Abstract 15427: Oxidative Stress From Altered Cardiac β3-adrenergic Receptor-activated Inducible Nitric Oxide Synthase/NO Pathways Promotes Cardiac Aging

Background: There is substantial evidence supporting oxidative stress as an important mechanism to age-related cardiac dysfunction and altered β 3 -adrenergic receptor (AR)-activated nitric oxide synthase (NOS) pathway contributing to this process. However, the NOS isoforms involved are controversial. The mechanism of how β 3 -AR stimulation impacts ROS, SERCA2a, and cardiac function in cardiac aging (CA) is unclear. We tested the hypothesis that oxidative stress from up-regulation of cardiac β 3 -AR-activated iNOS uncoupling promotes CA. Methods: We compared myocyte subtypes of β-ARs, three NOS, peroxynitrite (NT), NADPH, GTPCH and SERCA2a expressions and myocyte contractile and [Ca 2+ ] i transient ([Ca 2+ ] iT ) responses to β-AR stimulation with isoproterenol (ISO, 10 -8 M). LV myocytes were isolated from 2 young (Y) (~4 to 6 m) and 2 aged (A) (~28 to 31 m) groups (5/group) of wild-type (WT) and β 3 -AR knockout (β 3 KO) mice. Results: Compared to YWT, AWT myocytes had significantly increased protein levels of β 3 -AR (0.31 vs 0.16) and iNOS (0.48 vs 0.21) accompanied with increased oxidative stress indicated by significant increases in NT formation (increased 102%) and NADPH (P67 -phox , 35% and p22 -phox , 28%), but decreased GTPCH expression (41%, 0.48 vs 0.82) and activity. AWT myocytes had significantly decreased β 1 -AR protein (0.38 vs 0.59), SERCA2a (0.26 vs 0.72) and the ratios of SERCA2a/PLB. These changes were associated with reduced basal cell contraction (dL/dt max, 76.4 vs 122.8 μm/s), relaxation (dR/dt max , 58.9 vs 94.3 μm/s), and [Ca 2+ ] iT (0.16 vs 0.24) accompanied by diminished β-AR-stimulated positive inotropic response. Importantly, in contrast to age-caused changes in AWT, versus Yβ 3 KO, in Aβ 3 KO myocytes, there were no significant alterations in iNOS (0.19 vs 0.16), NT (increased 9%), β 1 -AR (0.60 vs 0.62), and SERCA2a (1.2 vs 1.4). Aβ 3 KO myocytes had normal cell contraction and relaxation with preserved ISO-stimulated positive inotropic response. Conclusions: Myocardial aging is associated with up-regulation of cardiac β 3 -AR-activated iNOS pathway, which leads to imbalance in NO/superoxide production, favoring subsequent iNOS uncoupling and directly contributes to age-associated deficits in LV myocyte function and [Ca 2+ ] i regulation.