pISSN 1226-4512 eISSN 2093-3827

Article

home Article View

Original Article

Korean J Physiol Pharmacol 2020; 24(2): 173-183

Published online March 1, 2020 https://doi.org/10.4196/kjpp.2020.24.2.173

Copyright © The Korean Journal of Physiology & Pharmacology.

The optimal model of reperfusion injury in vitro using H9c2 transformed cardiac myoblasts

Euncheol Son1,2,3,#, Dongju Lee1,2,4,#, Chul-Woong Woo5,*, Young-Hoon Kim1,2,*

1Department of Pharmacology, University of Ulsan College of Medicine, 2Bio-Medical Institute of Technology, University of Ulsan, 3Stem Cell Immunomodulation Research Center, Asan Medical Center, University of Ulsan College of Medicine, 4Department of Medical Science, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine, 5Asan Institute for Life Sciences, Asan Medical Center, Seoul 05505, Korea

Correspondence to:*Chul-Woong Woo
E-mail: cwwoo@amc.seoul.kr
*Young-Hoon Kim
E-mail: kimyh@amc.seoul.kr

#These authors contributed equally to this work.

Received: September 21, 2019; Revised: January 2, 2020; Accepted: January 2, 2020

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

An in vitro model for ischemia/reperfusion injury has not been well-established. We hypothesized that this failure may be caused by serum deprivation, the use of glutamine-containing media, and absence of acidosis. Cell viability of H9c2 cells was significantly decreased by serum deprivation. In this condition, reperfusion damage was not observed even after simulating severe ischemia. However, when cells were cultured under 10% dialyzed FBS, cell viability was less affected compared to cells cultured under serum deprivation and reperfusion damage was observed after hypoxia for 24 h. Reperfusion damage after glucose or glutamine deprivation under hypoxia was not significantly different from that after hypoxia only. However, with both glucose and glutamine deprivation, reperfusion damage was significantly increased. After hypoxia with lactic acidosis, reperfusion damage was comparable with that after hypoxia with glucose and glutamine deprivation. Although high-passage H9c2 cells were more resistant to reperfusion damage than low-passage cells, reperfusion damage was observed especially after hypoxia and acidosis with glucose and glutamine deprivation. Cell death induced by reperfusion after hypoxia with acidosis was not prevented by apoptosis, autophagy, or necroptosis inhibitors, but significantly decreased by ferrostatin-1, a ferroptosis inhibitor, and deferoxamine, an iron chelator. These data suggested that in our SIR model, cell death due to reperfusion injury is likely to occur via ferroptosis, which is related with ischemia/reperfusion-induced cell death in vivo. In conclusion, we established an optimal reperfusion injury model, in which ferroptotic cell death occurred by hypoxia and acidosis with or without glucose/glutamine deprivation under 10% dialyzed FBS.

Keywords: Ferroptosis, Ischemia, Lactic acidosis, Myocardial infarction, Reperfusion injury