The role of the Hsp90/Akt pathway in myocardial calpain-induced caspase-3 activation and apoptosis during sepsis

Abstract

Background: Recent studies have demonstrated that myocardial calpain triggers caspase-3 activation and myocardial apoptosis in models of sepsis, whereas the inhibition of calpain activity down-regulates myocardial caspase-3 activation and apoptosis. However, the mechanism underlying this pathological process is unclear. Therefore, in this study, our aim was to explore whether the Hsp90/Akt signaling pathway plays a role in the induction of myocardial calpain activity, caspase-3 activation and apoptosis in the septic mice.

Methods: Adult male C57 mice were injected with lipopolysaccharide (LPS, 4 mg/kg, i.p.) to induce sepsis. Next, myocardial caspase-3 activity and the levels of Hsp90/p-Akt (phospho-Akt) proteins were detected, and apoptotic cells were assessed by performing the TUNEL assay.

Results: In the septic mice, there was an increase in myocardial calpain and caspase-3 activity in addition to an increase in the number of apoptotic cells; however, there was a time-dependent decrease in myocardial Hsp90/p-Akt protein levels. The administration of calpain inhibitors (calpain inhibitor-Ш or PD150606) prevented the LPS-induced degradation of myocardial Hsp90/p-Akt protein and its expression in cardiomyocytes in addition to inhibiting myocardial caspase-3 activation and apoptosis. The inhibition of Hsp90 by pretreatment with 17-AAG induced p-Akt degradation, and the inhibition of Akt activity by pretreatment with wortmannin resulted in caspase-3 activation in wildtype C57 murine heart tissues.

Conclusions: Myocardial calpain induces myocardial caspase-3 activation and apoptosis in septic mice via the activation of the Hsp90/Akt pathway.

Figure 1

Panel A, The time course of myocardial Hsp90/p-Akt (Thy308) protein expression. Hsp90 and p-Akt/Akt protein expression was determined by Western blot at 0, 1, 2, 4, and 6 h after LPS (4 mg/kg) treatment. Panel B, Myocardial Hsp90/p-Akt expression is decreased in septic mice. In the mice were treated with LPS (4 mg/kg, i.p.) for 4 h, the myocardial Hsp90/p-Akt expression was detected. Panel C, Myocardial caspase-3 activity in the septic mice. Mice were treated with LPS (4 mg/kg, i.p.) for 4 h, the heart tissues were extracted and caspase-3 activity was determined. The data are shown as the mean ± SE (n = 5). LPS, lipopolysaccharide; CI, calpain inhibitor-III., PD, PD150606. * vs. sham, P < 0.05; # vs. LPS, P < 0.05.

Figure 2

The representative immunohistochemical photomicrographs of Hsp90/p-Akt (Thy308). The positive expression of Hsp90 was indicated by brown staining (original magnification × 400). Representative immunohisto- chemical data depicting cardiac Hsp90/p-Akt and cleaved caspase-3 expression in the LPS-treated mice (×400). Panels A and B, Representative pictures reveal a significant decrease in Hsp90 (A) and p-Akt (B) positive expression in the cytoplasm of the myocytes after LPS treatment. Panel C, Representative pictures of cleaved caspase-3 (the antibody was specific to detect the large fragment 17/19 KDa of activated caspase-3) expression in the cytoplasm after LPS treatment or LPS plus calpain inhibitors, calpain inhibitor III and PD150606 in the septic mice. Panel D: Representative quantitative data of Hsp90/p-Akt and cleaved caspase-3 expression in the septic mice and after treatment with calpain inhibitors. The values were from ten high power fields each section randomly selected and counted manually. CI, calpain inhibitor-III; PD, PD150606. * P < 0.05 vs. sham,# P < 0.05 vs. LPS.

Figure 3

Determination of apoptosis in the cardiomyocytes. Panel A, Four hours after LPS treatment (4 mg/kg), 4-mm paraffin- embeded tissue sections were prepared from LV apex and TUNEL staining of the nuclei of cardiomyocytes from control and LPS-treated mice was performed. Panel B, The apoptosis index among the sham-treated mice and mice treated with LPS or LPS plus calpain inhibitors is demonstrated. CI, calpain inhibitor-III; PD, PD150606. * P < 0.05 vs. sham, # P < 0.05 vs. LPS (n = 5).

Figure 4

The effects of treatment with 17AAG (Hsp90 inhibitor) on Akt activity and wortmannin (PI3K inhibitor) on caspase-3 activation are depicted. Mice were treated with either 17-AAG or wortmannin (3 mg/kg, i.p). Four hours after administration, the activation of Akt and caspase-3 was detected. * P < 0.05 vs. sham (n = 5).