Role of connexin 43 in ischemia-reperfusion injury: Effect of genetic connexin 43 manipulation on myocardial cell death and arrhythmias

Myocardial ischemia-reperfusion injury is one of the main causes of morbidity and mortality worldwide every year. Reperfusion is the treatment of choice in patients undergoing myocardial infarction in order to prevent cardiomyocyte death and improve patient? s survival and prognosis. However, reperfusion causes an additional injury, through mechanisms triggered by flow restoration, that can be prevented, at least in part and in the experimental setting, by drugs applied at the time of reperfusion. This fact has led to an intense research in order to identify therapeutic strategies able to limit myocardial infarction when applied at the time of reperfusion. One of the candidate targets is Connexin 43 (Cx43), and the channels that it forms (i.e, gap junction (GJ) channels). Cx43 is the main protein forming GJ in the ventricular myocardium, where it plays an essential role in electrical and chemical coupling between cardiomyocytes. However, previous studies have suggested that GJ play also a role in spreading of cell death during reperfusion, and that Cx43 may be involved in endogenous cardioprotective signaling during ischemic preconditioning (IPC). However, most of these studies have been conducted using GJ uncouplers having low specificity and lots of side effects. This made necessary to find new strategies to study the role of Cx43 in cell death during myocardial infarction and cardioprotection, including the use of transgenic mice models. Thus, the aims of this thesis were to assess the effects of Cx43 deficiency in isolated hearts from transgenic models on cardiac energetic metabolism, tolerance to ischemia-reperfusion injury, susceptibility to ischemic and pharmacological preconditioning, and on the incidence of ventricular arrhythmias. We used a knock-in mice model, in which Cx43 is replaced by Cx32, a connexin with lower conductivity and permeability, and a conditional knock-out model of Cx43 deficiency (Cx43Cre-ER(T)/fl). In this last model, isolated hearts from Cx43Cre/fl animals express, under normal conditions, half of the Cx43 content seen in their corresponding genetic controls (Cx43fl/fl). However, 14 days after tamoxifen treatment, hearts from these animals have only a residual Cx43 content, lower than 5%. We found that both genetic strategies alter myocardial energetic metabolism, with hearts from these mice having reduced ATP levels as compared with hearts from the corresponding genetic controls. Furthermore, isolated, Langendorff-perfused, hearts from homozygous (HOM) knock-in mice and from Cx43Cre/fl animals treated with tamoxifen, depicted an increased tolerance to ischemia-reperfusion injury, as denoted by a reduction in infarct size and in LDH release. In Cx43Cre/fl animals treated with tamoxifen, this effect seems to be more dependent on GJ communication than in HOM knock-in mice. Moreover, susceptibility to IPC and especially to diazoxide protection was abolished in both models of Cx43 deficiency. Furthermore, we have demonstrated that these effects were independent of a differential activation of cytosolic signaling cascades, including the RISK and SAFE pathways, which is suggestive that activation of these pathways during IPC is upstream of Cx43 deficiency. Finally, we have demonstrated that both models of Cx43 deficiency, mimicking a reduction in unitary GJ conductance and in the number of GJ channels, respectively, are associated with an increased incidence of both spontaneous and induced ventricular tachyarrhythmias. This finding indicates that the possibility of translation of these therapeutic strategies to the clinical arena requires that they could be applied locally at the area at risk. In conclusion, this thesis demonstrates an important, and previously unknown, specific role of Cx43 in myocardial energetic metabolism, tolerance to ischemia-reperfusion injury and in endogenous cardioprotection. GJ and Cx43 can become interesting pharmacological targets to improve the clinical outcome in patients with ischemic heart disease. However, translation should wait until the proarrhythmic effects of these treatments are solved.