Cardiovascular Pharmacology β-receptor antagonist treatment prevents activation of cell death signaling in the diabetic heart independent of its metabolic actions Vijay Sharma a , Arpeeta Sharma b , Varun Saran c , Pascal N. Bernatchez b , Michael F. Allard c , John H. McNeill a, ⁎ a Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, The University of British Columbia, 2146 East Mall, Vancouver, Canada b Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, and Providence Heart and Lung Institute at St. Paul's Hospital, Vancouver, British Columbia, Canada c Department of Pathology and Laboratory Medicine, Providence Heart and Lung Institute, St. Paul's Hospital, Vancouver, Canada abstract article info Article history: Received 22 May 2010 Received in revised form 15 January 2011 Accepted 25 January 2011 Available online 4 February 2011 Keywords: β-receptor antagonist Cell death Oxidative stress Cardiac metabolism Heart failure Diabetes We have previously shown that metoprolol improves function in the diabetic heart, associated with inhibition of fatty acid oxidation and a shift towards protein kinase B signaling. The aim of this study was to determine the relative importance of these metabolic and signaling effects to the prevention of cellular damage. Diabetes was induced in male Wistar rats by a single IV injection of 60 mg/kg streptozotocin, and treated groups received 15 mg/kg/day metoprolol delivered subcutaneously by osmotic pumps. Echocardiography was performed 6 weeks after streptozotocin injection, and the hearts immediately excised for histological and biochemical measurements of lipotoxicity, apoptosis, signaling and caveolin/caspase interactions. Metoprolol improved stroke volume and cardiac output, associated with attenuation of TUNEL staining and a more modest attenuation of caspase-3; however, the positive TUNEL staining was not associated with an increase in apoptosis or cell regeneration markers. Metoprolol inhibited CPT-1 without affecting CD36 translocation, associated with increased accumulation of triglycerides and long chain acyl CoA in the cytoplasm, and no effect on oxidative stress. Metoprolol induced a shift from protein kinase A to protein kinase B-mediated signaling, associated with a shift in the phosphorylation patterns of BCl-2 and Bad which favored BCl-2 action. Metoprolol also increased the interaction of activated caspase-3 with caveolins 1 and 3 outside caveolae. The actions of metoprolol on fatty acid oxidation do not prevent lipotoxicity; its beneficial effect is more likely to be due to pro-survival signaling and sequestration of activated caspase-3 by caveolins. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Cardiovascular disease is the leading cause of death among diabetic patients (King et al., 1998). Diabetes itself is an independent risk factor for cardiovascular death, and mortality following myocar- dial infarction is increased in diabetic patients (Cohn et al., 1991; CONSENSUS, 1987; Packer et al., 1999; SOLVD, 1991). The most common cause of this mortality is heart failure, produced by the cardiotoxic triad of hypertension, ischemic heart disease and diabetic cardiomyopathy. Diabetic cardiomyopathy has a distinct etiology, and there is a component of the condition which is related to disturbances in cardiac metabolism. Hyperglycemia (glucotoxicity) and the accumulation of toxic fatty acid oxidation intermediates such as ceramides (lipotoxicity) increase oxidative stress in the myocardium, resulting in the activation of pro-apoptotic, pro-fibrotic and pro- inflammatory pathways (Bielawska et al., 1997; Severson, 2004). Apoptosis is a highly regulated process in which pro-death and pro-survival cell signals are regulated and integrated to determine the fate of the cell. In the mitochondrial-mediated intrinsic pathway, stimuli such as oxidative stress stimulate the release of mitochondrial membrane proteins (cytochrome C, endonuclease G, apoptosis inducing factor). These proteins assemble into an apoptosome which activates caspases, the mediators of cell death (Crow et al., 2004). A key regulatory component of this process is the BCl-2 family, consisting of death antagonists such as BCl-2, and death agonists such as Bax and Bad (Adams and Cory, 2001). There has been increasing interest in lipotoxicity and glucotoxicity as inducers of cell damage and apoptosis in the diabetic heart. We have previously demonstrated that the β-receptor antagonist meto- prolol inhibits fatty acid oxidation and produces a modest improve- ment in glucose oxidation in the diabetic heart (Sharma et al., 2008a). The diabetic heart relies almost exclusively on fatty acids as an energy source (Carley and Severson, 2005; Garland and Randle, 1964), and we therefore expected that the ability of metoprolol to inhibit fatty acid oxidation would be especially beneficial in this setting. Metoprolol improved cardiac function as measured ex vivo in the European Journal of Pharmacology 657 (2011) 117–125 ⁎ Corresponding author. Tel.: + 1 604 822 9373; fax: + 1 604 822 8001. E-mail address: jmcneill@interchange.ubc.ca (J.H. McNeill). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.01.044 Contents lists available at ScienceDirect European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar