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. 2022 Feb 28:2022:4833791.
doi: 10.1155/2022/4833791. eCollection 2022.

T1AM Attenuates the Hypoxia/Reoxygenation-Induced Necroptosis of H9C2 Cardiomyocytes via RIPK1/RIPK3 Pathway

Bo Wei  1   2   3 , Hanbing Zhao  2 , Bailong Hu  4 , Lujun Dai  5 , Guoning Zhang  6 , Lili Mo  2 , Niwen Huang  7 , Changchao Zou  8 , Bei Zhang  2 , Haiyan Zhou  2 , Wei Li  2 , Xingde Liu  1   2   8
Affiliations

T1AM Attenuates the Hypoxia/Reoxygenation-Induced Necroptosis of H9C2 Cardiomyocytes via RIPK1/RIPK3 Pathway

Bo Wei et al. Biomed Res Int. .

Abstract

Purpose: To investigate the detailed mechanism of 3-iodothyronamine (T1AM) in cell apoptosis and programmed necrosis of hypoxia/reoxygenation- (H/R-) induced H9C2 injury.

Materials and methods: Cardiomyocyte H9C2 cells were cultured in vitro for the establishment of cardiomyocyte H/R models. Cells were randomly divided into four groups: the control group, H/R group, T1AM pretreatment group, T1AM pretreatment and H/R (6 μm T1AM+H/R) group. The degree of myocardial injury was determined by the detection of the cardiomyocyte inhibition rate by CCK8 and the detection of lactic dehydrogenase (LDH) activity. Cell apoptosis was assessed through TUNEL assay and flow cytometry analysis. The protein level and mRNA level of RIPK1, RIPK3, and CAMKII were detected by western blotting and qRT-PCR.

Results: Compared with the control group, the cell inhibition rate was dramatically elevated in the H/R group. LDH release of cardiomyocytes was significantly increased. Protein and mRNA expressions of RIPK1, RIPK3, and CAMKII were significantly enhanced. Compared with the H/R group, the cell inhibition rate, LDH release, cardiomyocyte necroptosis rate, and protein and mRNA levels of RIPK1, RIPK3, and CAMKII of the T1AM+H/R group were significantly decreased.

Conclusion: Pretreatment with T1AM could alleviate cardiomyocytes' H/R injury and inhibit necroptosis of cardiomyocytes, which might exert a protective function upon activation of the RIPK1/RIPK3 pathway.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
T1AM protects H/R-induced H9C2 cells injury. (a) The effects of T1AM on H/R-induced cell morphology. (b) CCK8 assay (n = 4) revealed that T1AM improved H/R-induced H9C2 cell viability. (c) LDH release (n = 4) in the culture medium of H9C2 cells exposed to hypoxia 24 h and reoxygenation 4 h treated with 6 μM T1AM. (d) TUNEL stain positivity and Hoechst stain condensed nuclei of H9C2 cells (n = 4). (e) Quantification of TUNEL-positive cells. Control: H9C2 cells cultured in normoxia; T1AM: H9C2 cells cultured in normoxia treated with 6 μM T1AM; H/R: H9C2 cells exposed to hypoxia 24 h and reoxygenation 4 h; H/R+T1AM: H9C2 cells exposed to hypoxia 24 h and reoxygenation 4 h treated with 6 μM T1AM.
Figure 2
Figure 2
T1AM protects H/R-induced H9C2 cells injury via RIPK1/RIPK3 pathway. (a) Annexin V/PI staining. (b) Quantification of PI-positive cells. (c) Western blot. (d) The band intensities are quantified.
Figure 3
Figure 3
mRNA expression of CAMKII, RIPK1, and RIPK3.
Figure 4
Figure 4
T1AM preconditioning alleviates cardiomyocytes necroptosis by the RIPK1/RIPK3 pathway which can be rescued by TNF-α. (a) LDH release. (b) Western blot. (c) The band intensities are quantified. H/R: H9C2 cells exposed to hypoxia 24 h and reoxygenation 4 h; H/R+T1AM: H9C2 cells exposed to hypoxia 24 h and reoxygenation 4 h treated with 6 μM T1AM; H/R+T1AM+TNF-α: H9C2 cells exposed to hypoxia 24 h and reoxygenation 4 h treated with 6 μM T1AM and 20 ng/ml TNF-α.
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