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Poster 7-31-16
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Poster 7-31-16
1.
RESEARCH POSTER PRESENTATION
DESIGN © 2012 www.PosterPresentations.com Cardiovascular diseases are the leading causes of death globally. We have previously found that mice with smooth muscle cell (SMC)-specific deletion of the mineralocorticoid receptor (MR-KO) lack the aging- associated rise in cardiac hypertrophy and fibrosis. We hypothesize that SMC-MR contributes to cardiac gene expression alterations with aging that contribute to cardiac hypertrophy and fibrosis and to the predisposition of the heart to atrial fibrillation (AF) with age and propose to use a mouse in which the MR is specifically deleted from SMCs (SMC-MR knockout mouse) to explore this hypothesis. The first aim was to characterize alterations in cardiac gene expression with aging in mice. Left ventricle tissue was isolated from young (3 mo.), old (9 mo.), and elderly (19 mo.) SMC-MR knockout and MR-intact littermates. RNA was extracted from left ventricle tissue and reverse transcribed to cDNA. Gene expression was analyzed by q/RT-PCR to determine the fold change in pro-fibrotic, hypertrophic, and cardiac function genes with aging. In aim two we explore the role of SMC-MR in cardiac dysfunction and atrial fibrillation in aged mice. Cardiac ultrasound was performed on 19 month old SMC-MR knockout and MR-intact littermates. Heart wall thickness, systolic and diastolic dimensions, and fractional shortening was obtained from the images to compare heart function in the two genotypes. Using a novel electrophysiology protocol in which we induced atrial fibrillation in aged anaesthetized mice, we compared the ability to induce atrial fibrillation in aged SMC-MR knockout and MR- intact littermate controls to determine if SMC-MR plays a role in aging- associated atrial fibrillation. These experiments have aided in the elucidation of the role of SMC-MR in cardiac gene expression with aging and investigated the role of SMC-MR in atrial fibrillation, which may lead to the identification of novel therapeutic targets in the treatment of these aging-associated cardiovascular diseases. ABSTRACT Methods & Results SUMMARY Acknowledgements Special thanks to Dr. Jen DuPont, Dr. Iris Jaffe, and everyone else at the MCRI for their help this summer with my project and presentations. I would also like to thank the BDBS program at Tufts University Sackler School of Biomedical Sciences for their mentoring and accommodations this summer. Patrick J. Donegan1,2, Jennifer J. DuPont1, Amy McCurley1, Mark Aronovitz1, Joseph McCarthy1, Robert Blanton1, Sami Noujaim1, Iris Z. Jaffe1 1Molecular Cardiology Research Institute, Tufts Medical Center, Boston, MA, USA 2Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, USA The Role of Smooth Muscle Cell Mineralocorticoid Receptor in Cardiac Aging & Atrial Fibrillation Figure 1. Cardiac gene expression protocol RNA was extracted from left ventricle tissue of young (3 mo.) (N=2 KO/4 Intact), old (9 mo.) (N=2 KO/2 Intact), and elderly (19 mo.) (N=2 KO/3 Intact) MR-intact and SMC-MR-KO mice, reverse transcribed to cDNA, plated on a 384 well plate, and quantified using qPCR. The genes tested for in this protocol can be classified as either “pro-fibrotic” or “hypertrophic”. Figure 5. Atrial fibrillation induction protocol. 19 month old MR- intact and SMC-MR-KO mice were anesthetized before four ECG probes were attached to the extremities to track sinus rhythm. A probe with multiple leads was inserted into the left atrium through the aorta, where it stimulated (paced) the atria with electrical pulses of varying frequency and duration to induce AF. Figure 9. Analyzing cardiac echocardiograms of 19 month old mice. Three MR-intact and two SMC-MR-KO mice were imaged with ultrasound to quantify cardiac function and structure. Figure 10. Results of echocardiography analysis. Anterior wall thickness (AWT), posterior wall thickness (PWT), end diastolic dimension (EDD), end systolic dimension (ESD), and heart rate were obtained from the images. Fractional shortening (FS) was calculated using EDD and ESD readings. Figure 6. Atrial fibrillation induction. Image of ECG and ELG showing heart stimulation/pacing (orange rectangle) and the resulting AF. A magnified portion shows disrupted p waves and rapid rate during AF. After ~7 seconds of AF, sinus rhythm returns. BACKGROUND HYPOTHESIS The pervasive effects of hypertension and atrial fibrillation (AF) have been well established, from heart and kidney failure to aneurysm and stroke. The mineralocorticoid receptor (MR) classically regulates blood pressure via control of renal sodium reabsorption. However, the role of MR in vascular smooth muscle cells (SMC) has recently been discovered, providing a new path in the study of hypertension and AF. It has been found that mice lacking MR in SMC have lower blood pressure and reduced ventricular hypertrophy and fibrosis throughout the aging process. This suggests that SMC-MR contributes to cardiac as well as vascular aging. Prior studies have also linked MR with AF, heart failure, and fibrotic remodeling. AF is an abnormal heart rhythm that is common in the elderly and when present, significantly raises the risk of a patient suffering heart failure or a stroke despite often not presenting any symptoms. However, the roles of SMC-MR in the regulation of genes that promote heart fibrosis, hypertrophy and failure and in promoting aging-associated atrial fibrillation and pump failure are currently unknown. We hypothesize that SMC-MR contributes to cardiac gene expression alterations with aging that contribute to cardiac hypertrophy, fibrosis, and pump failure and to the predisposition of the heart to AF with age and propose to use a mouse in which the MR is specifically deleted from SMCs (SMC-MR knockout mouse) to explore this hypothesis. Figure 2. Cardiac fibrosis gene expression. Compared expression of A) Col 1, B) Col 3, C) CTGF, and D) TGFB, four genes associated with cardiac fibrosis, in young (3 mo.), old (9 mo.), and elderly (19 mo.) MR-intact and SMC-MR-KO mice. *p<0.05 vs. Young MR-intact **p<0.05 vs. Young SMC-MR-KO Figure 3. Cardiac hypertrophy gene expression. Compared expression of ANP, BNP, and aMHC, three genes associated with cardiac hypertrophy, in young (3 mo.), old (9 mo.), and elderly (19 mo.) MR-intact and SMC-MR-KO mice. Figure 4. Cardiac function gene expression. Compared expression of SERCA and Calcineurin, two genes associated with cardiac function, in young (3 mo.), old (9 mo.), and elderly (19 mo.) MR-intact and SMC-MR-KO mice. *p<0.05 vs. Young MR-intact Figure 8. Body and organ weights of AF study mice. Mice were sacrificed and organ weights were corrected to tibia length. *p<0.05 • CTGF was upregulated with aging in MR-intact mice, but not in SMC-MR-KO mice. Collagen 3 was downregulated with aging in SMC-MR-KO mice, but not in MR-intact mice. This suggests a link between MR and cardiac fibrosis in aging. • Atrial fibrillation was induced in SMC-MR-KO mice, but not in their MR-intact (wild-type) littermate controls. • Overall we observed no difference in cardiac function between genotypes in 19 month old mice. Figure 7. Atrial fibrillation results. Atrial fibrillation was induced in SMC-MR-KO mice but not in MR-intact littermate controls. Cardiac FibrosisBlood Pressure %CardiacFibrosis
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