Hypothermia may attenuate ischemia/reperfusion-induced cardiomyocyte death by reducing autophagy

Objective: We sought to assess the effect of therapeutic hypothermia on the autophagy that occurred in ischemia-reperfused (IR) H9c2 cardiomyocytes. Methods: In control studies, the H9c2 cells at a density of 1 x 10(5) per culture dish in six-well plate were exposed to normoxic culture medium at 37 degrees C for 12 h. All assays contained appropriate controls and were performed in triplicate and repeated on three separately initiated cultures. In hypothermia-treated group, the ischemic and hypoxic cells were maintained in a 32 degrees C incubation. The trypan blue exclusion method was used to assess the cell viability. Autophagy was evaluated by determining both the microtubule-associated protein 1 light chain 3 [LC3] levels and punctuate distribution of the autophagic vesicle associated form [LC3-II]. Results: The results were mean +/- standard error of mean of triplicates. The viable cell percentage for control group, IR group, and IR group treated with hypothermia at the start of ischemia, or reperfusion were 100% +/- 9%, 20% +/- 1%, 32% +/- 3%, and 41% +/- 3%, respectively. The cell death in I/R H9c2 cells was positively associated with increased LC3 levels and punctuate distribution of (LC3-II). Mild hypothermia adopted at the start of ischemia or reperfusion significantly reduced both the cell death and the autophagy in H9c2 cells. Conclusion: Our data indicate that in H9c2, IR stimulates cell autophagy and causes cell death, which can be attenuated by mild hypothermia. Our results, if further confirmed in vivo, may have important clinical implications during IR injury. (C) 2013 Elsevier Ireland Ltd. All rights reserved.