Mild therapeutic hypothermic protection activates the PI3K/AKT signaling pathway to inhibit TRPM7 and suppress ferroptosis induced by myocardial ischemia‑reperfusion injury

Abstract

The present study aimed to investigate the role of PI3K‑mediated ferroptosis signaling induced by mild therapeutic hypothermia (MTH), which was defined as a temperature of 34˚C, in protecting against myocardial ischemia-reperfusion (I/R) injury (MIRI). To meet this aim, H9C2 cells underwent hypoxia‑reperfusion (H/R) and/or MTH. The MTT assay was used to assess cell viability, cytotoxicity was measured using a lactate dehydrogenase cytotoxicity assay, and Annexin V‑FITC/PI flow cytometric analysis was used to analyze early and late cell apoptosis. In addition, 84 healthy adult male Sprague‑Dawley rats were randomly divided into seven groups (n=12), and underwent I/R and various treatments. Hemodynamics were monitored, and the levels of myocardial injury marker enzymes and oxidative stress markers in myocardial tissue were measured using ELISA. The expression levels of PI3K, AKT, transient receptor potential cation channel subfamily M member 7 (TRPM7), glutathione peroxidase 4 (GPX4) and acyl‑CoA synthetase long chain family member 4 (ACSL4) in animals and cells were measured using western blot analysis. These experiments revealed that MTH could effectively reduce myocardial infarct size, improve hemodynamic performance following MIRI and suppress myocardial apoptosis, thereby contributing to the recovery from H/R injury. Mechanistically, MTH was revealed to be able to activate the PI3K/AKT signaling pathway in cells, upregulating GPX4, and downregulating the expression levels of TRPM7 and ACSL4. Treatment with 2‑aminoethoxydiphenyl borate (an inhibitor of TRPM7) could further strengthen the myocardial protective effects of MTH, whereas treatment with erastin (promoter of ferroptosis) and wortmannin (inhibitor of PI3K) led to the effective elimination of the myocardial protective effects of MTH. Compared with in the I/R group, the PI3K/AKT activation level and the expression levels of GPX4 were both significantly increased, whereas the expression levels of TRPM7 and ACSL4 were significantly decreased in the I/R + MTH group. Taken together, the results of the present study indicated that MTH may activate the PI3K/AKT signaling pathway to inhibit TRPM7 and suppress ferroptosis induced by MIRI.

Keywords: PI3K/AKT signaling pathway; mild therapeutic hypothermia; myocardial ischemia-reperfusion injury; transient receptor potential cation channel subfamily M member 7.

Figure 1.

(A) Schematic diagram of the cell experimental protocol. (B) Schematic diagram of the animal experimental protocol. 2-APB, 2-aminoethoxydiphenyl borate; Era, erastin; H/R, hypoxia-reperfusion; I/R, ischemia-reperfusion; MTH, mild therapeutic hypothermia; Wort, wortmannin.

Figure 2.

Cardiomyocyte viability, cytotoxicity and apoptosis. (A) Viability of H9C2 cells was examined using a MTT cell viability assay kit. (B) Cytotoxicity in H9C2 cells was examined using a lactate dehydrogenase cytotoxicity assay kit. (C) Flow cytometric analysis of the levels of H9C2 cell apoptosis. (D) Statistical analysis of apoptosis rate. *P<0.05 vs. control group; ^#P<0.05 vs. H/R group. H/R, hypoxia-reperfusion; MTH, mild therapeutic hypothermia.

Figure 3.

PI3K/AKT phosphorylation, TRPM7 expression levels and the expression level of ferroptosis-associated proteins in cells. Western blot analyses of (A) TRPM7, p-PI3K, PI3K, p-AKT and AKT; (B) GPX4 and ACSL4; and (C) FSP1. The expression levels of (D) TRPM7, (E) GPX4, (F) p-PI3K, (G) ACSL4, (H) p-AKT and (I) FSP1 are shown. *P<0.05 vs. control group; ^#P<0.05 vs. H/R group. ACSL4, acyl-CoA synthetase long chain family member 4; FSP1, ferroptosis suppressor protein 1; GPX4, glutathione peroxidase 4; H/R, hypoxia-reperfusion; MTH, mild therapeutic hypothermia; p-, phosphorylated; TRPM7, transient receptor potential cation channel subfamily M member 7.

Figure 4.

TTC staining results of myocardial infarction. (A) Myocardial sections following TTC staining. (B) Semi-quantitative analysis of the TTC staining results of myocardial infarction. *P<0.05 vs. sham group; ^#P<0.05 vs. I/R group; ^&P<0.05 vs. I/R + MTH group. 2-APB, 2-aminoethoxydiphenyl borate; Era, erastin; I/R, ischemia-reperfusion; MTH, mild therapeutic hypothermia; TTC, 2,3,5-triphenyltetrazolium chloride; Wort, wortmannin.

Figure 5.

Pathological changes of myocardial tissue and levels of myocardial enzymes. (A) Hematoxylin and eosin staining, arrows indicate the location of myocardial fiber rupture or dissolution. Levels of (B) CK-MB, (C) LDH and (D) cTnI. *P<0.05 vs. sham group; ^#P<0.05 vs. I/R group; ^&P<0.05 vs. I/R + MTH group. 2-APB, 2-aminoethoxydiphenyl borate; CK-MB, creatine kinase-MB; cTnI, cardiac troponin I; Era, erastin; I/R, ischemia-reperfusion; LDH, lactate dehydrogenase; MTH, mild therapeutic hypothermia; Wort, wortmannin.

Figure 6.

PI3K/AKT phosphorylation, TRPM7 expression levels and the expression level of ferroptosis-associated proteins. (A) Western blot analysis of TRPM7, p-PI3K, PI3K, p-AKT and AKT. Expression levels of (B) TRPM7, (C) p-PI3K and (D) p-AKT. (E) Western blot analysis of GPX4 and ACSL4. Expression levels of (F) GPX4 and (G) ACSL4. Levels of the oxidative stress indicators (H) GSH-Px, (I) MDA and (J) SOD. *P<0.05 vs. sham group; ^#P<0.05 vs. I/R group; ^&P<0.05 vs. I/R + MTH group. 2-APB, 2-aminoethoxydiphenyl borate; ACSL4, acyl-CoA synthetase long chain family member 4; Era, erastin; GPX4, glutathione peroxidase 4; GSH-Px, glutathione peroxidase; I/R, ischemia-reperfusion; MDA, malondialdehyde; MTH, mild therapeutic hypothermia; p-, phosphorylated; SOD, superoxide dismutase; TRPM7, transient receptor potential cation channel subfamily M member 7; Wort, wortmannin.

Figure 7.

Protective mechanism of MTH on myocardial IRI. MTH can reduce the ferroptosis induced by myocardial I/R via inhibiting the expression of TRPM7, thereby alleviating myocardial IRI. 2-APB, 2-aminoethoxydiphenyl borate; ACSL4, acyl-CoA synthetase long chain family member 4; Era, erastin; GPX4, glutathione peroxidase 4; I/R, ischemia-reperfusion; IRI, I/R injury; MTH, mild therapeutic hypothermia; p-, phosphorylated; ROS, reactive oxygen species; TRPM7, transient receptor potential cation channel subfamily M member 7; Wort, wortmannin.