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2017 Cardiovascular Research Day Abstract Book

89 Coarse-Grained

89 Coarse-Grained Molecular Dynamics Simulations of Kir2.2 Interactions with an Ensemble of Cholesterol Molecules Nicolas Barbera, MS 1 • Manuela Ayee, PhD 1 • Belinda Akpa, PhD 2 • Irena Levitan, PhD 3 1Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago • 2 Department of Molecular Biomedical Sciences, North Carolina State University • 3Department of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago Graduate Student Hypercholesterolemia, elevated plasma levels of cholesterol, is a major risk factor in the development of atherosclerosis. Our previous studies have shown that inwardly rectifying K+ (Kir) channels, which play an important role in endothelial function, are suppressed by enriching cells with cholesterol. Additionally, earlier work done by our group and others has shown that cholesterol regulates Kir2 channels, a sub-family of Kir channels expressed in endothelial cells, through direct interactions at non-annular interaction sites. However, while a putative binding site has been identified, the dynamics of the binding process and cholesterol’s structural effect on the protein remain poorly understood. To address these questions, we used coarse grained molecular dynamics simulations of Kir2.2, a subfamily of Kir2, in model membranes containing cholesterol and POPC to interrogate their molecular interactions at the microsecond timescale. Our simulations show that rather than single ligand-channel binding, interactions between cholesterol and the channel are both complex and numerous, with an average of 15-20 cholesterol molecules interacting with the protein at any given time. These interactions occur at a range of timescales and at distinct annular and non-annular sites on the surface of the protein. Additionally, we observed spontaneous diffusion between these sites and between the protein surface and the surrounding membrane. At present, we are exploring the functional significance of these various interactions and the impact of this collective action on the structure-function relationships governing Kir2 channel activity. 105

90 Reduced HDL in mice overexpressing SR-BI is associated with alterations in the acute inflammatory response to endotoxemia Yanzhang Li, PhD 1 • Ailing Ji, PhD 2 • Xuebing Wang 2 • Andrea Trumbauer 2 • Maria C. de Beer, PhD 3 • Frederick C. de Beer, MD 4 • Nancy R. Webb, PhD 5 1IM-Endocrinology, University of Kentucky • 2 Cardiovascular Research Center, University of Kentucky • 3 Physiology, University of Kentucky • 4 Internal Medicine, University of Kentucky • 5Pharmacology and Nutritional Sciences, University of Kentucky Faculty Objectives: Serum amyloid A (SAA) is an acute phase protein (APP) produced mainly by the liver during an acute phase response (APR). During the APR, virtually all of SAA circulating in the blood is associated with HDL. SR-BI is an HDL receptor and also can bind SAA. Previous reports showed that in SR-BI transgenic mice, hepatic uptake of SAA is increased, and this uptake is involved in the pro-inflammatory effects of SAA. Whether SR-BI regulates SAA expression in response to the inflammatory stimulation is not yet investigated. Methods/Results: Ten week-old male C57BL/6 mice were administered 1x1011 particles of a replication-defective adenoviral vector expressing mouse SR-BI (AdSR-BI) or an adenoviral vector containing no transgene (Adnull) as control. At 72h after adenovirus infusion, the mice were injected i.p. with 1mg /kg body weight LPS. Plasma and tissues were collected at selected time points after LPS injection. As expected, overexpression of SR-BI produced a dramatic reduction in HDL-C (56.52 ± 2.338 mg/dL vs 2.08 ± 1.555 mg/dL for Adnull versus AdSR-BI). Plasma levels of inflammatory cytokine IL-1b, TNFa and IL-6 were robustly higher in AdSR-BI-injected mice compared to control mice. Lympohcyte numbers were also signicantly higher in AdSR-BI-injected mice compared to control mice 12h after LPS injection. In contrast, SAA in plasma was much lower in AdSR-BI-injected mice compared to control mice. Before LPS injection, SAA could only be detected at very low levels in the plasma by ELISA (20.7±8.3 ug/ml). LPS induced a robust increase in plasma SAA levels as early as 2h post-LPS treatment in Adnull-treated mice (1100.0±108.7 ug/ml). Notably, increases in plasma SAA were significantly lower in AdSR-BI-treated mice (122.7±9.5 ug/ml) 2h after LPS injection. At 12h after LPS injection, plasma levels of SAA were 10- fold lower in AdSR-BI-treated mice compared to control mice (12.9±0.24 mg/ml vs 1.2±0.21 mg/ml). The results from western blot and real-time RT-PCR showed that hepatic SAA expression was significantly decreased in the liver of SR-BI overexpressed mice but significantly increased in hepatocytes of SR-BI knock out mice compared to that of control mice. Conclusions: Our results demonstrate that decreased levels of HDL in SR-BI-overexpressing mice are associated with alterations in acute inflammatory responses. Future studies will investigate the relationship between reduced SAA and increased inflammatory cytokines in mice with increased hepatic SR-BI overexpression. 106

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