The Protective Role of the TOPK/PBK Pathway in Myocardial Ischemia/Reperfusion and H₂O₂-Induced Injury in H9C2 Cardiomyocytes

Abstract

T-LAK-cell-originated protein kinase (TOPK) is a PDZ-binding kinase (PBK) that was recently identified as a novel member of the mitogen-activated protein kinase (MAPK) family. It has been shown to play an important role in many cellular functions. However, its role in cardiac function remains unclear. Thus, we have herein explored the biological function of TOPK in myocardial ischemia/reperfusion (I/R) and oxidative stress injury in H9C2 cardiomyocytes. I/R and ischemic preconditioning (IPC) were induced in rats by 3-hour reperfusion after 30-min occlusion of the left anterior descending coronary artery and by 3 cycles of 5-min I/R. Hydrogen peroxide (H₂O₂) was used to induce oxidative stress in H9C2 cardiomyocytes. TOPK expression was analyzed by western blotting, RT-PCR, immunohistochemical staining, and immunofluorescence imaging studies. The effects of TOPK gene overexpression and its inhibition via its inhibitor HI-TOPK-032 on cell viability and Bcl-2, Bax, ERK1/2, and p-ERK1/2 protein expression were analyzed by MTS assay and western blotting, respectively. The results showed that IPC alleviated myocardial I/R injury and induced TOPK activation. Furthermore, H₂O₂ induced TOPK phosphorylation in a time-dependent manner. Interestingly, TOPK inhibition aggravated the H₂O₂-induced oxidative stress injury in myocardiocytes, whereas overexpression relieved it. In addition, the ERK pathway was positively regulated by TOPK signaling. In conclusion, our results indicate that TOPK might mediate a novel survival signal in myocardial I/R, and that its effect on anti-oxidative stress involves the ERK signaling pathway.

Keywords: TOPK/PBK; ischemia/reperfusion; ischemic preconditioning; oxidative stress.

Figure 1

Ischemic preconditioning (IPC) treatment decreased myocardial ischemia/reperfusion (I/R) injury in vivo. (A) Representative photographs of Evans blue-TTC double staining and the quantitative analysis of myocardial infarct size between I/R (30-min ischemia/3-hour reperfusion) and IPC (three cycles of 5-min Ischemia/5-min Reperfusion)+I/R groups; (B) Hematoxylin and eosin (HE) staining of three groups, showing IPC mediated alleviation of the myocardium destruction; (C) Positive nuclear staining for myocardial apoptosis as assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and the quantitative analysis of apoptotic nuclei/total nuclei; (D) Analysis of Bcl-2 and Bax protein expression by western blotting and the corresponding quantitative analysis. β-actin was used as the loading control. n = 6. * p < 0.05 compared with I/R group.

Figure 1

Ischemic preconditioning (IPC) treatment decreased myocardial ischemia/reperfusion (I/R) injury in vivo. (A) Representative photographs of Evans blue-TTC double staining and the quantitative analysis of myocardial infarct size between I/R (30-min ischemia/3-hour reperfusion) and IPC (three cycles of 5-min Ischemia/5-min Reperfusion)+I/R groups; (B) Hematoxylin and eosin (HE) staining of three groups, showing IPC mediated alleviation of the myocardium destruction; (C) Positive nuclear staining for myocardial apoptosis as assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and the quantitative analysis of apoptotic nuclei/total nuclei; (D) Analysis of Bcl-2 and Bax protein expression by western blotting and the corresponding quantitative analysis. β-actin was used as the loading control. n = 6. * p < 0.05 compared with I/R group.

Figure 2

Ischemic preconditioning (IPC) treatment induced upregulation of TOPK in the rat myocardium after 30 min ischemia/3 h reperfusion. (A) Representative immunohistochemical staining of TOPK and p-TOPK in the ischemic area of left ventricular (LV) myocardial sections of rats subjected to sham operation, I/R, or IPC+I/R; (B) Detection of mRNA levels of TOPK by RT-PCR. GAPDH was used as the loading control; (C) Detection and quantitative analysis of TOPK and p-TOPK levels by western blotting. β-actin was used as the loading control. n = 6. * p < 0.05 compared with sham-operation group.

Figure 2

Ischemic preconditioning (IPC) treatment induced upregulation of TOPK in the rat myocardium after 30 min ischemia/3 h reperfusion. (A) Representative immunohistochemical staining of TOPK and p-TOPK in the ischemic area of left ventricular (LV) myocardial sections of rats subjected to sham operation, I/R, or IPC+I/R; (B) Detection of mRNA levels of TOPK by RT-PCR. GAPDH was used as the loading control; (C) Detection and quantitative analysis of TOPK and p-TOPK levels by western blotting. β-actin was used as the loading control. n = 6. * p < 0.05 compared with sham-operation group.

Figure 3

H₂O₂ treatment induced upregulation of p-TOPK in H9C2 cardiomyocytes. (A) Cultured H9C2 cardiomyocytes were stimulated with a different H₂O₂ concentrations (0–1000 µM) for various time points (1 or 2 h), and cell viability was evaluated by MTS assay; (B) Western blot analysis of TOPK activation by H₂O₂ treatment (750 µM for 0, 15, 30, 60, or 120 min); (C) Immunofluorescence staining of H9C2 cardiomyocytes with p-TOPK antibody (red) and DAPI for nuclei (blue) after treatment with 750 µM of H₂O₂ for 1 h. * p < 0.05 compared with nonstimulated controls.

Figure 4

Analysis of the effects of the TOPK specific inhibitor HI-TOPK-032 on cell viability, apoptosis, and protein expression. H9C2 cardiomyocytes were incubated with HI-TOPK-032 at concentrations of 0.5, 1.0, and 2.0 µM for 24 h and then stimulated with 750 µM of H₂O₂ for 1 h. Dimethyl sulfoxide (DMSO) was used as a solvent control. (A) Cell viability as detected by MTS assay; (B) Western blot analysis of Bcl-2, Bax, ERK, and p-ERK expression. β-actin was used as a loading control; (C) The Bcl-2/Bax ratio was calculated and compared. * p < 0.05 compared with nonstimulated controls; ** p < 0.05 compared with H₂O₂-stimulated controls.

Figure 5

Analysis of the effects of TOPK overexpression on cell viability, apoptosis, and protein expression. H9C2 cardiomyocytes were stimulated with 750 µM of H₂O₂ for 1 h after TOPK overexpression plasmid/empty plasmid transfection for 72 h. (A) Cell viability as detected by MTS assay; (B) Western blot analysis of Bcl-2, Bax, ERK, and p-ERK expression. β-actin was used as a loading control; (C) Comparison of the Bcl-2/Bax ratio. * p < 0.05 compared with nonstimulated controls with empty plasmid transfection; ** p < 0.05 compared with H₂O₂-stimulated controls with empty plasmid transfection.