Identification of a new modulator of the intercalated disc in a zebrafish model of arrhythmogenic cardiomyopathy
Angeliki Asimaki, Sudhir Kapoor, Eva Plovie, Anne Karin Arndt, Edward Adams, ZhenZhen Liu, Cynthia A James, Daniel P Judge, Hugh Calkins, Jared Churko, Joseph C Wu, Calum A MacRae, André G Kléber, Jeffrey E Saffitz, Angeliki Asimaki, Sudhir Kapoor, Eva Plovie, Anne Karin Arndt, Edward Adams, ZhenZhen Liu, Cynthia A James, Daniel P Judge, Hugh Calkins, Jared Churko, Joseph C Wu, Calum A MacRae, André G Kléber, Jeffrey E Saffitz
Abstract
Arrhythmogenic cardiomyopathy (ACM) is characterized by frequent cardiac arrhythmias. To elucidate the underlying mechanisms and discover potential chemical modifiers, we created a zebrafish model of ACM with cardiac myocyte-specific expression of the human 2057del2 mutation in the gene encoding plakoglobin. A high-throughput screen identified SB216763 as a suppressor of the disease phenotype. Early SB216763 therapy prevented heart failure and reduced mortality in the fish model. Zebrafish ventricular myocytes that expressed 2057del2 plakoglobin exhibited 70 to 80% reductions in I(Na) and I(K1) current densities, which were normalized by SB216763. Neonatal rat ventricular myocytes that expressed 2057del2 plakoglobin recapitulated pathobiological features seen in patients with ACM, all of which were reversed or prevented by SB216763. The reverse remodeling observed with SB216763 involved marked subcellular redistribution of plakoglobin, connexin 43, and Nav1.5, but without changes in their total cellular content, implicating a defect in protein trafficking to intercalated discs. In further support of this mechanism, we observed SB216763-reversible, abnormal subcellular distribution of SAP97 (a protein known to mediate forward trafficking of Nav1.5 and Kir2.1) in rat cardiac myocytes expressing 2057del2 plakoglobin and in cardiac myocytes derived from induced pluripotent stem cells from two ACM probands with plakophilin-2 mutations. These observations pinpoint aberrant trafficking of intercalated disc proteins as a central mechanism in ACM myocyte injury and electrical abnormalities.
Copyright © 2014, American Association for the Advancement of Science.
Figures
References
- Basso C, Corrado D, Marcus FI, Nava A, Thiene G. Arrhythmogenic right ventricular cardiomyopathy. Lancet. 2009;373:1289–1300.
- Saffitz JE. The pathobiology of arrhythmogenic cardiomyopathy. Annu Rev Pathol. 2011;6:299–321.
- Sen-Chowdhry S, Syrris P, Prasad SK, Hughes SE, Merrifield R, Ward D, Pennell DJ, McKenna WJ. Left-dominant arrhythmogenic cardiomyopathy: An under-recognized clinical entity. J Am Coll Cardiol. 2008;52:2175–2187.
- Asimaki A, Tandri H, Huang H, Halushka MK, Gautam S, Basso C, Thiene G, Tsatsopoulou A, Protonotarios N, McKenna WJ, Calkins H, Saffitz JE. A new diagnostic test for arrhythmogenic right ventricular cardiomyopathy. N Engl J Med. 2009;360:1075–1084.
- Asimaki A, Saffitz JE. Gap junctions and arrhythmogenic cardiomyopathy. Heart Rhythm. 2012;9:992–995.
- Sato PY, Musa H, Coombs W, Guerrero-Serna G, Patiño GA, Taffet SM, Isom LL, Delmar M. Loss of plakophilin-2 expression leads to decreased sodium current and slower conduction velocity in cultured cardiac myocytes. Circ Res. 2009;105:523–526.
- Rizzo S, Lodder EM, Verkerk AO, Wolswinkel R, Beekman L, Pilichou K, Basso C, Remme CA, Thiene G, Bezzina CR. Intercalated disc abnormalities, reduced Na+ current density, and conduction slowing in desmoglein-2 mutant mice prior to cardiomyopathic changes. Cardiovasc Res. 2012;95:409–418.
