Pim-1 Kinase Regulating Dynamics Related Protein 1 Mediates Sevoflurane Postconditioning-induced Cardioprotection

Jin-Dong Liu, Hui-Juan Chen, Da-Liang Wang, Hui Wang, Qian Deng, Jin-Dong Liu, Hui-Juan Chen, Da-Liang Wang, Hui Wang, Qian Deng

Abstract

Background: It is well documented that sevoflurane postconditioning (SP) has a significant myocardial protection effect. However, the mechanisms underlying SP are still unclear. In the present study, we investigated the hypothesis that the Pim-1 kinase played a key role in SP-induced cardioprotection by regulating dynamics-related protein 1 (Drp1).

Methods: A Langendorff model was used in this study. Seventy-two rats were randomly assigned into six groups as follows: CON group, ischemia reperfusion (I/R) group, SP group , SP+proto-oncogene serine/threonine-protein kinase 1 (Pim-1) inhibitor II group, SP+dimethylsufoxide group, and Pim-1 inhibitor II group (n = 12, each). Hemodynamic parameters and infarct size were measured to reflect the extent of myocardial I/R injury. The expressions of Pim-1, B-cell leukemia/lymphoma 2 (Bcl-2) and cytochrome C (Cyt C) in cytoplasm and mitochondria, the Drp1 in mitochondria, and the total Drp1 and p-Drp1ser637 were measured by Western blotting. In addition, transmission electron microscope was used to observe mitochondrial morphology. The experiment began in October 2014 and continued until July 2016.

Results: SP improved myocardial I/R injury-induced hemodynamic parametric changes, cardiac function, and preserved mitochondrial phenotype and decreased myocardial infarct size (24.49 ± 1.72% in Sev group compared with 41.98 ± 4.37% in I/R group; P< 0.05). However, Pim-1 inhibitor II significantly (P < 0.05) abolished the protective effect of SP. Western blotting analysis demonstrated that, compared with I/R group, the expression of Pim-1 and Bcl-2 in cytoplasm and mitochondria as well as the total p-Drp1ser637 in Sev group (P < 0.05) were upregulated. Meanwhile, SP inhibited Drp1 compartmentalization to the mitochondria followed by a reduction in the release of Cyt C. Pretreatment with Pim-1 inhibitor II significantly (P < 0.05) abolished SP-induced Pim-1/p-Drp1ser637 signaling activation.

Conclusions: These findings suggested that SP could attenuate myocardial ischemia-reperfusion injury by increasing the expression of the Pim-1 kinase. Upregulation of Pim-1 might phosphorylate Drp1 and prevent extensive mitochondrial fission through Drp1 cytosolic sequestration.

Conflict of interest statement

There are no conflicts of interest.

