To beat or not to beat: detrimental autophagy contributes to gap junctions degradation in ischemic heart

In the heart, gap junction intercellular communication (GJIC), through membrane channels formed by Connexins (Cx), ensures efficient action potential propagation, resulting in coordinated contraction. Several pathological conditions characterized by conduction block and arrhythmogenesis have been associated with dysfunction of GJ regulation. We have recently shown that GJIC can be regulated through autophagy(definition)-mediated degradation of Cx43-containing GJ, however the physiological relevance of such mechanism and its implications in pathological conditions remain largely unknown. However, in the heart, autophagy plays a dual role, being either protective or detrimental, depending on the nature, extent and severity of the stimuli or insult. The main objective of this study was to evaluate the effect of autophagy mediated degradation of GJ in the ischemic heart. To address this we used a rat heart Langendorff retrograde perfusion system. Control hearts were subjected to perfusion with Krebs-Henseleit buffer, while ischemia was induced by 30 min of no-flow. A 15, 30 and 60 min of further reperfusion (I/R) period was performed at the same flow rate used before ischemia. To investigate the involvement of autophagy in degradation of Cx43 triggered by ischemia and I/R, hearts were perfused with KH solution containing pepstatin A and NH4Cl. The effect of ischemia and I/R on the total levels of Cx43 was evaluated by Western Blot, while the subcellular distribution of Cx43 was determined by confocal microscopy. The involvement of autophagy in the degradation of GJ was determined by the interaction between Cx43 and autophagy markers, such as LC3 and p62 by colocalization and immunoprecipitation (IP) assays. The results obtained showed that activation of autophagy occurs in ischemia, and is further increased during I/R. On the other hand, the levels of Cx43 do not significantly vary during ischemia, but gradually decrease during reperfusion, being partially reverted in the presence of pepstatin A and NH4Cl. Microscopy data showed that ischemia and I/R results in a dramatic subcellular redistribution of Cx43, with a decrease of Cx43 at the intercalated discs and an accumulation at the lateral membrane. Moreover, colocalization and IP assays demonstrated an increased interaction between Cx43 and autophagy markers during ischemia and I/R, particularly in hearts perfused with pepstatin A and NH4Cl. Altogether, this data demonstrates that degradation of GJ protein Cx43 in I/R results from an increased activation of harmful autophagy. This study followed the "Principles of laboratory animal care".