- Noorman M, Hakim S, Kessler E, Groeneweg JA, Cox MG, Asimaki A, van Rijen HV, van Stuijvenberg L, Chkourko H, van der Heyden MA, Vos MA, de Jonge N, van der Smagt JJ, Dooijes D, Vink A, de Weger RA, Varro A, de Bakker JM, Saffitz JE, Hund TJ, Mohler PJ, Delmar M, Hauer RN, van Veen TA. Remodeling of the cardiac sodium channel, connexin43, and plakoglobin at the intercalated disk in patients with arrhythmogenic cardiomyopathy. Heart Rhythm. 2013;10:412–419.
- Garcia-Gras E, Lombardi R, Giocondo MJ, Willerson JT, Schneider MD, Khoury DS, Marian AJ. Suppression of canonical Wnt/β-catenin signaling by nuclear plakoglobin recapitulates phenotype of arrhythmogenic right ventricular cardiomyopathy. J Clin Invest. 2006;116:2012–2021.
- Ruiz P, Brinkmann V, Ledermann B, Behrend M, Grund C, Thalhammer C, Vogel F, Birchmeier C, Günthert U, Franke WW, Birchmeier W. Targeted mutation of plakoglobin in mice reveals essential functions of desmosomes in the embryonic heart. J Cell Biol. 1996;135:215–225.
- Gallicano GI, Kouklis P, Bauer C, Yin M, Vasioukhin V, Degenstein L, Fuchs E. Desmoplakin is required early in development for assembly of desmosomes and cytoskeletal linkage. J Cell Biol. 1998;143:2009–2022.
- Grossman KS, Grund C, Huelsken J, Behrend M, Erdmann B, Franke WW, Birchmeier W. Requirement of plakophilin 2 for heart morphogenesis and cardiac junction formation. J Cell Biol. 2004;167:149–160.
- Panáková D, Werdich AA, Macrae CA. Wnt11 patterns a myocardial electrical gradient through regulation of the L-type Ca2+ channel. Nature. 2010;466:874–878.
- Becker JR, Deo RC, Werdich AA, Panàkovà D, Coy S, MacRae CA. Human cardiomyopathy mutations induce myocyte hyperplasia and activate hypertrophic pathways during cardiogenesis in zebrafish. Dis Model Mech. 2011;4:400–410.
- McKoy G, Protonotarios N, Crosby A, Tsatsopoulou A, Anastasakis A, Coonar A, Norman M, Baboonian C, Jeffery S, McKenna WJ. Identification of a deletion in plakoglobin in arrhythmogenic right ventricular cardiomyopathy with palmoplantar keratoderma and woolly hair (Naxos disease) Lancet. 2000;355:2119–2124.
- Protonotarios N, Tsatsopoulou A. Naxos disease and carvajal syndrome: Cardiocutaneous disorders that highlight the pathogenesis and broaden the spectrum of arrhythmogenic right ventricular cardiomyopathy. Cardiovasc Pathol. 2004;13:185–194.
- Becker JR, Robinson TY, Sachidanandan C, Kelly AE, Coy S, Peterson RT, MacRae CA. In vivo natriuretic peptide reporter assay identifies chemical modifiers of hypertrophic cardio-myopathy signalling. Cardiovasc Res. 2012;93:463–470.
- Eldar-Finkelman H, Martinez A. GSK-3 inhibitors: Preclinical and clinical focus on CNS. Front Mol Neurosci. 2011;4:32.
- Mallat Z, Tedgui A, Fontaliran F, Frank R, Durigon M, Fontaine G. Evidence of apoptosis in arrhythmogenic right ventricular dysplasia. N Engl J Med. 1996;335:1190–1196.
- Asimaki A, Tandri H, Duffy ER, Winterfield JR, Mackey-Bojack S, Picken MM, Cooper LT, Wilber DJ, Marcus FI, Basso C, Thiene G, Tsatsopoulou A, Protonotarios N, Stevenson WG, McKenna WJ, Gautam S, Remick DG, Calkins H, Saffitz JE. Altered desmosomal proteins in granulomatous myocarditis and potential pathogenic links to arrhythmogenic right ventricular cardiomyopathy. Circ Arrhythm Electrophysiol. 2011;4:743–752.
- Milstein ML, Musa H, Balbuena DP, Anumonwo JM, Auerbach DS, Furspan PB, Hou L, Hu B, Schumacher SM, Vaidyanathan R, Martens JR, Jalife J. Dynamic reciprocity of sodium and potassium channel expression in a macromolecular complex controls cardiac excitability and arrhythmia. Proc Natl Acad Sci USA. 2012;109:E2134–E2143.
