Connexin 43 mimetic peptide Gap26 confers protection to intact heart against myocardial ischemia injury

Abstract

Unapposed connexin 43 hemichannels (Cx43Hc) are present on sarcolemma of cardiomyocytes. Whereas Cx43Hc remain closed during physiological conditions, their opening under ischemic stress contributes to irreversible tissue injury and cell death. To date, conventional blockers of connexin channels act unselectively on both gap junction channels and unapposed hemichannels. Here, we test the hypothesis that Gap26, a synthetic structural mimetic peptide deriving from the first extracellular loop of Cx43 and a presumed selective blocker of Cx43Hc, confers resistance to intact rat heart against ischemia injury. Langendorff-perfused intact rat hearts were utilized. Regional ischemia was induced by 40-min occlusion of the left anterior descendent coronary and followed by 180 min of reperfusion. Gap26 was applied either 10 min before or 30 min after the initiation of ischemia. Interestingly, myocardial infarct size was reduced by 48% and 55% in hearts treated with Gap26 before or during ischemia, respectively, compared to untreated hearts. Additionally, myocardial perfusate flow was increased in both groups during reperfusion by 37% and 32%, respectively. Application of Gap26 increased survival of isolated cardiomyocytes after simulated ischemia-reperfusion by nearly twofold compared to untreated cells. On the other hand, superfusion of tsA201 cells transiently expressing Cx43 with Gap26 caused 61% inhibition of Cx43Hc-mediated currents recorded using the patch clamp technique. In summary, we demonstrate for the first time that Cx43 mimetic peptide Gap26 confers protection to intact heart against ischemia-reperfusion injury whether administered before or after the occurrence of ischemia. In addition, we provide unequivocal evidence for the inhibitory effect of Gap26 on genuine Cx43Hc.

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).…”

Role of Gap Junction Channel in the Development of Beat-to-Beat Action Potential Repolarization Variability and Arrhythmias

“…In ischaemia uncontrolled ATP release through hemichannels may result in cellular ATP depletion. Administration of GAP26 before or after the ischaemia protected heart cells against hypoxia and reperfusion and decreased infarct size [161]. Increased hemichannel function could be a side effect of gap junction channel enhancer treatment, via greater loss of ATP [162].…”

Connexin 43 Hemichannels and Pharmacotherapy of Myocardial Ischemia Injury

“…Since peptides exert their effect by binding to the extracellular loops, these were added to the extracellular bath solution (0.5 M). To determine the sensitivity of isolated myocytes to simulated ischemia/reperfusion, freshly isolated cardiac myocytes were incubated with saline, Gap26 or scrambled GAP26 (sGap26) 10 min before or 30 min after the initiation of ischemia as described (Hawat et al, 2010). Briefly, cells were pelleted by low speed centrifugation for 1 min and were washed twice with incubation buffer.…”

“…Briefly, cells were pelleted by low speed centrifugation for 1 min and were washed twice with incubation buffer. Under these conditions, most of the non-myocyte cells remain in the supernatant, producing a pellet that contains >95% myocytes Hawat et al, 2010). To simulate ischemic stress, cells were transferred to microcentrifuge tubes, washed twice with degassed glucose-free incubation buffer and pelleted as before.…”

“…The Cx43 structural mimetic peptide (0.5 M) was administered either 10 min prior to the onset of ischemia or 30 min after (this is 10 min before reperfusion), as described (Hawat et al, 2010). For control, s Gap26 was utilized.…”