University of Cincinnati

UNIVERSITY OF CINCINNATI Date:___________________ I, _________________________________________________________, hereby submit this work as part of the requirements for the degree of: in: It is entitled: This work and its defense approved by: Chair: _______________________________ _______________________________ _______________________________ _______________________________ _______________________________ Transcriptional Regulation of Cardiac Hypertrophy and Heart Failure A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in the Department of Pharmacology and Cell Biophysics of the College of Medicine 2006 by Jian Xu B.S. Peking University, 2000 Committee Chair: Dr. Jeffery D. Molkentin Abstract Cardiac hypertrophy and dilatation are mediated by neuro-endocrine factors, internal stretch and stress sensitive signaling pathways, which in turn transduce alterations in cardiac gene expression through specific transcription factors. This dissertation will, in the first section, provide direct evidence for transcription factor myocyte enhancer factor 2 (MEF2) in the regulation of cardiac dilation and fibrosis through reprogramming cardiac gene expression; in the second section, introduce a novel secreted factor growth differentiation factor 15 (GDF15) as a cardiac anti-hypertrophic and protective factor. The MEF2 family of transcription factors have been indirectly implicated as a downstream mediator of hypertrophic signaling pathways. In this dissertation, we demonstrate directly that MEF2 induce dilated cardiomyopathy and the lengthening of myocytes without a primary induction of cardiac hypertrophy. Cardiac-specific overexpression of MEF2A or MEF2C showed spontaneous cardiomyopathy, which was not altered by activated calcineurin, or de- veloped more fulminant disease following pressure overload. In cultured cardiomyocytes, MEF2A and MEF2C overexpression induced sarcomeric disorganization and focal elon- gation. Mechanistically, MEF2A and MEF2C programmed similar alteration in gene expression that included extracellular matrix remodeling, ion handling, and metabolic genes. Indeed, cultured cardiomyocytes overexpressing MEF2A, or adult myocytes from MEF2A transgenic hearts, showed reduced transient outward currents, suggesting a proximal mech- anism underlying MEF2-dependent cardiomyopathy. During the analysis of gene reprogramming by MEF2, we noted dramatic induction of GDF15. GDF15 is induced by conditions that promote hypertrophy and dilation. Trans- genic mice with cardiac-specific overexpression of GDF15 were normal, but were partially resistant to induced hypertrophy. GDF15 antagonized induced hypertrophy in cultured cardiomyocyte. Transient expression of GDF15 by intravenous adenoviral delivery, or by direct injection of recombinant protein, attenuated ventricular dilation and heart failure in muscle lim protein null mice through an endocrine effect. Conversely, Gdf15 null mice showed enhanced cardiac hypertrophic growth, and a pronounced loss in ventricular performance following stimulation. Mechanistically, GDF15 promoted activation of Smad2/3, which was partially responsible for the anti-hypertrophic effects. These results identify GDF15 as a novel endocrine factor that antagonizes the hypertrophic response and loss of ventricular performance. To my dearest parents Acknowledgment It is impossible to express my sincere gratitude for Dr. Jeff Molkentin, who has been a wonderful mentor and role model. He has shown me not only with words, but also with his enthusiasm and devotion in science, how to become a scientist. I truly appreciate all the time and energy he has invested in leading me to be a confident and independent scientist. I would also like to thank the members of my dissertation committee Dr. Jo El Schultz, Dr. Keith Jones, Dr. Jun Ma and Dr. Mark Olah for their continuous guidance, support and encouragement throughout my graduate training. I would like to acknowledge Dr. Orlando Bueno, Dr. Qiangrong Liang and Dr. Yanshan Dai for teaching me molecular and cellular experimental skills, Dr. Benjamin Wilkins for teaching me transverse aorta constriction surgery, Dr. Nicole Purcell and Dr. Marjorie Maillet for teaching me adult cardiomyocytes isolation, Dr. Nicole Purcell and Dr. Jaime Me- lendez for their help with confocal microscope imaging, and Dr. Julian Braz for teaching me mini-pump implantation. I would also like to acknowledge those collaborators who have contributed in obtaining data presented in this dissertation: Dr. Robert Kaiser for myocardial infarction and ischemic/reperfusion surgery, Dr. Raisa Klevitsky and Dr. Timothy Hewett, for the working heart studies, Dr. Ilona Bodi for the in vivo ion channel measurement, Dr. Bruce Aronow