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Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. Bell & Howell Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA UIVLI800-521-0600 ENHANCED IN VIVO AND IN VITRO RESPONSE TO BETA-2 ADRENERGIC RECEPTOR STIMULATION IN ANIMALS SUSCEPTIBLE TO VENTRICULAR FIBRILLATION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Melanie Taghon Houle, M.S. ***** The Ohio State University 1999 Dissertation Committee: Approved By Professor George E. Billman, Adviser Professor Ruth A. Altschuld Professor Patrick Ward Professor Jack Rail Adviser Graduate Program in Physiology UMI Number: 9951667 UTVLI 9951667 Copyright 2000 by Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Biformation and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 ABSTRACT Sudden, cardiac death is an unexpected natural death from a cardiac cause within a short period of time from the onset of symptoms. A canine model of sudden cardiac death has been developed such that dogs with a healed myocardial infarct (MI) develop ventricular fibrillation (VF) induced by a 2 min coronary occlusion during the last min of exercise and maintained one min after exercise. An enhanced (3%-adrenergic receptor (P 2- AR) response was noted in dogs that were susceptible to VF. It was previously demonstrated that the pz-AR agonist zinterol elicited larger increases in the transient amplitude in myocytes obtained from susceptible (S) as compared to resistant (R) dogs. The contractile response to Pz-AR stimulation had not been determined in the intact animal. Therefore, the contractile response to P-adrenergic receptor (p-AR) stimulation was evaluated using echocardiography (velocity o f circumferential fiber shortening (Vcf) both before (n=35, S=19 & R=16) and after anterior wall infarction (MI, n=31, S=12 & R=19). Before MI, increasing doses of isoproterenol (ISO) provoked similar responses in both groups of dogs. The Pi-adrenergic receptor (Pi-AR) or Pz-AR antagonists (bisoprolol or ICI 118,551 respectively) had similar effects on the ISO response in both groups with a larger reduction noted after Pi-AR blockade. In contrast, after Ml, the susceptible dogs displayed a significantly (ANOVA p<0.01) larger Vcf response to ISO which was eliminated by both Pa-AR and Pi-AR blockade. The single cell unloaded shortening response to ISO was also larger in cells obtained from susceptible animals as compared to resistant dogs. As was noted in the intact animal, the Pa-AR antagonist ICI 118,551 elicited a greater reduction in the susceptible dog myocytes (S, -48%, n = 6 and R, -15%, n=9). The number o f Pa-ARs, as determined by radioligand binding, as well as the Kd value, was similar in both groups. As such, these data suggest that the enhanced Pa-AR response provoked by infarction in the susceptible dogs may result from altered Pa-AR coupling to G proteins resulting in cytosolic Ca“^ changes. The p44/42 mitogen activated protein (MAP) kinase cascade is coupled to a Gi protein and has been implicated as a possible alternative non-arrhythmogenic signaling pathway for the Pa-AR. Therefore, the hypothesis that the resistant animals activate the p44/42 MAP kinase pathway while the susceptible animals activate the Gs/cAMP pathway was explored. With Pa-AR as well as a i-adrenergic receptor (ai-AR) activation, the susceptible animals (n= 6 ) had a significantly greater upregulation of the p44/42 MAP kinase pathway as compared to the resistant animals (n= 6 ). Therefore, preferential activation of the p44/42 MAP kinase system cannot explain the observed differences in susceptibility to ventricular fibrillation. However, the possibility still exists for stronger coupling to the Gs protein of the cardiac pz-AR in susceptible animals thereby contributing to abnormalities in calcium handling in these animals. Ill DEDICATION “There are no secrets to success: Don’t waste time looking for them. Success is the result of perfection, hardwork, learning from failure, loyalty to those with whom you work and persistence.” General Colin Powell I would like to dedicate this thesis to my husband, Tom, who never let me give up, and to my son, Sam, who never let me forget what is truly important. IV ACKNOWLEDGMENTS I wish to thank my adviser. Dr. George Billman, for many years of support and understanding as well as his patience in guiding me through the formation of this dissertation. I would also like to thank Dr. Ruth Altschuld for the insightfid conversations, guidance and encouragement through the (at times) exasperating single cell and MAP kinase experiments. 