Downregulation of CuZn-superoxide dismutase contributes to beta-adrenergic receptor-mediated oxidative stress in the heart.
OBJECTIVE
Sustained beta-adrenergic receptor (beta-AR) activation augments oxidative stress in the heart; whether alterations in antioxidant enzymes contribute to this effect is unknown.


RESULTS
Adult male Wistar rats were implanted with osmotic minipumps to infuse either l-isoproterenol (ISO, 25 microg/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 approximately 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 YinYang1 (YY1) are transcription factors that positively and negatively regulate CuZn-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 beta1-AR antagonist, but not by ICI118,551, a beta2-AR antagonist. CuZn-SOD downregulation paralleled the increase in ROS, and were similarly blocked by beta1- but not beta2-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 approximately 40-50% (analogous to the in vivo changes) induced cellular apoptosis.


CONCLUSIONS
Sustained beta-AR stimulation transcriptionally downregulates CuZn-SOD in myocardium via the beta1-AR, thereby contributing to beta-AR-mediated oxidative stress.
Srivastava
Sanjay
S
Louisville VAMC and Institute of Molecular Cardiology, Department of Medicine, University of Louisville, Louisville, KY 40202, United States.
Chandrasekar
Bysani
B
Gu
Yan
Y
Luo
Jianzhu
J
Hamid
Tariq
T
Hill
Bradford G
BG
Prabhu
Sumanth D
SD
D058665:Adrenergic beta-1 Receptor Agonists; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D015536:Down-Regulation; D005978:Glutathione / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D007150:Immunohistochemistry; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D018384:Oxidative Stress; D034622:RNA Interference; D012333:RNA, Messenger / Q000032:analysis; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D013268:Stimulation, Chemical; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D050986:YY1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D050807:ets-Domain Protein Elk-1 / Q000235:genetics / Q000378:metabolism
D058665:Adrenergic beta-1 Receptor Agonists; D000318:Adrenergic beta-Agonists; D007093:Imidazoles; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011943:Receptors, Adrenergic, beta; D050986:YY1 Transcription Factor; D050807:ets-Domain Protein Elk-1; D004247:DNA; C049763:CGP 20712A; D013482:Superoxide Dismutase; D005978:Glutathione; D007545:Isoproterenol