for genechip data analysis, Dr. John Lorenz for hemodynamics analysis, Mr. Allen York for echocardiography analysis and adult cardiomyocytes isolation, Mr. Scott Blair for adenovirus purification, Dr. Peter Backx for the in vitro ion channel measurement, Veterinary Surgical Suite at Children’s hospital for their assistance during mouse surgery, Transgenic and Gene Targeting Core at Children’s hospital for the microinjection, Affymetrix GENECHIP Microarray Core at Children’s hospital for genechip processing, Di- vision of Cardiovascular Imaging at Children’s Hospital for echocardiography analysis and Department of Biomedical Informatics for statistical consultation. I would like to thank everyone in the Department of Pharmacology and Cell Biophysics for their support and kind help. Last but not least, I would like to acknowledge all my colleagues in Dr. Jeff Molkentin’s laboratory with whom I have shared many wonderful memories and received a great deal of support and advice throughout the years: Orlando Bueno, Stephenie Parsons, Qiangrong Liang, Yanshan Dai, Benjamin Wilkins, Julian Braz, Joerg Heineke, Christopher Baines, Robert Kaiser, Nicole Purcell, Scott Blair, Toru Oka, Hiroyuki Nakayama, Marjorie Mail- let, Bastiano Sanna, Michael Hambleton, Mannix Auger-Messier, Qinghang Liu, Douglas Millay, Matt Benard, Jaime Melendez, Michelle Sargent, Jeff Lynch, Kevin Tymitz, Ulrike Delling, Dawinder Sohal. Contents 1 Introduction 1 1.1 Heart failure and cardiac hypertrophy . 1 1.2 Myocyte Enhancing Factor 2 (MEF2) . 4 1.2.1 Structure - Function of MEF2 . 4 1.2.2 Signaling pathways regulating MEF2 . 8 1.2.3 Co-factors regulating MEF2 . 14 1.3 Nuclear factor of activated T cells (NFAT) . 18 1.3.1 Structure - Function of NFAT . 18 1.3.2 Signaling pathways regulating NFAT . 20 1.3.3 Co-factors regulating NFAT . 22 1.4 GATA transcription factors . 23 1.4.1 Structure - Function of GATA . 23 1.4.2 Signaling pathways regulating GATA . 24 1.4.3 Co-factors regulating GATA . 25 1.5 Serum Response Factor (SRF) . 26 1.5.1 Structure - Function of SRF . 26 1.5.2 Co-factors regulating SRF . 28 1.6 Nuclear Factor ·B (NF·B) . 29 1.6.1 Structure - Function of NF·B .................... 29 1.6.2 Cytokines and signaling kinases regulating NF·B . 30 1.7 SMA/MAD related proteins (Smads): Role of GDF-15 signaling . 30 1.7.1 Growth differentiation factor 15 (GDF-15) . 30 1.7.2 Structure - Function of Smads . 31 1.7.3 Smad modulation by kinases . 39 1.7.4 Smad modulation by transcription factors . 40 1.8 The unknowns about cardiac transcription factors . 41 2 Methods and Reagents 45 2.0.1 Animal models . 45 2.0.2 Surgical procedure . 46 2.0.3 Echocardiography . 47 2.0.4 Working heart analysis . 48 2.0.5 Primary cardiomyocyte isolation . 49 i 2.0.6 Adult cardiomyocyte isolation . 49 2.0.7 Replicant deficient recombinant adenovirus generation and infection 50 2.0.8 [3H]-leucine incorporation . 50 2.0.9 Affymetrix gene expression profiling and bioinformatics . 51 2.0.10 Electrophysiological measurements . 52 2.0.11 Immunocytochemistry . 54 2.0.12 Histology and immunostaining . 55 2.0.13 Western blotting . 56 2.0.14 Reverse transcription-polymerase chain reaction (RT-PCR) . 56 2.0.15 Antibodies and reagents . 59 2.0.16 Statistical analysis . 60 3 Results 61 3.1 Regulation of cardiac hypertrophy and contractile function by transcription factor MEF2 . 61 3.1.1 Cardiac characterization of MEF2A and MEF2C transgenic mice . 61 3.1.2 MEF2A and MEF2C do not phenotypically interact with calcineurin in the heart . 79 3.1.3 Global assessment of altered gene expression in MEF2A and MEF2C hearts . 89 3.2 Regulation of cardiac hypertrophy, contractile function and heart failure by a secreted factor, GDF-15 . 100 3.2.1 Identification of GDF-15 as a cardiac inducible factor . 100 3.2.2 GDF-15 transgenic mice are protected from pressure overload hypertrophy. 109 3.2.3 Analysis of cardiac hypertrophy and function in Gdf15 null mice . 121 3.2.4 GDF-15 attenuates hypertrophy in humoral-neuronal agonists induced hypertrophy . 129 3.2.5 GDF-15 attenuates hypertrophy in neonatal cardiomyocytes . 134 3.2.6 Analysis of signaling pathways downstream of GDF-15 in neonatal cardiomyocytes . 136 3.2.7 GDF-15 protects from myocardial infarction and ischemia reperfusion145 4 Discussion 148 4.1 Regulation of cardiac hypertrophy and contractile function by transcription factor MEF2 . 148 4.1.1 Evidence for MEF2 as a Hypertrophic Mediator . 148 4.1.2 Non-Hypertrophic Functions of MEF2 . 154 4.2 Regulation of cardiac hypertrophy, contractile function and heart failure by a secreted factor, GDF-15 . 161 4.2.1 Evidence of GDF-15 as an inducible factor produced in the heart. 161 4.2.2 Evidence of

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