Abstract 1243: Extracellular Superoxide Dismutase (ecSOD) Modulates Cardiac Beta-Adrenergic Responsiveness: Studies in ecSOD Null Mice
Extracellular superoxide dismutase (ecSOD) is expressed in the sarcolemma and provides a unique antioxidant role in the cell membrane and interstitium. We hypothesized that ecSOD, via local sarcolemmal control of O2−, dynamically regulates the β-adrenergic receptor (β-AR) response in the heart. Echocardiography in wild-type (WT) and ecSOD knockout (KO) mice (n = 8 –18) revealed no differences in baseline cardiac structure and function. However, after isoproterenol (ISO, 300 ng/g IP), KO mice exhibited blunted chronotropic (HR 659 ± 22 vs 694 ± 23 bpm, p < 0.01) and inotropic (Vcf 13.5 ± 0.5 vs 16.7 ± 0.8 circ/s, p<0.001) responses compared to WT. Although overall tissue oxidative stress (protein-MDA immunoblotting) was unchanged in KO mice compared to WT, in vivo pretreatment with the thiol donor N-acetylcysteine (NAC, 0.4 mg/g/d IP for 10 d) normalized the β-AR response, suggesting the importance of regional, rather than global, changes in redox state in this effect. Studies in isolated myocytes indicated augmented ISO-induced ROS generation in KO myocytes (normalized DCF fluorescence: 0.049 ± 0.0005 vs 0.024 ± 0.0002, p < 0.001), localized to both the β1- and β2-AR with studies using the selective antagonists CGP and ICI (β1- and β2- blocker, respectively). Cell contraction studies confirmed reduced β-AR stimulated contraction in KO myocytes (% increase with 50 nM ISO 163 ± 11 vs. 426 ± 20 %, p < 0.05); this was related to both a significantly (p < 0.05) blunted β1-AR response and the uncovering of a negatively inotropic β2-AR response. Pretreatment of KO myocytes with NAC for 1 h improved both the β1- and β2-AR responses. Further, the response of KO myocytes to the β2-AR/Gs selective agonist fenoterol was comparable to WT, but the response to the direct AC stimulator forskolin was significantly reduced. Molecular evaluation of the β-AR components revealed no significant changes in Gi and Gs, GRK2, or β-arrestin expression in KO hearts. Immunoprecipitation in WT hearts demonstrated co-localization of ecSOD with caveolin-3.
Conclusion: ecSOD moderates ISO-induced ROS generation, and thereby maintains β1- and β2-AR responsiveness. The localization of ecSOD in the caveolae suggests a localized ecSOD microdomain that can dynamically regulate β-AR receptor responses.