1 am grateful for the technical help and encouragement of Lou Castillo who helped with the myocyte isolation procedure. Bob Kelley for help with the edge detection studies. Dr. Tomohiro Nakayama for his expert echocardiography skills, and Dr. Beth Holycross for her insightful conversations. I would also like to thank my husband, Tom Houle, for his expert computer assistance. Finally, I must acknowledge the generous contributions of Merck Parmaceutical for supplying the bisoprolol as well as Proctor and Gamble for supplying some of the susceptible animals used for the single cell and MAP kinase studies. VITA September 8 , 1968 .......................................................Bom - South Bend, Indiana May 1990 ..................................................................... B.S. Biology, Saint Mary’s College, South Bend, Indiana 1990-1992 ...................................................................AT.S. Biology, University o f Denver. Denver, Colorado 1992-1994 .....................................................................Instructor, Chemistry University of Nebraska, Omaha Omaha, Nebraska 1995-present .................................................................Graduate Research Associate The Ohio State University Columbus, Ohio PUBLICATIONS 1. Taghon, M. S. and S.E. Sadler, Insulin-like growth factor 1 receptor mediated endocytosis in Xenopus laevis oocytes: A role for receptor tyrosine kinase activity. Developmental Biology 163(l):66-74, 1994. 2. Houle, M. S. and G. E. Billman. Low frequency component of the heart rate variability spectrum: a poor marker of sympathetic activity. Am J Physiol. 276(Heart Circ. Physiol. 45)H215-H223, 1999 3. Lynch, J. J., M. S. Houle, G. L. Stump, A. A. Wallace, D. B. Gilberto, H. Jahansouz, G. R. Smith, A. J. Tebbens, N. J. Liverton, H. G. Seinick, D. A. Claremon, and G. E. Billman. Antiarrhythmic efficacy of selective blockade o f the cardiac slowly activating delayed rectifier current Iks in canine models of malignant ischemic ventricular arrhythmias. Circulation 1999;100:1917-1923. VI ABSTRACTS 1 . Houle, M. S. and G. E. Billman. The autonomic response to exercise in animals susceptible to ventricular fibrillation. Physiologist 39: A17, 1996. 2. Houle, M. S., G. E. Billman, and P. Ward. In Vivo biological activities of angiotensin and kinin metabolites in dog vasculature. FASEB /II: A501, 1997. 3. Billman, G. E. and M. S. Houle. Heart rate variability in animals susceptible to ventricular fibrillation: low firequency power is a poor marker of sympathetic activity. JAuton Nerv Sys 65: 87, 1997. 4. Houle, M. S., G. E. Billman, J. Hensley, and R. A. Altschuld. Effects of calcium channel antagonists on Ca^^ transients in canine atrial cardiomyocytes. FASEB J 12: A74, 1998. 5. Billman, G. E., M. S. Houle, and J. J. Lynch. Selective Iks blockade protects against ventricular fibrillation induced by myocardial ischemia. Enr Heart J 19(Abstract Suppl): 17, 1998. 6 . Houle, M. S., L. Castillo, J. Hensley, C. M. Hohl, P. F. Binkley, R. A. Altschuld, and G. E. Billman. Enhanced in vivo response to Pi-adrenergic receptor activation in post- infarcted canines susceptible to ventricular fibrillation. Circulation 98:1-553, 1998. 7. Billman, G. E., M. S. Houle, and J. J. Lynch. Selective Iks, but not Ikt blockade protects against ventricular fibrillation induced by myocardial ischemia. Circulation 98: 1-52, 1998. 8 . Houle, M. S., T. Nakayama, R. A. Altschuld, and G. E. Billman. Enhanced in vivo and in vitro contractile responses to Pa-adrenergic