Cytoprotection of rat hepatocytes by desipramine in a model of simulated ischemia/reperfusion

We investigated the cytoprotective effect of desipramine (DMI) during in vitro simulated ischemia/reperfusion (I/R) of rat hepatocytes. Primary hepatocytes isolated from male Sprague-Dawley rats were subjected to 4 h of anoxia at pH 6.2 followed by normoxia at pH 7.4 for 2 h to simulate ischemia and reperfusion, respectively. During simulated reperfusion, some hepatocytes were reoxygenated using media containing 5 μM DMI. Necrotic cell death and the onset of mitochondrial permeability transition (MPT) were assessed using fluorometry and confocal microscopy. Changes in autophagic flux and autophagy-related proteins (ATGs) were analyzed by immunoblotting. DMI was shown to substantially delay MPT onset and suppress I/R related cell damage. Mechanistically, DMI treatment during reperfusion increased the expression level of the microtubule-associated protein 1A/1B-light chain 3 (LC3) processing enzymes, ATG4B and ATG7. Genetic knockdown of ATG4B abolished the cytoprotective effect of DMI. Together, these results indicate that DMI is a unique agent which enhances LC3 processing in an ATG4B-dependent way.