Cardioprotective effect of levosimendan against homocysteine- induced mitochondrial stress and apoptotic cell death in H9C2 Azadeh Aminzadeh a, b, * , Saeed Mehrzadi c a Department of Pharmacology and Toxicology, School of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran b Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran c Razi Drug Research Center, Iran University of Medical Sciences, Tehran, Iran article info Article history: Received 5 November 2018 Accepted 9 November 2018 Available online xxx Keywords: H9C2 cells Homocysteine Apoptosis Oxidative stress Levosimendan abstract Levosimendan is a cardiac inotropic and vasodilator agent that has been reported to have anti-oxidative, anti-inflammatory, and smooth muscle vasodilatory properties. The purpose of this study was to examine the effect of levosimendan on homocysteine-induced cardiomyocyte injury and to explore its underlying mechanisms. H9C2 myocardial cells were incubated with levosimendan 30 min before exposure to homocysteine (Hcy) for 24 h. The effect of levosimendan on cell viability was assessed using the MTT assay. Biological markers of oxidative stress were examined by assessment of lipid peroxidation (LPO), total antioxidant power (TAP), and total thiol groups. Moreover, the expression of caspase-3, Bcl-2, and Bax proteins was determined by western blot analysis. These results showed that levosimendan increased survival of cardiomyocytes in Hcy condition. Treatment with levosimendan decreased lipid peroxidation level. It also enhanced the TAP and total thiol groups. Further, levosimendan pretreatment upregulated the expression of Bcl-2 and downregulated the expression of Bax. The experiments also demonstrated that levosimendan could decrease the expression and activity of caspase-3, which is a key factor in regulating apoptosis. Taken together, these results indicated that levosimendan protects H9C2 myocardial cells against Hcy-induced oxidative stress and apoptosis by scavenging free radicals and modulating the mitochondrial-mediated apoptotic signaling pathway. © 2018 Published by Elsevier Inc. 1. Introduction Cardiovascular disease is one of the most common diseases in both developed and developing countries due to its high morbidity and mortality [1]. Recent studies have indicated that cardiomyocyte apoptosis is implicated in the pathological advancement of various cardiovascular diseases such as atherosclerosis, heart failure, cor- onary heart disease and diabetic cardiomyopathy [2e4]. Homocysteine (Hcy) is an independent risk factor for cardio- vascular disorders [5]. Recent studies have indicated that Hcy may cause cardiomyocyte dysfunction by inducing programmed cell death and oxidative stress [5,6]. Oxidative stress causes excessive formation of reactive oxygen species (ROS), which has been implicated in the development of cardiovascular diseases. It is shown that myocardial cells have a high density of mitochondria. Thus, the consumption of oxygen in myocardial cells is relatively higher that predisposes cells to oxidative stress [7]. Antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) play important role in neutralization of elevated ROS. Oxidative stress refers to increased levels of ROS that cannot be counteracted by the function of antioxidants [8]. Therefore, control of oxidative stress and modification of ROS production may prevent apoptosis in cardio- vascular diseases. Levosimendan is a positive inotropic agent widely used for the treatment of heart failure and low-output syndrome. Levosi- mendan enhances myocardial contraction by stabilizing troponin C without increasing calcium concentration in myocardial cells. In addition, it has a vasodilating effect by opening ATP-sensitive po- tassium channels and increasing endothelial nitric oxide synthase (eNOS)-dependent NO release [9, 10]. Interestingly, recent studies have shown that levosimendan has anti-inflammatory and anti- apoptotic properties [11e 13]. Previous studies have reported that levosimendan reduced LPS-induced upregulation of IL-6, inducible * Corresponding author. Department of Pharmacology and Toxicology, School of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran. E-mail addresses: Azadehaminzadeh@yahoo.com, a.aminzadeh@kmu.ac.ir (A. Aminzadeh). Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc https://doi.org/10.1016/j.bbrc.2018.11.049 0006-291X/© 2018 Published by Elsevier Inc. Biochemical and Biophysical Research Communications xxx (xxxx) xxx Please cite this article as: A. Aminzadeh, S. Mehrzadi, Cardioprotective effect of levosimendan against homocysteine-induced mitochondrial stress and apoptotic cell death in H9C2, Biochemical and Biophysical ResearchCommunications, https://doi.org/10.1016/j.bbrc.2018.11.049