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Introduction: In post-menopausal women, small coronary microvessel dysfunction is the predominant heart disease presentation.There is currently no single treatment to tackle the multifactorial etiology of micro vessel dysfunction including oxidative stress, hyper constricted state, endothelial dysfunction, and blunted beta-adrenergic function. In the heart, G protein-coupled receptors (GPCRs), such as the b-adrenergic receptor (bADR) investigated in this experiment, are active in mediating vessel contractility vasodilation to facilitate blood flow. The presence of GPCR kinase 2 (GRK2), which inhibit bADRs, increase in old age and may mediate blunted capacity to vasodilate. Furthermore, GRK2 can be translocated to the mitochondria via heat shock protein 90 (HSP90), where it is responsible for accumulation of damaging reactive oxygen species (ROS). Increased ROS, in turn, may also contribute to blunted BADR response via desensitization, which is why understanding this mechanism is valuable in order to understand why treatments, such as the injection of adipose-derived Stomal Vascular Fraction (SVF), works in improving the coronary flow reserve. Our hypothesis is that coronary microvascular b-adrenergic desensitization in old age is influenced in part by oxidative stress mediated by GRK2 translocation to the mitochondria via HSP90, reversible by 17-DMAG (HSP90 inhibitor) or Paroxetine (GRK2 inhibitor) treatment. Furthermore, b-adrenergic desensitization, we hypothesize, is inducible in young age via exogenous ROS treatment. Methods: Female rat coronary arterioles isolated from young (3 months) and old (24 months) were mounted in a pressure myography system. Arterioles were infused with Mitosox (superoxide) and 2,3-diaminonapthalene (nitric oxide) and the vessels were incubated in 17-DMAG or Paroxetine for one hour. Changes in mean fluorescence intensity (MFI) were monitored to compare concentration of ROS within the arterioles. Old vessels were also subjected to bADR agonists(dobutamine (b1), salbutamol (b2), BRL (b3), isoproterenol (b1 and b2), and norepinephrine (b1 and a1/a2) in a dose response fashion in the presence or absence of paroxetine or 17-DMAG. Dose responses were also evaluated in young vessels with or without the presence of menadione (superoxide donor, 10 mM) and hydrogen peroxide (1 mM). Results:Paroxetine and 17-DMAG significantly reduced superoxide levels in OC compared to YC vessels. Paroxetine increases while 17-DMAG reduces NO density. Exogenous ROS treatment of young vessel attenuated vasodilation response to all beta agonists. For old vessels, the incubation in 17-DMAG did not restore vasodilation capacity, whereas previously we have shown that paroxetine significantly improves vasodilation response to norepinephrine. Conclusions: Coronary microvessel bADRfunction is blunted by elevated ROS, and is restored upon reduction of ROS through GRK2, but not HSP90 inhibition in the aged female rat. The presented data suggests microvessel bADR dysfunction may be linked to increased oxidative stress in age. Understanding mechanisms behind the pathophysiology of microvessel dysfunction including oxidative stress-linked bADR dysfunction is critical for designing therapies to target this disease.
aging, reactive oxygen species, microvascular dysfunction, adrenergic signaling, mitochondria, receptor desensitization
Dukes, Michaela N; Tracy, Evan P. B.S.; and LeBlanc, Amanda Jo Ph.D., "Inhibition of GRK2, but not HSP90 Reduces Mitochondrial Superoxide and Improves Vasodilation Capacity of Coronary Arterioles from Aged Female Rats" (2020). Undergraduate Arts and Research Showcase. 15.