Hemichannels in cardiomyocytes open transiently during ischemia and contribute to reperfusion injury following brief ischemia

Abstract

The aim of this study was to investigate changes in hemichannel activity during in vitro simulated ischemia [oxygen-glucose deprivation (OGD)] and the contribution of hemichannels to ischemia-reperfusion injury in rat neonatal cardiomyocytes. Dye uptake assays showed that hemichannels opened as OGD progressed, peaking after 1 h, and then closed, returning to the pre-OGD state after 2 h of OGD. The increase in dye uptake after 1 h of OGD was inhibited by hemichannel blockers (lanthanum chloride and a connexin 43 mimetic peptide, Gap26). During OGD, intracellular Ca(2+) concentration ([Ca(2+)](i)) began to increase after 1 h and reached several micromolar after 2 h. After 1 h of OGD, Gap26 inhibited the increases in hemichannel activity and [Ca(2+)](i). In contrast, dantrolene [an endo(sarco)plasmic reticulum Ca(2+) release inhibitor] suppressed the increase in [Ca(2+)](i), but not in hemichannel activity. After 2 h of OGD, the combined administration of 2',4'-dichlorobenzamil and dantrolene reduced [Ca(2+)](i) to <1 microM and increased hemichannel activity to the level attained after 1 h of OGD. Simulated ischemia-reperfusion, induced by 1 h of OGD followed by 2 h of recovery, reduced cell viability to 54% of the control level. The addition of Gap26 to OGD medium improved viability to 80% of the control level. In conclusion, this study demonstrated that 1) hemichannels open transiently during OGD, 2) closure of hemichannels, but not their opening, is regulated by an increase in [Ca(2+)](i) during OGD, and 3) open hemichannels contribute to cell injury during recovery from OGD.

RETRACTED: Glycogen synthase kinase 3β transfers cytoprotective signaling through connexin 43 onto mitochondrial ATP-sensitive K + channels

“…Inhibition of gap junctions during ischemia or reperfusion reduced infarct size (30,31), suggesting a possible protective effect by impeding spread of damaging metabolites via cell-cell contacts, which, however, remains controversial, given that ischemia, per se, initiates closure of gap junctions (32). Ischemia induces redistribution of Cx43 to the lateral wall of cardiomyocytes and may open these Cx43 hemichannels, which has been proposed to contribute to reperfusion injury (33)(34)(35). Notably, IP also suppresses gap junction permeability and induces Cx43 lateralization (31,33).…”

Currently Used Methods for Identification and Characterization of Hemichannels

“…A Px1 mimetic peptide with apparent specific blocking effect was also recently reported ( 110 pxn1) (Pelegrin and Surprenant 2006). A large number of publications demonstrate specific effects of some of these peptides on connexin hemichannels by using in parallel scrambled peptide versions, additional hemichannel blockers and/or studies of their effects on connexin-specific expression systems (Braet et al 2003a(Braet et al , 2003bPearson et al 2005;Gomes et al 2005;Eltzschig et al 2006;Takeuchi et al 2006;DeVuyst et al 2006;De Vuyst et al 2007;Retamal et al 2006Retamal et al , 2007bShintani-Ishida et al 2007). However, it was recently reported that some Cx32, Cx43, and Px1 mimetic peptides cause non-specific blockade of hemichannels formed by Px1, Cx46, and a connexin Cx32 chimera carrying the first extracellular loop of Cx43 (Cx32E143) in the oocyte expression system (Wang et al 2007).…”

Negative-feedback regulation of ATP release: ATP release from cardiomyocytes is strictly regulated during ischemia

“…1C). Previous studies have suggested that cardiomyocytes release ATP in response to cell stress via a variety of factors such as maxi-anion channels [7], cystic fibrosis transmembrane conductance regulator (CFTR) [17], and connexin and/or pannexin hemichannels [8,18]. In addition, in cultured astrocytes, mechanical stress to cells results in the release of ATP via exocytosis (vesicular ATP release) [19].…”

“…In neonatal rat cardiomyocytes, exposure of cultures to ischemic stress mimicked by oxygen-glucose deprivation (OGD) results in the release of ATP via maxi-anion channels [7]. In addition, Shintani-Ishida et al [8] have recently demonstrated that brief ischemic stress to neonatal cardiomyocytes transiently opens Cx43 hemichannels, leading to the release of ATP via hemichannels. At present, however, it is almost completely unknown whether the release of ATP from cardiomyocytes during ischemia is strictly regulated, and if so, how cardiomyocytes 5 regulate the release.…”