Establishing a Therapeutically Modifiable ROS/RNS-Beta 1 Adrenergic Receptor Desensitization and Internalization Axis in Aging Coronary Microvessels

Introduction: Coronary Microvascular Disease (CMD) causes hyperconstriction, reduced perfusion, and chronic angina in aging. We previously showed increased coronary microvascular ROS and reduced NO/glutathione in aging rats correlating with reduced β1 Adrenergic receptor (β1ADR) dilation. Adipose Stromal Vascular Fraction (SVF) therapy reverses oxidative stress in CMD to improve β1ADR function. Our objective is to elucidate a mechanism for SVF’s recovery of β1ADR function in pursuit of a cell therapy to treat CMD. Hypothesis: β1ADR dilation is reduced in aging due to ROS-mediated receptor desensitization & internalization, reversible by inhibition of GRK2/dynamin & SVF antioxidation. Methods: Rat coronary microvessels were isolated from young (YC, 3-6 months), old (OC, 24 months), and old + SVF injection (OSVF, 24 months). RNAseq (n = 3 per group) and immunofluorescence (n ≥ 5 per group) were used to analyze pro/antioxidant gene & protein expression. Using pressure myography, dilation to isoproterenol was evaluated at baseline and with ROS (hydrogen peroxide and menadione) or RNS (sodium nitroprusside, SNP) +/- inhibitors of receptor desensitization (GRK2 via paroxetine) and internalization (dynamin via dynasore). Significance was set at p < .05 utilizing Two-Way Repeated Measures ANOVA with Holm-Sidak post-hoc analysis. Results: Aging (n = 16) reduced dilation to isoproterenol, reversed to youthful (n = 17) levels by SVF (n = 16). In YC/OSVF, ROS (n = 10 & 6) attenuated dilation reversed by dynasore (n = 8) and paroxetine (n = 7). In OC, SNP (n = 7), dynasore (n = 10), and paroxetine (n = 5) restored dilation. Aging increased prooxidant genetic (xanthine oxidase) and protein (NOX4) expression and increased antioxidant genetic (catalase) but not protein expression. SVF reduced prooxidant genetic (dynamin related protein 1) and protein (NOX4) expression with increased antioxidant genetic (glutathione synthetase) and protein (SOD2) expression. Conclusion: ROS in aging causes β1ADR dysfunction via desensitization & internalization, as their inhibition preserves β1ADR function. We name this interaction the ROS/RNS- β1ADR Desensitization & Internalization Axis , which was therapeutically modulated by SVF antioxidation to restore β1ADR function.