I.E.-B., S.L., B.R., R.S., I.A., M.B., X.-B.Z.). , Matthias KarckMatthias Karck Department of Cardiac Surgery, University Hospital Heidelberg (M.F., C.J.B., M.K.) , Martin BorggrefeMartin Borggrefe HCR (Heidelberg Center for Heart Rhythm Disorders), University Hospital Heidelberg (C.S., F.W., A.R. H.A.K., D.T.) First Department of Medicine, University Medical Center Mannheim, Germany (I.E.-B., S.L., B.R., R.S., I.A., M.B., X.-B.Z.). , Hugo A. KatusHugo A. Katus Department of Cardiology, University Hospital Heidelberg (C.S., F.W., A.R., H.A.K., D.T.) DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, University Hospital Heidelberg (C.S., F.W., I.E.-B., S.L., B.R., R.S., I.A., M.B., H.A.K., X.-B.Z., D.T.) HCR (Heidelberg Center for Heart Rhythm Disorders), University Hospital Heidelberg (C.S., F.W., A.R. H.A.K., D.T.) , Xiao-Bo ZhouXiao-Bo Zhou Xiao-Bo Zhou, MD, First Department of Medicine, University Medical Center Mannheim, Theodor-Kutzer-Ufer 1-3, Mannheim 68167, Germany, Email E-mail Address: [email protected] DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, University Hospital Heidelberg (C.S., F.W., I.E.-B., S.L., B.R., R.S., I.A., M.B., H.A.K., X.-B.Z., D.T.) First Department of Medicine, University Medical Center Mannheim, Germany (I.E.-B., S.L., B.R., R.S., I.A., M.B., X.-B.Z.). and Dierk ThomasDierk Thomas Dierk Thomas, MD, Department of Cardiology, University of Heidelberg, Im Neuenheimer Feld 410, Heidelberg D-69120, Germany, Email E-mail Address: [email protected] Department of Cardiology, University Hospital Heidelberg (C.S., F.W., A.R., H.A.K., D.T.) DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, University Hospital Heidelberg (C.S., F.W., I.E.-B., S.L., B.R., R.S., I.A., M.B., H.A.K., X.-B.Z., D.T.) HCR (Heidelberg Center for Heart Rhythm Disorders), University Hospital Heidelberg (C.S., F.W., A.R. H.A.K., D.T.) Originally published2 Nov 2018https://doi.org/10.1161/CIRCGEN.118.002263Circulation: Genomic and Precision Medicine. 2018;11:e002263We assessed the pathophysiology of Brugada syndrome (BrS) in primary cardiomyocytes obtained from a 64-year-old male patient. At age 54 the patient had experienced syncope and presented with type 1 ST-segment elevation on the surface ECG during a febrile infection. Whole exome sequencing was performed to identify the genetic basis of BrS, yielding 88 325 variants in the index patient. Filtering out noncoding, synonymous, and benign variants yielded 11 nonsynonymous variants. In silico analyses predicted potential pathogenicity for 4 out of 11 variants, located in genes encoding for SNTB2 (β-2-syntrophin), obscurin, the α-1H subunit of the voltage-dependent T-type calcium channel, and LRP1 (low-density lipoprotein receptor-related protein-1). The present work represents a candidate gene study, focusing on the role of SNTB2 in BrS pathophysiology. Potential mechanistic contribution of other variants to BrS, for example through interaction between obscurin and Nav1.5 via ankyrin, remains to be investigated. Syntrophins interact directly with the cardiac Nav1.5 channel at a binding domain consisting of an SIV (Ser-Ile-Val) motif.1,2 A BrS-associated SCN5A missense mutation in the SIV motif decreased Nav1.5 surface expression and Na+ currents in human embryonic kidney (HEK298T) cells.3 In the BrS patient, direct sequencing confirmed the presence of a heterozygous SNTB2-N167K mutation located in the Nav1.5 binding region. Sanger sequencing was extended to an independent cohort of 115 unrelated patients with BrS. No SNTB2 mutations were detected in these patients, reflecting low frequency of the mutated allele.To assess the molecular pathophysiology of BrS associated with SNTB2-N167K, right atrial tissue samples were obtained from the index patient during coronary artery bypass grafting at age 64. Reduced cardiac abundance of SNTB2 and Nav1.5 protein in the BrS patient is illustrated by immunoblots of right atrial protein lysates of the index patient in comparison with 3 unrelated control subjects undergoing open heart surgery for coronary artery bypass grafting (Figure [A]).Download figureDownload PowerPointFigure. Identification and characterization of SNTB2 (β-2-syntrophin) mutation associated with Brugada syndrome (BrS). A, Representative SNTB2 and Nav1.5 immunoblots are shown for human right atrial tissue lysates obtained from the BrS patient and from 3 unaffected control subjects (probands No.: 1–3), respectively. