Propofol protects against hydrogen peroxide-induced injury in cardiac H9c2 cells via Akt activation and Bcl-2 up-regulation

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Abstract

Propofol is a widely used intravenous anesthetic agent with antioxidant properties secondary to its phenol based chemical structure. Treatment with propofol has been found to attenuate oxidative stress and prevent ischemia/reperfusion injury in rat heart. Here, we report that propofol protects cardiac H9c2 cells from hydrogen peroxide (H2O2)-induced injury by triggering the activation of Akt and a parallel up-regulation of Bcl-2. We show that pretreatment with propofol significantly protects against H2O2-induced injury. We further demonstrate that propofol activates the PI3K–Akt signaling pathway. The protective effect of propofol on H2O2-induced injury is reversed by PI3K inhibitor wortmannin, which effectively suppresses propofol-induced activation of Akt, up-regulation of Bcl-2, and protection from apoptosis. Collectively, our results reveal a new mechanism by which propofol inhibits H2O2-induced injury in cardiac H9c2 cells, supporting a potential application of propofol as a preemptive cardioprotectant in clinical settings such as coronary bypass surgery.

Introduction

Propofol (2,6-diisopropylphenol), an intravenous anesthetic, has been found to attenuate oxidative stress induced mechanical and metabolic derangements in the isolated rat heart and prevent ischemia/reperfusion injury [1], [2], [3]. The antioxidant mechanism mainly relates to its phenolic chemical structure. Given its relatively weak antioxidant effect in vitro [4], we postulate a previously unappreciated mechanism of action to prevent myocardial ischemia/reperfusion injury. We believe the presence of the drug during the first few minutes of tissue reperfusion might serve to offset the cellular response to the ischemic insult, initiating the cell signaling events whereby cardiomyocytes opt for cell survival.

Phosphoinositide 3-kinase (PI3K)–Akt signaling pathway is one of the important signal transduction pathways controlling cardiomyocytes survival and function [5]. Various growth factors and cellular stresses activate Akt through phosphorylation of threonine 308 or serine 473 residues. Once activated, Akt proceeds to phosphorylate its downstream targets, in various subcellular locations including in the nucleus, which contribute to its anti-apoptotic effects [6], [7]. A wide variety of putative downstream effectors have been identified that could contribute to the anti-apoptotic effects of Akt, including transcription factor NF (nuclear factor)-κB, B-cell lymphoma-2 (Bcl-2) family pro-apoptotic protein Bad, caspase family protein caspase 9, and endothelial nitric oxide synthase (eNOS) [7].

We previously reported that propofol attenuated tumor necrosis factor-α and hydrogen peroxide (H2O2)-induced apoptosis in human umbilical vein endothelial cells, and that this attenuation was associated with up-regulation of eNOS expression, increase in intracellular nitric oxide (NO) release and preservation of Bcl-2 expression [8], [9]. Endothelial cell apoptosis is known to precede myocyte apoptosis in the setting of myocardial ischemia/reperfusion injury [10]. However, the effects of propofol on cardiomyocyte apoptosis have yet to be fully elucidated.

Recently, propofol was reported to protect cardiomyocytes against H2O2-induced apoptosis and the anti-apoptotic effect of propofol was attributed to the increased expression of heme oxygenase-1 [11] in the study. Of interest, it is believed that the cytoprotective effects of heme oxygenase-1 are mediated via interaction with and activation of the PI3K–Akt pathway [12]. However, whether propofol activates cell survival signaling through a PI3K–Akt signaling pathway is currently unclear and remains speculative. Therefore, the present study was designed to determine propofol’s effect on PI3K–Akt signaling pathway as a potential alternative mechanism of propofol-mediated cytoprotection.

Section snippets

Materials and methods

Reagents. Propofol, wortmannin, dimethyl sulfoxide (DMSO) and H2O2 were from Sigma Chemical. The Fluorometric CaspACE™ Assay System and Cell Titer 96® Aqueous Non-radioactive Cell Proliferation Assay were from Promega Corporation. All culture reagent, Hoechst 33258 and Trizol reagent were from Invitrogen. All SDS–PAGE reagents and RT-PCR reagents were from Bio-Rad Laboratories, Inc. Rabbit antibodies against Akt, phospho-Akt, Bcl-2 and horseradish peroxidase (HRP) conjugated goat anti-rabbit

Effects of H2O2 and propofol alone on H9c2 cells viability

We first determined the dose at which cytotoxicity develops in a period of 24 h upon H2O2 exposure in H9c2 cells through the MTS assay. Cells were treated with increasing doses of H2O2 for 24 h. As shown in Fig. 1A, H2O2 impaired cell viability in a concentration-dependent manner over the tested concentration range (50–500 μm). A maximum reduction of 51 ± 8% was observed with 500 μm of H2O2. We chose the level of H2O2 400 μm for use in our subsequent experiments based on these results.

In order to

Discussion

Consistent with the observed anti-apoptotic effect of propofol reported previously, we showed that propofol significantly protects cardiac H9c2 cells from cell death and apoptosis induced by H2O2 as determined by the MTS cell viability assay, caspase-3 activity assay and apoptotic nuclear morphological Hoechst staining. In addition, we found for the first time that propofol activates the PI3K–Akt signaling pathway as demonstrated by the suppression of the effect of propofol on cardiac H9c2

Acknowledgments

This work was supported by Vancouver Coastal Health Research Institute Interim Bridge Funding Program, Canadian Institutes of Health Research Operating Grant #82757, and the Natural Sciences and Engineering Research Council of Canada. Dr. Honglin Luo is a Michael Smith Foundation for Health Research Scholar. Koen Raedschelders is supported by Canada Graduate Scholarship from Canadian Institutes of Health Research.

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