University of Louisville ThinkIR: The University of Louisville's Institutional Repository Electronic Theses and Dissertations 12-2012 Hyperhomocysteinemia alters sinoatrial and atrioventricular nodal function : role of the cardiac NMDA receptor. Chirag V. Soni University of Louisville Follow this and additional works at: https://ir.library.louisville.edu/etd Recommended Citation Soni, Chirag V., "Hyperhomocysteinemia alters sinoatrial and atrioventricular nodal function : role of the cardiac NMDA receptor." (2012). Electronic Theses and Dissertations. Paper 1360. https://doi.org/10.18297/etd/1360 This Doctoral Dissertation is brought to you for free and open access by ThinkIR: The University of Louisville's Institutional Repository. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of ThinkIR: The University of Louisville's Institutional Repository. This title appears here courtesy of the author, who has retained all other copyrights. For more information, please contact [email protected]. HYPERHOMOCYSTEINEMIA ALTERS SINOATRIAL AND ATRIOVENTRICULAR NODAL FUNCTION: ROLE OF THE CARDIAC NMDA RECEPTOR By Chi rag V. Soni B.A., Northwestern University, 1999 M.S., University of Louisville, 2005 A Dissertation Submitted to the Faculty of the School of Medicine of the University of Louisville In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy Department of Physiology and Biophysics University of Louisville Louisville, KY December 2012 Copyright 2012 by Chirag V. Soni All rights reserved HYPERHOMOCYSTEINEMIA ALTERS SINOATRIAL AND ATRIOVENTRICULAR NODAL FUNCTION: ROLE OF THE CARDIAC NMDA RECEPTOR By Chirag V. Soni B.A., Northwestern University, 1999 M.S., University of Louisville, 2005 A Dissertation Approved on August 27th, 2012 By the Following Dissertation Committee: Claudio Maldonado, Ph.D., Mentor Suresh Tyagi, Ph.D. Irving Joshua, Ph.D. William Wead, Ph.D. Aruni Bhatnagar, Ph.D. ii DEDICATION This dissertation is dedicated to my late grandfather Shantilal Soni, my grandmother Shardaba Soni, my grandfather Pujalal Soni, my grandmother Susilaba Soni and my foi Kantaben Soni. Foremost, this dissertation is dedicated to my loving parents Dr. Vinod and Geeta Soni, who worked very hard to be able to provide opportunities for their children which they did not have growing up in rural India, my sisters Hina and Amita who always keep me in line, and my baby niece Ava, the light of my life. iii ACKNOWLEDGEMENTS I would like to acknowledge and thank everyone who contributed to this work, without whom this would not have been possible. First and foremost, I would like to acknowledge and thank my mentor, Dr. Claudio Maldonado. His unwavering and unceasing support, patience, guidance, and sacrifices made this work possible. Most importantly, he never gave up on me. He is the exemplar of what distinguishes an outstanding and dedicated mentor. I would like to thank my committee members, Dr. Irving Joshua, Dr. William Wead, Dr. Suresh Tyagi, and Dr. Aruni Bhatnagar for all their help and guidance. I would like to thank the faculty and staff of the Department of Physiology and Biophysics. I would especially like to acknowledge Drs. Joshua and We ad for their invaluable support and guidance. I would like to thank Dr. Tyagi and Dr. Bhatnagar for allowing me to work in their laboratories, and I would like to thank everyone in both labs for all their help. I would especially like to acknowledge Dr. Sathnur Pushpakumar, Dr. Gustavo Perez­ Abadia, Dr. Nathaniel Todnem, Dr. Srikanth Givvimani, Dr. Naira Metrevelli, and Dr. Phanikumar Patibandla for all their contributions to this work. Additionally I would like to thank Dr. Srinivas Tipparaju and Peter Kilfoil for teaching me about whole-cell and patch-clamp electrophysiology. Finally, I would like to thank the members of the Pathology Core, as well as Dr. Rong Wan for assistance with immunohistochemistry. iv ABSTRACT HYPERHOMOCYSTEINEMIA ALTERS SINOATRIAL AND ATRIOVENTRICULAR NODAL FUNCTION: ROLE OF THE CARDIAC NMDA RECEPTOR Chirag V. Soni August 27,2012 Introduction: Hyperhomocysteinemia (HHcy) is a pathological condition characterized by excessive levels of plasma homocysteine (Hcy). Patients with HHcy are reported to be at risk for arrhythmias like atrial fibrillation and sudden cardiac death (SCD); however, the causative mechanisms remain unknown. The effects of HHcy on sinus node function, atrioventricular conduction and ventricular vulnerability were investigated by in vivo electrophysiological (EP) analysis, and the role of the cardiac N-methyl-D-aspartate receptor (NMDA-R) in promoting Hcy-induced conduction abnormalities was explored. Materials and Methods: Anesthetized wild-type control mice (WT), mice receiving Hcy in the drinking water for 12 weeks (DW), and