Abstract 3590: Extracellular Superoxide Dismutase Modulates the Cardiac β-adrenergic Response in Caveolar Microdomains
We have previously shown that extracellular superoxide dismutase (ecSOD) modulates β-adrenergic receptor (β-AR) responses in heart. However, the subcellular localization of ecSOD and its implications for such effects are not defined. Caveolae are sarcolemmal microdomains rich in signaling molecules and supported by caveolar resident protein caveolin-3 (Cav-3). We hypothesized that ecSOD regulation of the β-AR is facilitated by co-localization with the β-AR macromolecular signaling complex in caveolar microdomains. Immunofluorescence studies of isolated ventricular myocytes using confocal microscopy demonstrated sarcolemmal and T-tubular co-localization of Cav-3 and ecSOD that was abolished upon caveolar disruption by β-methyl cyclodextrin. Using density gradient centrifugation, we isolated cardiac caveolar and noncaveolar membrane fractions and found that ecSOD is predominantly localized to the caveolae. Co-immunoprecipitation experiments using the caveolar membrane fraction revealed that Cav-3, the β2-AR, Gαi, and adenylyl cyclase 5/6 are closely associated, and that all of these components were in turn associated with ecSOD. A less pronounced association was also present for ecSOD and the β1-AR and Gαs. To determine the functional impact of the β-AR/ecSOD signaling complex, wild-type (WT) or ecSOD−/− myocytes were stimulated with isoproterenol (ISO) with or without CGP or ICI (β1-and β2-selective blockers, respectively). There was significantly (p<0.05) diminished ISO-stimulated contraction in ecSOD−/− myocytes secondary to both a blunted inotropic β1-AR response and new negatively inotropic β1-AR response, contractile responses that were normalized upon pretreatment with either MnTBAP, an SOD mimetic, or N-acetylcysteine, a thiol donor. Studies in DCF-loaded myocytes revealed comparable levels of reactive oxygen species (ROS) at baseline, but significantly augmented ROS generation with either β1- or β 2-AR stimulation in ecSOD−/− myocytes (ISO fluorescence fold changes 2.26±0.005 vs WT 1.23±0.004, p<0.0001). We conclude that ecSOD fundamentally and dynamically modulates β-AR-induced ROS production, and thereby the ensuing contractile response, by virtue of its caveolar co-localization with the β1- and β2-AR.