Korean J Physiol Pharmacol.  2020 Mar;24(2):173-183. 10.4196/kjpp.2020.24.2.173.

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

Affiliations
  • 1Department of Pharmacology, University of Ulsan College of Medicine, Seoul 05505, Korea. kimyh@amc.seoul.kr
  • 2Bio-Medical Institute of Technology, University of Ulsan, Seoul 05505, Korea.
  • 3Stem Cell Immunomodulation Research Center, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Korea.
  • 4Department of Medical Science, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Korea.
  • 5Asan Institute for Life Sciences, Asan Medical Center, Seoul 05505, Korea. cwwoo@amc.seoul.kr

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.

Keyword

Ferroptosis; Ischemia; Lactic acidosis; Myocardial infarction; Reperfusion injury

MeSH Terms

Acidosis
Acidosis, Lactic
Anoxia
Apoptosis
Autophagy
Cell Death
Cell Survival
Deferoxamine
Glucose
Glutamine
In Vitro Techniques*
Iron
Ischemia
Myoblasts, Cardiac*
Myocardial Infarction
Reperfusion Injury*
Reperfusion*
Deferoxamine
Glucose
Glutamine
Iron
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