- Zhang SS, Shaw RM. Trafficking highways to the intercalated disc: New insights unlocking the specificity of connexin 43 localization. Cell Commun Adhes. 2014;21:43–54.
- Kaplan SR, Gard JJ, Protonotarios N, Tsatsopoulou A, Spiliopoulou C, Anastasakis A, Squarcioni CP, McKenna WJ, Thiene G, Basso C, Brousse N, Fontaine G, Saffitz JE. Remodeling of myocyte gap junctions in arrhythmogenic right ventricular cardio-myopathy due to a deletion in plakoglobin (Naxos disease) Heart Rhythm. 2004;1:3–11.
- Yamada K, Green KG, Samarel AM, Saffitz JE. Distinct pathways regulate expression of cardiac electrical and mechanical junction proteins in response to stretch. Circ Res. 2005;97:346–353.
- James CA, Bhonsale A, Tichnell C, Murray B, Russell SD, Tandri H, Tedford RJ, Judge DP, Calkins H. Exercise increases age-related penetrance and arrhythmic risk in arrhythmogenic right ventricular dysplasia/cardiomyopathy-associated desmosomal mutation carriers. J Am Coll Cardiol. 2013;62:1290–1297.
- Petitprez S, Zmoos AF, Ogrodnik J, Balse E, Raad N, El-Haou S, Albesa M, Bittihn P, Luther S, Lehnart SE, Hatem SN, Coulombe A, Abriel H. SAP97 and dystrophin macromolecular complexes determine two pools of cardiac sodium channels Nav1.5 in cardiomyocytes. Circ Res. 2011;108:294–304.
- Gutman GA, Chandy KG, Grissmer S, Lazdunski M, McKinnon D, Pardo LA, Robertson GA, Rudy B, Sanguinetti MC, Stühmer W, Wang X. International Union of Pharmacology. LIII. Nomenclature and molecular relationships of voltage-gated potassium channels. Pharmacol Rev. 2005;57:473–508.
- Ai Z, Fischer A, Spray DC, Brown AM, Fishman GI. Wnt-1 regulation of connexin43 in cardiac myocytes. J Clin Invest. 2000;105:161–171.
- Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. Stages of embryonic development of the zebrafish. Dev Dyn. 1995;203:253–310.
- Shin JT, Pomerantsev EV, Mably JD, MacRae CA. High-resolution cardiovascular function confirms functional orthology of myocardial contractility pathways in zebrafish. Physiol Genomics. 2010;42:300–309.
- Thomas SP, Bircher-Lehmann L, Thomas SA, Zhuang J, Saffitz JE, Kléber AG. Synthetic strands of neonatal mouse cardiac myocytes: Structural and electrophysiological properties. Circ Res. 2000;87:467–473.
- Neher E. Correction for liquid junction potentials in patch clamp experiments. Methods Enzymol. 1992;207:123–131.
- Gavillet B, Rougier JS, Domenighetti AA, Behar R, Boixel C, Ruchat P, Lehr HA, Pedrazzini T, Abriel H. Cardiac sodium channel Nav1.5 is regulated by a multiprotein complex composed of syntrophins and dystrophin. Circ Res. 2006;99:407–414.
- Dobrev D, Graf E, Wettwer E, Himmel HM, Hála O, Doerfel C, Christ T, Schüler S, Ravens U. Molecular basis of downregulation of G-protein–coupled inward rectifying K+ current (IK,ACh) in chronic human atrial fibrillation: Decrease in Girk4 mRNA correlates with reduced IK,ACh and muscarinic receptor–mediated shortening of action potentials. Circulation. 2001;104:2551–2557.
- Nemtsas P, Wettwer E, Christ T, Weidinger G, Ravens U. Adult zebrafish heart as a model for human heart? An electrophysiological study. J Mol Cell Cardiol. 2010;48:161–171.
- Zhuang J, Yamada KA, Saffitz JE, Kléber AG. Pulsatile stretch remodels cell-to-cell communication in cultured myocytes. Circ Res. 2000;87:316–322.
- Churko JM, Burridge PW, Wu JC. Generation of human iPSCs from human peripheral blood mononuclear cells using non-integrative Sendai virus in chemically defined conditions. Methods Mol Biol. 2013;1036:81–88.
Source: PubMed