Figures

Figure 1
Figure 1
Hemodynamic changes at different points in rats subjected to myocardial ischemia-reperfusion injury. (a) The changes of HR at T0–T4; (b) the changes of LVDP at T0–T4; (c) the changes of LVEDP at T0–T4; (d) the changes of + dp/dtmax at T0–T4; (e) and the changes of −dp/dtmax at T0–T4. Data were reported as mean ± SD (n = 6 for each group). *P < 0.05 versus CON; †P < 0.05 versus I/R; ‡P < 0.05 versus Sev (repeated measures ANOVA). CON: Control group; I/R: Ischemia/reperfusion; Sev: Sevoflurane postconditioning; Sev+PIM-Inh: Sevoflurane postconditioning + Pim-1 inhibitor II; Sev–DMSO: Sevoflurane postconditioning + dimethyl sulfoxide; PIM-Inh: Pim-1 inhibitor II; T0: The end of equilibration; T1: 30 min of reperfusion; T2: 60 min of reperfusion; T3: 90 min of reperfusion; T4: 120 min of reperfusion; HR: Heart rate; LVDP: Left ventricular developed pressure; LVEDP: Left ventricular end-diastolic pressure; +dp/dtmax: Maximum rate of left ventricular development pressure; −dp/dtmax: Maximum rate of left ventricular fall pressure; SD: Standard deviation; ANOVA: Analysis of variance.
Figure 2
Figure 2
Western blotting analysis of cytosolic and mitochondrial fraction of Pim-1, Bcl-2, and Cyt C in myocardium. The Pim-1 (44,000 Da) (a and b) was analyzed by Western blotting with specific Pim-1 antibody at 1 h of reperfusion; the Bcl-2 (26,000 Da) (c and d) was analyzed by Western blotting with specific Bcl-2 antibody at 1 h of reperfusion; and the Cyt C (15,000 Da) (e and f) was analyzed by Western blotting with specific Cyt C antibody at 1 h of reperfusion. (n = 3 for each group). Data were reported as mean ± SD. *P < 0.05 versus CON; †P < 0.05 versus I/R; ‡P < 0.05 versus Sev (repeated measures ANOVA). CON: Control group; I/R: Ischemia/reperfusion; Sev: Sevoflurane postconditioning; Sev+PIM-Inh: Sevoflurane postconditioning + Pim-1 inhibitor II; Sev+DMSO: Sevoflurane postconditioning + dimethyl sulfoxide; PIM-Inh: Pim-1 inhibitor II; Pim-1: Proto-oncogene serine/threonine-protein kinase 1; Bcl-2: B-cell leukemia/lymphoma 2; Cyt C: Cytochrome C; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; COX-IV: Cytochrome C oxidase IV; SD: Standard deviation; ANOVA: Analysis of variance.
Figure 3
Figure 3
Western blotting analysis of total Drp1 and p-Drp1ser637 in myocardium as well as Drp1 translation to the mitochondria. The total Drp1 (80,000 Da) (a) and the Drp1 in mitochondria (c) were analyzed by Western blotting with specific Drp1 antibody at 1 h of reperfusion; The p-Drp1ser637 (80,000 Da) (b) was analyzed by Western blotting with specific p-Drp1ser637 antibody at 1 h of reperfusion (n = 3 for each group). Data were reported as mean ± SD. *P < 0.05 versus CON; †P < 0.05 versus I/R; ‡P < 0.05 versus Sev (one-way ANOVA). CON: Control group; I/R: Ischemia/reperfusion; Sev: Sevoflurane postconditioning; Sev+PIM-Inh: Sevoflurane postconditioning + Pim-1 inhibitor II; Sev+DMSO: Sevoflurane postconditioning + dimethyl sulfoxide; PIM-Inh: Pim-1 inhibitor II; Drp1: Dynamics-related protein 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; COX-IV: Cytochrome C oxidase IV; SD: Standard deviation; ANOVA: Analysis of variance.
Figure 4
Figure 4
Myocardial infarct size of isolated hearts in six groups. (a) Representative cross sections of rat hearts from six groups after I/R and staining with TTC to visualize the infarcted area; (b) infarct size expressed as percentage of the left ventricular area (×100%) for each group. Values are represented as mean ± SD (n = 6 for each group), *P < 0.05 versus CON; †P < 0.05 versus I/R; ‡P < 0.05 versus Sev (one-way ANOVA). CON: Control group; I/R: Ischemia/reperfusion; Sev: Sevoflurane postconditioning; Sev+PIM-Inh: Sevoflurane postconditioning + Pim-1 inhibitor II; Sev+DMSO: Sevoflurane postconditioning + dimethyl sulfoxide; PIM-Inh: Pim-1 inhibitor II; SD: Standard deviation; ANOVA: Analysis of variance.
Figure 5
Figure 5
Transmission electron microscope (uranyl acetate-lead citrate stained, original magnification ×30,000) performance of the myocardial mitochondria in six groups. (a) In the CON group, the mitochondria were normal (b) in the I/R group, the mitochondria appeared small, irregular, and less dense cristae (c) in the Sev group, the severity of mitochondrial injury decreased compared with the I/R group (d) in the Sev+PIM-Inh group, the severity of mitochondrial injury increased compared with the Sev group (e) in the Sev+DMSO group, no significant changes were found compared with the Sev group (f) in the PIM-Inh group, no significant changes were found compared with the I/R group (n = 3 for each group, scale bar = 1 μm). The black arrow shows normal mitochondria, and the red arrow shows fragmented mitochondria. CON: Control group; I/R: Ischemia/reperfusion; Sev: Sevoflurane postconditioning; Sev+PIM-Inh: Sevoflurane postconditioning + Pim-1 inhibitor II; Sev+DMSO: Sevoflurane postconditioning + dimethyl sulfoxide; PIM-Inh: Pim-1 inhibitor II.

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