Levosimendan Postconditioning Attenuates Cardiomyocyte Apoptosis after Myocardial Infarction

Abstract

Background: Levosimendan preconditioning has been shown to attenuate myocardial apoptosis in animal models. However, protective effects of levosimendan postconditioning against myocardial apoptosis following myocardial infarction (MI) have not been evaluated. Therefore, we investigated the effects of levosimendan postconditioning on myocardial apoptosis in MI rat models.

Methods: In an anoxia/reoxygenation (A/R) model, H9c2 cells were pretreated with or without levosimendan postconditioning after which their apoptosis rates were assessed by flow cytometry, RT-qPCR, and western blot analyses. Then, postconditioning was performed with or without levosimendan in MI rat models. Myocardiocyte apoptosis was evaluated by echocardiography, TTC staining, TUNEL staining, immunohistochemical staining, RT-qPCR, and western blot analysis.

Results: Levosimendan postconditioning inhibited H9c2 cell apoptosis in A/R models by elevating Bcl-2 while suppressing Caspase-3 and Bax at both mRNA and protein levels. Moreover, it improved cardiac functions and reduced the left ventricle infarction area in MI rat models. Compared to the MI control group, cardiomyocyte apoptosis rates in the levosimendan postconditioning group were low. The reduced cardiomyocyte apoptosis rates were associated with downregulation of Bax and Caspase-3 as well as with upregulation of Bcl-2 at mRNA and protein levels.

Conclusions: Levosimendan postconditioning of MI rat models protected against cardiomyocyte apoptosis, implying that it is a potential strategy for preventing cardiomyocyte apoptosis in the treatment of cardiac dysfunction following MI.

Figure 1

(a, b) Apoptosis of H9c2 cells in each group 24 h after A/R induction, as determined by flow cytometry (n = 5). (c) mRNA expressions of Caspase-3, Bax, and Bcl-2 in H9c2 cells in each group 24 h after A/R induction, as determined by RT-qPCR (n = 5). (d, e) Representative western blot images and quantitative analyses of Caspase-3, Bax, and Bcl-2 protein levels in H9c2 cells in each group 24 h after A/R induction (n = 5). A/R: anoxia-reoxygenation; Levo: levosimendan. #p < 0.05, A/R + Levo group versus A/R group; △p < 0.05, A/R + Levo group versus control group; ^∗p < 0.05, A/R group versus control group.

Figure 2

(a, b) The left ventricular infarct area was detected by TTC staining in each group 7 days after MI model establishment (n = 3). (a, c) Representative TUNEL staining analysis of cardiomyocyte apoptosis in rats from each group 7 days after MI model establishment (n = 4). Normal cell nuclei are stained blue, while apoptotic cell nuclei are stained brown (magnification: × 200). (a) Immunohistochemical staining of Caspase-3, Bax, and Bcl-2 in rats from each group 7 days after MI model establishment (n = 4). Normal and apoptotic cell nuclei are stained blue and brown, respectively (magnification: × 100). (d) Caspase-3, Bax, and Bcl-2 mRNA levels in rats of each group 7 days after MI model establishment, as determined by qRT-PCR analysis (n = 4). (e, f) Representative western blot images and quantitative analyses of Caspase-3, Bax, and Bcl-2 protein levels in rats from each group 7 days after MI model establishment (n = 4). MI: myocardial infarction; Levo: levosimendan; ^#p < 0.05, MI + Levo group versus MI group; ^Δp < 0.05, MI + Levo group versus control group; ^∗p < 0.05, MI group versus control group.