Role of connexin 43 in ischemic preconditioning does not involve intercellular communication through gap junctions

Abstract

Connexin 43 (Cx 43) has recently been implicated in protection of ischemic preconditioning. Cx 43 colocalization with protein kinase C and p38 mitogen-activated protein kinase is increased in preconditioned myocardium, Cx 43 phosphorylation is preserved in preconditioned myocardium, and hearts from Cx 43-deficient mice cannot be preconditioned. It is, however, unclear whether the important role of Cx 43 relates to intercellular communication through gap junctions or its function in volume homeostasis. To address this issue, we used isolated cardiomyocytes, which no longer-form gap junctions, from wild-type (n = 5) and heterozygous Cx 43-deficient mice (n = 8) and subjected them to 2 h simulated ischemia (hypoxia, acidosis) and an additional challenge by extracellular hypo-osmolarity (from 310 to 250 mOsm/l). Viability (trypan blue exclusion) was well maintained in normoxic wild-type cardiomyocytes (54 ± 5% at baseline vs. 46 ± 4 (mean ± S.D.) % at 2 h). With simulated ischemia, viability was reduced to 17 ± 5%. Preconditioning by a preceding exposure to 10 min simulated ischemia and 15 min reoxygenation preserved viability after 2 h simulated ischemia (36 ± 1%, P < 0.001 vs. simulated ischemia). In Cx 43-deficient cardiomyocytes, viability was also well maintained in normoxia (56 ± 10% vs. 44 ± 10%). Viability was also reduced to 17 ± 6% with 2 h simulated ischemia. In contrast to wild-type cells, preconditioning did not prevent the reduction in viability (18 ± 8%). In conclusion, Cx 43 is essential for preconditioning in the absence of gap junctions, supporting its function through improved volume regulation.

Introduction

Myocardial gap junctions and/or their constitutive proteins, i.e. the connexins, have recently been implicated in the mechanism of ischemic preconditioning. Connexin 43 (Cx 43) is colocalized in signaling complexes with protein kinases that are centrally involved in the protection of ischemic preconditioning, i.e. protein kinase C [1], [2] and p38 mitogen-activated protein kinase [2]. Accordingly, in anesthetized pigs the phosphorylation of Cx 43 is preserved in preconditioned myocardium whereas it is decreased in non-preconditioned myocardium during ischemia [2]. These findings were confirmed in isolated rat [3] and rabbit hearts [4] and extended in that the effects of ischemic preconditioning on Cx 43 phosphorylation were mimicked by the mitochondrial K_ATP-opener diazoxide and abolished by the mitochondrial K_ATP-blocker 5-hydroxydecanoate [3]. In the isolated rat heart the protein kinase C inhibitors chelerythrine and calphostin did not affect the preservation of Cx 43 phosphorylation [3], but in the isolated rabbit heart calphostin did prevent the preservation of Cx 43 phosphorylation [4]. Uncoupling of gap junctions by heptanol abolished the infarct size reduction by ischemic preconditioning in isolated mouse hearts [5]. We have recently shown that in anesthetized heterozygous Cx 43-deficient mice the myocardium cannot be preconditioned, even when the preconditioning stimulus is increased from one to three cycles of ischemia/reperfusion [6], [7]. Whereas a central role for Cx 43 in ischemic preconditioning is thus established, it is unclear whether this relates to intercellular communication through gap junctions. A recent study in isolated rat hearts and in situ pig hearts found no effects of ischemic preconditioning on the ischemia-induced changes in electrical impedance, suggesting that intercellular coupling is not important for protection [8]. Alternatively, Cx 43 hemichannels have a central role in cellular volume regulation [9], and improved cellular volume regulation is one potential end-effector of protection of ischemic preconditioning [10]. To address this issue more directly, we have now utilized the isolated cardiomyocyte model of ischemic preconditioning [11] in Cx 43-deficient mice.