This is the revised version of an application for competitive renewal of grant RO1-GM57691-13. Since its inception (in 1999), we focused on the mechanisms that regulate gap junctions formed by Connexin43 (Cx43). For this application, we take the reverse angle, and look at the ability of Cx43 to be the regulator of other molecular complexes. In its classical definition, the function of Connexin43 (Cx43) is to form gap junctions. While this description is certainly correct, it does not exclude the possibility that Cx43 exerts other actions, separate from that of gap junction pore formation. Here, we propose that a) specific amino acids within the Cx43 carboxyl terminal domain modulate the function of sodium channels, b) this regulation occurs within the confines of the "perinexus," that is, the area surrounding a gap junction plaque, and c) Cx43-mediated regulation of cell electrophysiology is necessary for proper cardiac rhythm.
Under Aim 1, we will characterize the cardiac electrophysiological profile of mice after loss of Cx43 expression (Cx43-CKO), or after deletion of the last five amino acids of Cx43 (line Cx43D378stop). Both of these mice present with ventricular fibrillation and sudden death. A fundamental difference is that mutation D378stop does not prevent formation of gap junction plaques. We propose that Cx43 is necessary for proper function of other ion channels.
In aim 2, we will define the structure of the perinexus in relation to the sodium channel complex, and the importance of preservation of region 378-382 of Cx43 to control the distance between molecules. We implement a unique combination of modern imaging/recording techniques (scanning ion conductance microscopy, SICM-guided patch clamp, tomographic electron microscopy, direct stochastic reconstruction microscopy, proximity ligation assays), and novel animal models, to address new hypotheses that directly impact our understanding of the molecular mechanisms of cardiac arrhythmias. Overall, our experiments challenge the prevailing concept that, in a structurally normal heart, Cx43-dependent arrhythmias are only consequent to the loss of gap junction channels between cells.
Arrhythmias are a major cause of morbidity and mortality, at great economic and societal cost. A role of Cx43 in the function of non---junctional ion channels remains largely unexplored. Yet, Cx43 is seen as a potential target for arrhythmia therapy, and Cx43 remodeling is considered an important arrhythmia substrate. We seek to shift the paradigm, from the perception of Cx43 as a single---function molecule, to that of a multi---tasking component of a protein interacting network that includes the sodium channel complex at the cardiac intercalated disc.
|Cerrone, Marina; Lin, Xianming; Zhang, Mingliang et al. (2014) Missense mutations in plakophilin-2 cause sodium current deficit and associate with a Brugada syndrome phenotype. Circulation 129:1092-103|
|Cerrone, Marina; Delmar, Mario (2014) Desmosomes and the sodium channel complex: implications for arrhythmogenic cardiomyopathy and Brugada syndrome. Trends Cardiovasc Med 24:184-90|
|Agullo-Pascual, Esperanza; Cerrone, Marina; Delmar, Mario (2014) Arrhythmogenic cardiomyopathy and Brugada syndrome: diseases of the connexome. FEBS Lett 588:1322-30|
|Agullo-Pascual, Esperanza; Lin, Xianming; Leo-Macias, Alejandra et al. (2014) Super-resolution imaging reveals that loss of the C-terminus of connexin43 limits microtubule plus-end capture and NaV1.5 localization at the intercalated disc. Cardiovasc Res 104:371-81|
|Meens, Merlijn J; Pfenniger, Anna; Kwak, Brenda R et al. (2013) Regulation of cardiovascular connexins by mechanical forces and junctions. Cardiovasc Res 99:304-14|
|Lubkemeier, Indra; Requardt, Robert Pascal; Lin, Xianming et al. (2013) Deletion of the last five C-terminal amino acid residues of connexin43 leads to lethal ventricular arrhythmias in mice without affecting coupling via gap junction channels. Basic Res Cardiol 108:348|
|Agullo-Pascual, Esperanza; Reid, Dylan A; Keegan, Sarah et al. (2013) Super-resolution fluorescence microscopy of the cardiac connexome reveals plakophilin-2 inside the connexin43 plaque. Cardiovasc Res 100:231-40|
|Bhargava, Anamika; Lin, Xianming; Novak, Pavel et al. (2013) Super-resolution scanning patch clamp reveals clustering of functional ion channels in adult ventricular myocyte. Circ Res 112:1112-20|
|Chkourko, Halina S; Guerrero-Serna, Guadalupe; Lin, Xianming et al. (2012) Remodeling of mechanical junctions and of microtubule-associated proteins accompany cardiac connexin43 lateralization. Heart Rhythm 9:1133-1140.e6|
|Makita, Naomasa; Seki, Akiko; Sumitomo, Naokata et al. (2012) A connexin40 mutation associated with a malignant variant of progressive familial heart block type I. Circ Arrhythm Electrophysiol 5:163-72|
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