No ischemic preconditioning in heterozygous connexin43-deficient mice

Abstract

Protein kinase C⑀ (PKC⑀) plays a central role in ischemic preconditioning (IP) in mice and rabbits, and activated PKC⑀ colocalizes with and phosphorylates connexin43 (Cx43) in rats and humans. Whether or not Cx43 contributes to the mechanism(s) of IP in vivo is yet unknown. Therefore, wild-type (n ϭ 8) and heterozygous Cx43-deficient mice (n ϭ 8) were subjected to 30 min occlusion and 120 min reperfusion of the left anterior descending coronary artery. IP was induced by one cycle of 5 min occlusion and 10 min reperfusion (n ϭ 8/8 mice) before the sustained occlusion. Infarct size was reduced by IP in wild-type mice [11.3 Ϯ 3.4% vs. 23.7 Ϯ 7.2% of the left ventricle (LV), P Ͻ 0.05] but not in Cx43-deficient mice (26.0 Ϯ 6.0% vs. 25.1 Ϯ 3.8% of LV). Also, three cycles of 5 min occlusion and 10 min reperfusion (n ϭ 5) did not induce protection in Cx43-deficient mice (27.6 Ϯ 5.5 % of LV). Thus Cx43 contributes to the protection of IP in mice in vivo. mouse heart in situ; infarct size; gap junctions ISCHEMIC PRECONDITIONING (IP) by brief episodes of ischemia-reperfusion protects the myocardium from the damage induced by a subsequent more prolonged ischemia. Several triggers and mediators of IP have been identified, whereas the final end effector is still unknown (15). Protein kinase C⑀ (PKC⑀) is an established mediator of IP in mice and rabbits, whereas other PKC isoforms may be more important in other species (15). PKC⑀ is involved in signaling complexes with at least 36 proteins (1, 9, 18), among them connexin43 (Cx43) (10), an integral protein of myocardial gap junctions. Activated PKC⑀ colocalizes with Cx43 and contributes to phosphorylation of Cx43 in rats (3) and humans (2), which might then modulate gap junction transmission characteristics and intercellular communication. Indeed, in isolated mouse hearts, uncoupling of gap junctions using heptanol abolished infarct size reduction by IP (8). The data on the effect of gap junction uncoupling on infarct size per se are controversial. Pretreatment with the gap junction uncoupler heptanol had no effect on infarct size in isolated rabbit hearts (6), but heptanol given during early ischemia decreased infarct size in isolated rabbit hearts (16). Also, in pigs in vivo heptanol given during early reperfusion decreased infarct size (4). Data on the importance of Cx43 for IP in vivo, however, are lacking. We therefore studied whether or not Cx43 is involved in the cardioprotection by IP using heterozygous Cx43-deficient mice. EXPERIMENTAL PROCEDURESThe experimental protocols were approved by the bioethics committee of the district of Dü sseldorf, Germany. Mice were handled according to the guidelines of the American Physiological Society.We used the in situ mouse heart model developed by Guo et al. (5). Briefly, male and female C57BL/6J wild-type and heterozygous Cx43-deficient mice (B6.129-Gja1 tm1Kdr , JAX mice; Bar Harbor, ME) (12) (weight: 30.5 Ϯ 4.5 g, age: 14-20 wk) were anesthetized with pentobarbital sodium (80 mg/kg ip) and atropine sulfate (0.04 mg/kg ip). Ele...

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

“…Consistent with previous results, infarct size with vehicle control was comparable in C57BL/6J mice, Cx43-deficient mice, and Cx43 WT littermates ( Fig. 4A) (17). SB216763 (70 μg/kg) significantly reduced infarct size in C57BL/6J mice [46.9 ± 4.6%, n = 7, vs. control (57.8 ± 3.5%, n = 7); P < 0.05] without altering Cx43 protein levels in plasma membrane and mitochondria (Fig.…”

“…We used the in situ mouse heart model, as described previously (9,17). Briefly, male mice, as indicated, were anesthetized with pentobarbital sodium (80 mg/kg administered i.p.).…”

“…The area at risk was measured by Evans blue, and the infarct size was determined by 1% 2,3,5-triphenyl tetrazolium chloride staining. Hearts were cut into four to five transverse slices, and infarct size was measured by planimetry and expressed as a percentage of the left ventricle (17).…”

Repeated Simulated Ischemia and Protection Against Gap Junctional Uncoupling

“…The presence of Cx43 in mitochondria from rat, mouse, pig, and human myocardium has been demonstrated (2,15,34). The first evidence that Cx43 might play a role in ischemic preconditioning came from studies by Schwanke et al (40,41), and it has been demonstrated that ischemic preconditioning increases the amount of mitochondrial Cx43, before the prolonged ischemia (2,34). In the present study, we did not demonstrate an increased Cx43 expression in cell homogenates after sublethal ischemic stress…”

“…In addition, substantial evidence supports the hypothesis that the protective effect may be explained, at least in part, by contribution from gap junction-mediated intercellular communication (8,14,16,19,39). Schwanke et al (40) showed that a 50% reduction in Cx43 content was sufficient to abolish the infarct-sparing effect of preconditioning strongly indicating a role for Cx43.…”