Downregulation of CuZn-superoxide dismutase contributes to β-adrenergic receptor-mediated oxidative stress in the heart

Objective: Sustained p-adrenergic receptor (beta-AR) activation augments oxidative stress in the heart; whether alterations in antioxidant enzymes contribute to this effect is unknown. Methods and results: Adult male Wistar rats were implanted with osmotic minipumps to infuse either L-isoproterenol (ISO, 25 mu g/kg/h) or saline (SAL). After 7-days, ISO-treated hearts exhibited significant (p<0.005): 1) concentric hypertrophy and augmentation of systolic function, 2) reductions of end-systolic wall stress, and 3) augmentation of oxidative stress, with a similar to 3-fold increase in 4-hydroxy-2-nonenal-and malondialdehyde-protein adducts. ISO-treated hearts also exhibited significant (p<0.01) reductions of CuZn-superoxide dismutase (SOD) enzyme activity (30%), protein (40%), and mRNA (60%), without changes in Mn-SOD, catalase, or glutathione peroxidase. Elk-1 and YinYang 1 (YY1) are transcription factors that positively and negatively regulate CurZn-SOD expression, respectively. ISO-treated hearts exhibited a 3-fold increase in YY1 and a 2-fold reduction in Elk-1 DNA binding activity, strongly favoring CuZn-SOD gene repression. In isolated cardiomyocytes, sustained (24 h) ISO stimulation significantly (p<0.01) increased reactive oxygen species (ROS), an effect blocked by CGP20712A, a beta(1)-AR antagonist, but not by ICI118,551, a beta(2)-AR antagonist. CuZn-SOD downregulation paralleled the increase in ROS, and were similarly blocked by beta(1)-but not beta(2)-AR blockade. There were no changes in CuZn-SOD mRNA stability or myocyte size with ISO treatment. However, nuclear run-on revealed a 40% reduction in CuZn-SOD mRNA expression (p<0.01), consistent with transcriptional repression. ISO also depressed total cellular antioxidant capacity, reduced glutathione (GSH) levels, and the GSH:GSSG ratio. Moreover, CuZn-SOD siRNA transfection of H9c2 cardiomyocytes to suppress CuZn-SOD protein by similar to 40-50% (analogous to the in vivo changes) induced cellular apoptosis. Conclusions: Sustained beta-AR stimulation transcriptionally downregulates CuZn-SOD in myocardium via the beta(1)-AR, thereby contributing to beta-AR-mediated oxidative stress. (C) 2007 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.