Dopamine Treatment of Postischemic Contractile Dysfunction Rapidly Induces Calcium-Dependent Pro-Apoptotic Signaling

Abstract

Background Ischemia and adrenergic stimulation of cardiomyocyte cultures have been shown to induce apoptotic cell death. We hypothesized that in a model of contractile dysfunction following ischemia, a commonly used catecholamine such as dopamine augments cardiomyocyte apoptosis via activation of calcium-dependent signaling cascades. Methods and Results Isolated perfused rabbit hearts were subjected to 45 minutes of normothermic ischemia with cardioplegic arrest. Hearts were reperfused for 120 minutes with unmodified perfusate (control), perfusate containing 20 nM dopamine, dopamine+2,3-butanedione monoxime (BDM), a MgATPase-inhibitor, or the calcium-sensitizing inotrope ORG 30029. Ischemia-reperfusion alone caused contractile dysfunction without significant myocardial necrosis (left ventricular pressure-volume curves; 1% triphenyltetrazolium chloride staining; creatine kinase release) or apoptosis (terminal deoxynucleotidyl transferase-mediated nick end labeling [TUNEL] analysis; immunoblotting for poly(ADP-ribose) polymerase [PARP] cleavage; activation of caspases-3, -8, and -9; expression of Bax/Bcl-2). Intracellular calcium [Ca 2+ ] i measured by rhod-2 spectrofluorometry was increased in dopamine-reperfused hearts. Although postischemic dopamine treatment improved contractility, the number of apoptotic cardiomyocytes was significantly higher than in untreated postischemic hearts (32.5±9 versus 5.5±1.6/1000 nuclei, P <0.01). Further evidence of dopamine-stimulated apoptosis included PARP cleavage, activation of mitochondrial-derived caspase-9, and the terminal effector caspase-3. Dopamine also increased cellular content of pro-apoptotic Bax while decreasing anti-apoptotic Bcl-2. Simultaneous treatment with BDM suppressed contractility without affecting [Ca 2+ ] i and did not reduce dopamine-stimulated apoptotic markers. When contractility was increased without elevating [Ca 2+ ] i using ORG 30029, no activation of pro-apoptotic signaling cascades was found. Dopamine infusion in nonischemic hearts did not result in cardiomyocyte apoptosis. Conclusions Postischemic dopamine treatment of contractile dysfunction activates pro-apoptotic signal cascades, most likely via a calcium-dependent process and mitochondrial damage.