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is blotted for comparison. B, Sodium currents in native human atrial cardiomyocytes (inset, microscopic view) of the BrS index patient and of an unaffected proband. Currents were generated by 400 ms depolarizations to −50 mV, holding potential: −100 mV. C, Mean peak INa densities in cardiomyocytes obtained from the index patient and from control subjects, quantified at −50 mV using the protocol described in (B). D, Late cardiac sodium current (INa-L) in human cardiomyocytes, activated by depolarizing pulses from −100 to −50 mV (400 ms) and quantified at 200 ms. Superimposed representative currents recorded from the affected BrS patient and from a control subject are shown. Inset, a summary of INa-L current densities in cells obtained from the index patient and from controls. E, Representative atrial action potentials (AP) recorded at 1 Hz from the BrS patient and from a proband are shown. F, Corresponding AP durations (APD) at 50% (APD50) and 90% repolarization (APD90). Mean data are provided±SEM, n/N indicates number of cardiomyocytes/patients. Independent sample t tests were used for statistical comparisons. G and H, Immunofluorescence analysis of SNTB2 and Nav1.5 expression in human embryonic kidney (HEK293T) cells. Green fluorescence in photomicrographs reflects anti-FLAG antibody staining wild-type (WT; G) or mutant (H) SNTB2 proteins carrying FLAG epitopes (i.e. the octapeptide DYKDDDDK), and red staining indicates anti-HA epitope detection of Nav1.5-HA protein. Scale bar, 10 µm. I, Current-voltage relationships for peak INa produced by expression of indicated subunits in Xenopus oocytes. Sodium currents were evoked by step depolarizations (5 ms) to voltages between −80 mV and −10 mV in 10 mV increments. Mean data are provided±SEM. J, Peak sodium currents. K, Representative families of currents analyzed in (I and J). L, Coimmunoprecipitation (IP) of Nav1.5 with SNTB2 in HEK293T cells. Anti-FLAG antibodies detecting SNTB2-FLAG and anti-HA antibodies binding Nav1.5-HA were used for IP and immunoblotting (IB), and vice versa. Coprecipitation of SNTB2 and Nav1.5 was not observed in the presence of SNTB2-N167K. M, Cellular mechanism of BrS associated with SNTB2-N167K-mutation. Mutation of SNTB2 in a BrS patient disrupts regional and functional interaction with Nav1.5 in cardiac myocytes, resulting in reduced cardiac sodium current compared with a healthy subject. For scatter dot plots, center line shows mean, error bars indicate SEM (*P<0.001 and †P<0.05).Cardiac myocytes were isolated from right atrial samples and subjected to electrophysiological recordings to assess functional consequences of the PDZ domain mutation in SNTB2 (Figure [B]). Peak INa density at −50 mV was reduced by 86.7% in the BrS patient (n=10) compared with control subjects (n=8 cells obtained from N=3 individuals; P=0.0008; Figure [C]). Current reduction was accompanied by a depolarizing shift of the half-maximal activation voltage from −57.9±1.7 mV (probands; n/N=8/3) to −43.5±1.0 mV (BrS patient; n=5; P<0.0001). In addition to peak INa, the late sodium current (INa-L) has been implicated in arrhythmogenesis and antiarrhythmic therapy and was modulated by interaction with α-1-syntrophin in vitro.4,5INa-L density was reduced by 72.1% (n=8) in the BrS patient compared with non-affected controls (n/N=8/3; P=0.049; Figure [D]). Reduced late sodium current is expected to shorten cardiac action potential duration (APD). In cardiomyocytes of the BrS patient, APD at 90% repolarization (APD90) was 46.9% shorter (115±14.6 ms; n=6) compared with 217±11.6 ms in probands (n/N=8/6; P=0.0003; Figure [E and F]). APD at 50% of repolarization (APD50) was similarly shortened.To further elucidate the cellular mechanism underlying SNTB2-N167K-associated BrS, wild-type (WT), or mutant SNTB2 were coexpressed with human Nav1.5 in HEK293T cells. Colocalization of WT SNTB2 and Nav1.5 (Figure [G]) was not observed when SNTB2-N167K was expressed (Figure [H]), suggesting defective interaction between the proteins. Reduced sodium current upon coexpression of Nav1.5 with WT SNTB2 and SNTB2-N167K (to mimic heterozygous conditions in the index patient) or with SNTB2-N167K alone (to model homozygous conditions) was recapitulated in the Xenopus oocyte expression system (Figure [I through K]). Significant Na+ current reduction in homozygous conditions and similar Na+ current levels in oocytes mimicking the heterozygous situation suggest a dominant-negative effect of mutant SNTB2-N167K on its WT counterpart. Sodium currents produced by coexpression of Nav1.5 and SNTB2-N167K (−11.0±2.5 µA; n=7) were 58.2% lower compared with coexpression of Nav1.5 with WT SNTB2 (−26.2±3.9 µA; n=5; P=0.01). Physical interaction between WT SNTB2 and Nav1.5 was finally demonstrated by coimmunoprecipitation of both proteins (Figure [L]). By contrast, mutant SNTB2-N167K was not isolated when Nav1.5 was immunopurified (and vice versa), confirming loss of SNTB2-Nav1.5 interaction caused by the BrS-associated mutation.Limitations arise from the use of atrial cardiomyocytes because ventricular tissue was not available. Nonetheless, functional and regulatory similarities between atrial and ventricular INa indicate that the findings of the present work may be extrapolated to ventricular electrophysiology. Further limitations include the lack of functional rescue or knockin experiments (potentially including genome-edited induced pluripotent stem cell (iPSC)-derived cardiomyocytes) that would be required to establish the causal nature of the SNTB2 variant. In addition, the study represents a report from a single patient without appropriately matched control cells. Further studies are needed to validate this hypothesis-driven study in a larger number of BrS patients and appropriate controls.In conclusion, reduced INa underlying BrS was functionally demonstrated in native human cardiomyocytes. SNTB2 was identified as a novel gene associated with BrS. Mutation of SNTB2 reduced Nav1.5 current through defective functional Nav1.5-SNTB2 interaction (Figure [M]).AcknowledgmentsWe gratefully acknowledge the excellent technical support of Katrin Kupser and Kai Sona. We thank Stefan Kallenberger for helpful discussions and critical comments. The data, analytic methods, and study materials will be made available to other researchers for purposes of reproducing the results or replicating the procedure upon reasonable request.Sources of FundingThis study was supported in part by research grants from the University of Heidelberg, Faculty of Medicine (Rahel Goitein-Straus Scholarship and Olympia-Morata Scholarship to Dr Schmidt), from the DZHK (German Center for Cardiovascular Research; Excellence Grant to Dr Schmidt), from the German Heart Foundation/German Foundation of Heart Research (F/41/15 to Dr Schmidt, F/08/14 to Dr Thomas), from the Else Kröner-Fresenius-Stiftung (2014_A242 to Dr Thomas), from the Joachim Siebeneicher Foundation (to Dr Thomas), from the Deutsche Forschungsgemeinschaft (German Research Foundation; SCHM 3358/1-1 to Dr Schmidt, TH 1120/7-1 to Dr Thomas), and from the Ministry of Science, Research and the Arts Baden-Wuerttemberg (Sonderlinie Medizin to Dr Thomas). Dr Wiedmann was supported by the Otto-Hess-Scholarship and a Fellowship of the German Cardiac Society. A. Ratte was supported by the Kaltenbach-Scholarship of the German Heart Foundation/German Foundation of Heart Research.DisclosuresNone.Footnotes*Drs Schmidt, Wiedmann, and El-Battrawy are joint first authors.†Drs Zhou and Thomas contributed equally to this work.Xiao-Bo Zhou, MD, First Department of Medicine, University Medical Center Mannheim, Theodor-Kutzer-Ufer 1-3, Mannheim 68167, Germany, Email Xiaobo.[email protected]uni-heidelberg.deDierk Thomas, MD, Department of Cardiology, University of Heidelberg, Im Neuenheimer Feld 410, Heidelberg D-69120, Germany, Email dierk.[email protected]uni-heidelberg.deReferences1. Ou Y, et al. Syntrophin gamma 2 regulates SCN5A gating by a PDZ domain-mediated interaction.J Biol Chem. 2003; 278:1915–1923. doi: 10.1074/jbc.M209938200CrossrefMedlineGoogle Scholar2. Gavillet B, et al. 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Zimmermann P, Aberer F, Braun M, Sourij H and Moser O (2022) The Arrhythmogenic Face of COVID-19: Brugada ECG Pattern in SARS-CoV-2 Infection, Journal of Cardiovascular Development and Disease, 10.3390/jcdd9040096, 9:4, (96) Daimi H, Lozano-Velasco E, Aranega A and Franco D (2022) Genomic and Non-Genomic Regulatory Mechanisms of the Cardiac Sodium Channel in Cardiac Arrhythmias, International Journal of Molecular Sciences, 10.3390/ijms23031381, 23:3, (1381) El-Battrawy I, Lang S, Kowitz J, Zhou X and Akin I (2022) Human induced pluripotent stem cells for modeling Brugada syndrome Current Topics in iPSCs Technology, 10.1016/B978-0-323-99892-5.00011-6, (361-372), . 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November 2018Vol 11, Issue 11 Advertisement Article InformationMetrics © 2018 American Heart Association, Inc.https://doi.org/10.1161/CIRCGEN.118.002263PMID: 30571189 Originally publishedNovember 2, 2018 Keywordschannelopathiessodium channelsexomeBrugada syndromemutationPDF download Advertisement SubjectsArrhythmiasBasic Science ResearchIon Channels/Membrane TransportSudden Cardiac DeathVentricular Fibrillation
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