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

• Son E
• Lee D
• Woo CW
• Kim YH

• KMID: 2471039
• DOI: http://doi.org/10.4196/kjpp.2020.24.2.173

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

Figure

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  Fig. 1 Experimental schedule. SI stands for simulated ischemia and SR stands for simulated reperfusion. SI, simulated ischemia; SR, simulated reperfusion.

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  Fig. 2 Effect of fetal bovine serum (FBS) deprivation and 10% dialyzed FBS on H9c2 cell viability. To investigate the effect of serum deprivation and 10% dialyzed FBS on cell viability, MTT assay was performed. H9c2 cells were seeded in 6-well plates at 60,000 cells per well and cultured for four days. A media change was carried out once two days after seeding. After the medium was removed and cells were washed twice with PBS, DMEM was changed with FBS or dialyzed FBS at the concentration of the above experimental conditions. After culturing for 24 h, the medium was removed and 0.5 mg/ml Thiazolyl Blue Tetrazolium Blue (MTT) powder suspended in DMEM containing 10% FBS was treated. After 2 h, medium was removed, and cells were washed twice with PBS and subsequently treated with DMSO. The formazan solution was transferred to a 96-well plate and absorbance was measured at 562 nm. Data are expressed as means ± S.D. (n = 3) (**p < 0.01 vs. 10% FBS, and #p < 0.05 or ##p < 0.01 vs. 10% diFBS).

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  Fig. 3 Reperfusion damage under 1% serum condition. Ischemia/reperfusion simulation was performed under 1% fetal bovine serum (FBS) conditions. The control group was cultured in DMEM containing 1% FBS for 24 h under normoxia. The hypoxia group was cultured for 24 h under hypoxia (1% oxygen). To simulate extreme ischemia, cells were cultured under hypoxia, glucose/glutamine deprivation (GGD), and lactic acidosis (pH 6.4). Cells were cultured for 24 h in these conditions. After the ischemic period, the medium was changed with DMEM containing 1% FBS and incubated under normoxia. Seventeen hours after the media change, lactate dehydrogenase (LDH) release was measured. Cell photographs were taken as soon as ischemia was over and as soon as the reperfusion was over. Data are expressed as means ± S.D. (n = 3). Scale bar: 200 µm.

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  Fig. 4 Under 10% dialyzed fetal bovine serum (FBS), reperfusion damage was significantly increased when both glucose and glutamine were deprived. SnakeSkin wrinkle dialysis tubing was used to remove material ≤ 10 kDa in FBS, including nutrients such as glucose and glutamine. Dialyzed FBS was used for ischemia simulation with DMEM at a concentration of 10%. After culturing for 24 h under the specified conditions, cells were photographed. The medium was changed with DMEM containing 10% FBS. After 17 h incubation under normoxia, cells were photographed and lactate dehydrogenase (LDH) release was measured. Cell passage used in these experiments was below 20. Data are expressed as means ± S.D. (n = 3) (++p < 0.01 vs. control under normoxia, **p < 0.01 vs. control under hypoxia, #p < 0.05 vs. glucose/glutamine deprivation under hypoxia). Scale bar: 200 µm.

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  Fig. 5 Acidosis during hypoxia significantly induced reperfusion damage. To produce media with pH 6.4 under 5% CO2, 2.4 mM NaHCO3, obtained from the Henderson-Hasselbach equation, was added to DMEM (D5030). In the control group, 24 mM NaHCO3 was added to achieve pH 7.4. Therefore, we added 21.6 mM Na-lactate to match the osmolality of the media used to induce acidosis in the control group. As a result, we used the media, which is made for simulating lactic acidosis. Media pH was measured 7.4–7.5 in the control group and 6.4–6.5 in the acidosis group. Cell passage used in these experiments was below 20. Data are expressed as means ± S.D. (n = 3) (*p < 0.05 and **p < 0.01vs. control under hypoxia, #p < 0.05 vs. glucose/glutamine deprivation + acidosis under hypoxia). GGD, glucose/glutamine deprivation; N.S., not significant. Scale bar: 200 µm.

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  Fig. 6 High passage H9c2 cells obtained resistance to reperfusion damage. SIR was performed using high-passage H9c2 cells grown for 5 months or more. The passage used in the high passage data in (A) above was 25–29, and the passage of H9c2 used in the photograph of (B) was 27. Cells were maintained by subculturing twice weekly (Monday, Thursday). During subculturing, cell density did not exceed 90%, and media changed were also performed periodically (Wednesday, Saturday). There were no special events to mention while we maintain cells, such as extensive cell death or sudden changes in cell number. Data are expressed as means ± S.D. (high passage n = 4, low passage n = 3) (**p < 0.01 vs. low passage, ##p < 0.01vs. acidosis with hypoxia). LDH, lactate dehydrogenase; SIR, simulated ischemia/reperfusion; GGD, glucose/glutamine deprivation. Statistical analyses were conducted using an unpaired t-test. Scale bar: 200 µm.

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  Fig. 7 Reperfusion induced iron-dependent cell death, which was likely ferroptosis. Before the media change to simulate the reperfusion, a media change was performed after adjusting the temperature and pH by incubating for 3–4 h in the incubator. At this time, 100 µM deferoxamine, 1 µM ferrostatin-1, and 20 µM necrostatin-1 were added to the media before incubation. Data are expressed as means ± S.D. (n = 3) (*p < 0.05 and **p < 0.01 vs. control). LDH, lactate dehydrogenase. Scale bar: 200 µm.

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