Involvement of HSP90 in ischemic postconditioning-induced cardioprotection by inhibition of the complement system, JNK and inflammation

Abstract

Purpose: To investigate whether heat shock protein 90 (HSP90) is involved in complement regulation in ischemic postconditioning (IPC).

Methods: The left coronary artery of rats underwent 30 min of occlusion, followed by 120 min of reperfusion and treatment with IPC via 3 cycles of 30s reperfusion and 30s occlusion. The rats were injected intraperitoneally with 1 mg/kg HSP90 inhibitor geldanamycin (GA) after anesthesia. Eighty rats were randomly divided into four groups: sham, ischemia-reperfusion (I/R), IPC and IPC + GA. Myocardial infarct size, apoptosis index and the expression of HSP90, C3, C5a, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1β and c-Jun N-terminal kinase (JNK) were assessed.

Results: Compared with the I/R injury, the IPC treatment significantly reduced infarct size, release of troponin T, creatine kinase-MB, and lactate dehydrogenase, and cardiomyocyte apoptosis. These beneficial effects were accompanied by a decrease in TNF-α, IL-1β, C3, C5a and JNK expression levels. However, all these effects were abrogated by administration of the HSP90 inhibitor GA.

Conclusion: HSP90 exerts a profound effect on IPC cardioprotection, and may be linked to the inhibition of the complement system and JNK, ultimately attenuating I/R-induced myocardial injury and apoptosis.

Figure 1. Effects of IPC on HSP90 protein expression. (A) Representative Western blots showing the expression of HSP90. (B) Relative expression of HSP90 protein. Values are presented as the mean ± standard deviation. #P < 0.05 vs. I/R group; n=5 for each group.

Figure 2. Effects of GA and IPC on myocardial infarct size after cardiac I/R injury (IFA/LV). (A) Sham group, (B) I/R group, (C) IPC group, (D) IPC+GA group, (E) Effects of GA and IPC on myocardial infarct size after cardiac I/R injury. GA = geldanamycin; I/R = ischemia - reperfusion; IPC = ischemic postconditioning; the results presented the mean ± standard deviation. #P < 0.05 vs. I/R group; *P < 0.05 vs. IPC group; n=5 for each group.

Figure 3. Effects of GA and IPC on apoptosis after myocardial I/R. (A) Sham group, (B) I/R group, (C) IPC group, (D) IPC+GA group. Apoptotic cardiomyocyte nuclei appear brown stained, whereas TUNEL-negative nuclei appear blue. Mean apoptotic index was counted in each group (A-D), results presented in a bar graph (E) are the mean ± standard deviation. Arrow indicates TUNEL-positive cells. TUNEL stain ×400, all bars = 20μm. Arrow indicates TUNEL - positive cells. #P < 0.05 vs. I/R group; *P < 0.05 vs. IPC group; n=5 for each group.

Figure 4. Effects of GA and IPC on TNF-α, IL-1β mRNA and protein expression. (A) Representative Western blots showing the expression of TNF-α and IL-1β. (B)(C) The mRNA levels of TNF-α and IL-1β were measured using qPCR in different groups. (D) Relative expression of TNF-α protein. (E) Relative expression of IL-1β protein. Values are presented as the mean ± standard deviation. #P < 0.05 vs. I/R group; *P < 0.05 vs. IPC group; n=5 for each group.

Figure 5. Effects of GA and IPC on C3, C5a and JNK protein expression. (A) Representative Western blots showing the expression of C3, C5a and JNK. (B) Relative expression of C5a protein. (C) Relative expression of C3 protein. (D) Relative expression of JNK protein. Values are presented as the mean ± standard deviation. #P < 0.05 vs. I/R group; *P < 0.05 vs. IPC group; n=5 for each group.