Dephosphorylation and Intracellular Redistribution of Ventricular Connexin43 During Electrical Uncoupling Induced by Ischemia

Abstract

Electrical uncoupling at gap junctions during acute myocardial ischemia contributes to conduction abnormalities and reentrant arrhythmias. Increased levels of intracellular Ca(2+) and H(+) and accumulation of amphipathic lipid metabolites during ischemia promote uncoupling, but other mechanisms may play a role. We tested the hypothesis that uncoupling induced by acute ischemia is associated with changes in phosphorylation of the major cardiac gap junction protein, connexin43 (Cx43). Adult rat hearts perfused on a Langendorff apparatus were subjected to ischemia or ischemia/reperfusion. Changes in coupling were monitored by measuring whole-tissue resistance. Changes in the amount and distribution of phosphorylated and nonphosphorylated isoforms of Cx43 were measured by immunoblotting and confocal immunofluorescence microscopy using isoform-specific antibodies. In control hearts, virtually all Cx43 identified immunohistochemically at apparent intercellular junctions was phosphorylated. During ischemia, however, Cx43 underwent progressive dephosphorylation with a time course similar to that of electrical uncoupling. The total amount of Cx43 did not change, but progressive reduction in total Cx43 immunofluorescent signal and concomitant accumulation of nonphosphorylated Cx43 signal occurred at sites of intercellular junctions. Functional recovery during reperfusion was associated with increased levels of phosphorylated Cx43. These observations suggest that uncoupling induced by ischemia is associated with dephosphorylation of Cx43, accumulation of nonphosphorylated Cx43 within gap junctions, and translocation of Cx43 from gap junctions into intracellular pools.

Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function

“…Cx43 function is strongly regulated by its localization and protein stability [30][31][32] . Therefore, we examined Cx43 levels and localization in control and Tmem65 knockdown cells.…”

“…41). We assessed electrical conduction in the scrambled control and Tmem65 shRNA cardiomyocytes using optical mapping with the electrically sensitive imaging dye, di-4-ANEPPS, and by direct electrical conduction recordings using microelectrode arrays.…”

Key Connexin 43 Phosphorylation Events Regulate the Gap Junction Life Cycle

“…When cardiac tissue is immunoblotted for Cx43, only slower migrating "phosphorylated" isoforms are observed. Myocardial ischemia leads to Cx43 "dephosphorylation" (i.e., the loss of P1, P2, etc and gain of P0) and loss of localization from the intercalated disk, which likely contributes to contractile failure and arrhythmias (Beardslee et al, 2000;Schulz et al, 2003). We have shown that Cx43 localized to intercalated disks is phosphorylated at S325, S328 and/or S330 and that ischemia leads to loss of this phosphorylation and re-localization of the protein (Lampe et al, 2006).…”

A possible mechanism of halocarbon-induced cardiac sensitization arrhythmias

“…This protein is regulated by phosphorylation, with the unphosphorylated version of the channel generally showing a reduced open probability that results in reduced gap junction conductance [23]. Because of the rapidity of the Epi+halon effect and its similarity to that of heptanol, we hypothesized that Cx43 dephosphorylation may have contributed to the changes in conduction velocity.…”