Ischemic preconditioning preserves connexin 43 phosphorylation during sustained ischemia in pig hearts in vivo

Abstract

During myocardial ischemia, connexin 43 (Cx43) is dephosphorylated in vitro, and the subsequent opening of gap junctions formed by two opposing Cx43 hexamers was suggested to propagate ischemia/reperfusion injury. Reduction of infarct size (IS) by ischemic preconditioning (IP) involves activation of protein kinase C (PKC) and p38 mitogen activated protein kinase (MAPK), both of which can phosphorylate Cx43. We now studied in anesthetized pigs whether IP impacts on Cx43 phosphorylation by measuring the density of non-phosphorylated and total Cx43 (confocal laser) during normoperfusion and 90-min ischemia in non-preconditioned and preconditioned hearts. Co-localization of PKCalpha, p38MAPKalpha, and p38MAPKbeta with Cx43 and the activity of p38MAPK were assessed. IP by 10 min ischemia and 15 min reperfusion reduced IS. Non-phosphorylated Cx43 remained unchanged during ischemia in preconditioned hearts, while it increased from 35+/-3 to 75+/-8 AU (P<0.05) in non-preconditioned hearts. Co-localization of PKCalpha, p38MAPKalpha, and p38MAPKbeta with Cx43 during ischemia increased only in preconditioned hearts. While the ischemia-induced increase in p38MAPKalpha activity was comparable in preconditioned and non-preconditioned hearts, p38MAPKbeta activity was increased only in preconditioned hearts. Blockade of p38MAPK by SB203580 attenuated the IS-reduction and the increased p38MAPK-Cx43 co-localization by IP. We conclude that IP increases co-localization of protein kinases with Cx43 and preserves phosphorylation of Cx43 during ischemia.

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).…”

Connexin 43 Hemichannels and Pharmacotherapy of Myocardial Ischemia Injury

“…2) (Contreras et al, 2002). While Cx43 is phosphorylated under physiological conditions van Veen et al, 2001) and remains so in the first few minutes of ischemia , subsequent dephosphorylation of Cx43 occurs with increasing duration of myocardial ischemia (Beardslee et al, 2000;Jain et al, 2003;Jeyaraman et al, 2003;Miura et al, 2004;Schulz et al, 2003) a process that has been associated with the opening of unapposed Cx43Hc (John et al, 1999b;Li et al, 2001) which results in metabolic stress and cell death. Based on single Hc conductance measurements, it was suggested that opening of only 50 Hc is sufficient to drown the cell with Na + (John et al, 1999b).…”

“…Cx43Hc dephosphorylation (Beardslee et al, 2000;Jain et al, 2003;Jeyaraman et al, 2003;Miura et al, 2004;Schulz et al, 2003) and opening (John et al, 1999b;Kondo et al, 2000;Shintani-Ishida et al, 2007) have been proposed as key mechanisms underlying cell injury in response to simulated ischemia in cardiomyocytes. Preservation of Cx43 phosphorylation by selective activation of the ɛPKC isoform occurs in response to ischemic pre- (Chou & Messing, 2005;Liu et al, 1999;Ytrehus et al, 1994) and postconditioning (Penna et al, 2006a;Philipp et al, 2006;Zatta et al, 2006), in which brief episodes of ischemia reduce the adverse effects of subsequent or preceding myocardial ischemia, respectively.…”

“…This effect has been related to www.intechopen.com increase in Cx43 phosphorylation (Hatanaka et al, 2004;Hund et al, 2007;Miura et al, 2004;Schulz et al, 2003). Interestingly, different studies pointed to the isozyme PKC as a principal mediator of ischemic preconditioning-mediated cardioprotection (Gray et al, 1997;Hund et al, 2007;Inagaki et al, 2003;Liu et al, 1999;Ping et al, 1997;Saurin et al, 2002).…”

Repeated Simulated Ischemia and Protection Against Gap Junctional Uncoupling

“…Studies in cultured neonatal rat cardiomyocytes have identified multiple sites on Cx43 for phosphorylation by several kinases (22,37,47). Schultz et al (38) reported that colocalization of PKCα, p38MAPKα, and p38MAPKβ with Cx43 during ischemia increased only in preconditioned pig hearts and preserved phosphorylation of Cx43 during ischemia.…”