P145Degradation of gap junction protein Cx43 by autophagy in ischemic heart is determined by the triggering signal: the role of AMPK Vs Beclin1

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Abstract

Several cardiomyopathies, including myocardial ischemia, have been associated with an impairment of gap junction intercellular communication (GJIC) between cardiomyocytes, due to an increased internalization and/or degradation of Connexin43 (Cx43). Recent data of our group, demonstrates that ischemia-induced activation of autophagy results in degradation of Cx43 in HL-1 cells. However, the molecular mechanisms underlying Cx43 degradation and the involvement of autophagy machinery remain unknown. It is broadly accepted that autophagy have a dichotomous role likely depending on the nature, extent and severity of the stimuli, as well as on the autophagy players involved. The emerging theory stands for a cardioprotective role of ischemia-induced autophagy via AMPK, being up-regulation of Beclin1 associated with exacerbated autophagy and cell death. Hence, it is conceivable to suggest that according to the stimuli (ischemia vs ischemia/reperfusion (I/R)), the triggering signals and mechanisms whereby autophagy is activated may differ and, consequently, determine the degradation levels of Cx43. To test this hypothesis, we used HL-1 cardiomyocytes subjected to different periods of ischemia and I/R, after which the levels and subcelullar distribution of Cx43 were determined by WB and confocal microscopy, respectively. Data show that Cx43 is degraded gradually during ischemia, and that reperfusion further increases its degradation. Furthermore, using chemical and genetic approaches to manipulate AMPK and Beclin-1 pathways, we showed that activation of AMPK occurs in the initial periods of ischemia, when an inhibition of AMPK protects Cx43 from degradation. On the other hand, for long periods of ischemia and during reperfusion, the protective effect regarding Cx43 degradation is mainly conferred by knockdown of Beclin 1. Concerning autophagy activation, we show that conversion of LC3-I in LC3-II occurs mainly during I/R, which is consistent with the increased degradation of Cx43 under these conditions. Interaction of Cx43 with autophagy adaptors during ischemia and I/R was also evaluated by immunoprecipitation assays and confocal microscopy. Increased interaction with autophagy players p62 and LC3 is mainly detected upon ischemia, which is likely required for the subsequent robust degradation of Cx43 observed during reperfusion. Altogether, our data led us to suggest a model in which differential activation of autophagy in ischemia and I/R, involving either AMPK or Beclin1, respectively, determines the final fate of Cx43.

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