heterozygous cystathionine-~-synthase 1 knockout mice (CBS+ -) were subjected to electrocardiographic (ECG) analysis and programmed electrophysiological (EP) studies. To examine the role of the NMDA-R in eliciting conduction changes in HHcy, animals from the three groups were subjected to paired-design repeat EP studies before and after intraperitoneal injection of magnesium sulfate (MgS04, 20mg/kg), an endogenous blocker of NMDA-R. Another set of studies v utilized a specific NMDA-receptor antagonist, dizocilpine (MK-801), in similarly paired­ design studies in an acute, intraperitoneal infusion model of HHcy. Expression levels of cardic NMDA-R in all three groups of animals were quantified by immunoblotting, and immunohistochemical identification of cardiac NMDA-Rs at the mouse atrioventricular (A V) junction was undertaken. Results: DW compared to WT had significantly shorter RR, PR, QT, and HV intervals, corrected sinus node recovery times (CSNRT), Wenckebach periodicity (WP), AV nodal effective refractory periods (A VNERP), and right ventricular effective refractory periods (RVERP). No ventricular arrhythmias were induced in either WT or DW. In the paired­ design studies, blockade of cardiac NMDA-R with MgS04 had no effect on any ECG or EP variables in WTbut significantly prolonged RR, QT, HV, CSNRT, WP, and 1 AVNERP in DWand CBS+ - animals. Immunopositive staining for cardiac NMDA-R at the mouse A V junction was detected. Conclusions: Significant changes in conduction were observed between wild-type mice and mice with diet-induced or genetically-induced hyperhomocysteinemia (HHcy). Low­ dose magnesium administration did not alter ECG or EP conduction variables in wild­ type mice, but had a profound effect on mice with HHcy. These results suggest that shortened sinoatrial and atrioventricular conduction time in mild HHcy may involve cardiac NMDA-Rs. vi TABLE OF CONTENTS TITLE PAGE .............................................................................................. i SIGNATURE PAGE ........................................................................................................ .ii DEDICATION .......................................................................................... .iii ACKNOWLEDGMENTS ............................................................................. .iv ABSTRACT .............................................................................................. v LIST OF TABLES ........................................................................" .............. x LIST OF FIGURES ...................................................................................... xi CHAPTER I: INTRODUCTION AND BACKGROUND I. Introduction .................................................................................................................... 1 II. Background .................................................................................................................... 3 A. Homocysteine ........................................................................................................... 3 B. Accumulation of homocysteine ............................................................................... .4 C. Homocysteine and disease ........................................................................................ 5 D. Cardiac arrhythmias in hyperhomocysteinemia ....................................................... 6 E. Cardiac action potentials ........................................................................................... 7 F. Glutamate receptors and the NMDA receptor. .......................................................... 8 G. Homocysteine and oxidative stress .......................................................................... lO H. Connexins ................................................................................................................. 11 I. Cardiac remodelling and connexin dysfunction ...................................................... 13 vii J. Remodelling at the sinoatrial and atrioventricular nodes ........................................ 15 K. Cardiac Electrophysiological Pacing and Recording ............................................. 18 CHAPTER II: HYPOTHESIS AND SPECIFIC AIMS A. Hypothesis and Specific Aims ................................................................................ 20 CHAPTER III: AIM 1: HYPERHOMOCYSTEINEMIA ALTERS SINOATRIAL AND ATRIOVENTRICULAR CONDUCTION A. Introduction ....................................................................................... 22 B. Materials and methods ........................................................................... 23 C. Results ..................................................................................................