published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

The adrenergic hormones norepinephrine (NE) and epinephrine (EPI) are essential for cardiovascular development as embryos lacking NE/EPI begin to die abruptly between embryonic days 10.5 and 11.5 due to apparent cardiac failure. The objective of this research aims to elucidate the mechanism of the embryonic fatality observed in the NE/EPI deficient mouse model. Here we utilized the dopamine β- hydroxylase knockout (Dbh-/-) mouse model, which lacks the gene and subsequent enzyme necessary for the conversion of dopamine to norepinephrine. Embryonic mouse hearts were extracted from Dbh+/+ (control) and Dbh-/- (experimental model) mice for mRNA transcript expression profiling. These studies were performed using the Affymetrix Mouse Genome 430A 2.0 Arrays and quantitative real-time RT-PCR. Gene expression data suggest a novel connection between the ability of the heart to synthesize adrenergic hormones and the gene expression of enzymes involved in the synthesis of retinoic acid. Specifically, we found a statistically significant change in transcriptional expression of the retinol binding protein-1 (Rbp-1), retinol dehydrogenase 12 (Rdh-12) and beta carotene monooxygenase-1 (Bcmo-1) genes in the E10.5 Dbh-/- mouse heart. The gene expression of Rbp-1 and Rdh-12 were increased 1.4 fold and 2.1 fold on the microarray, respectively. The proteins translated from these genes play central roles in the transport and enzymatic conversion of precursor molecules in the pathway of retinoic acid biosynthesis. Additionally, we found that the expression of Bcmo-1, an enzyme responsible for the breakdown of beta carotene to the retinoic acid iii precursor retinal, was down regulated 2.7 fold in the Dbh-/- heart based on microarray assessment. Bcmo-1 is a well known retinoic acid responsive gene, suggesting that the loss of adrenergic hormones in the Dbh-/- mouse heart may result in a deregulation of retinoic acid synthesis and further an alteration in the concentration of retinoic acid present in the embryonic tissue of adrenergic hormone deficient embryos. In addition, we utilized a genetic mouse model that expresses β-galactosidase (β-Gal) in cells capable of synthesizing epinephrine in order to identify the spatial and temporal distribution of adrenergic-derived cells in the developing heart. The model was designed so that cells capable of expressing the gene phenylethanolamine Nmethyltransferase (Pnmt), which is responsible for the synthesis of epinephrine, also produce the enzyme β-Gal as a reporter. The resulting presence of the β-Gal enzyme can be visualized using a β-Gal substrate called XGAL, which is converted into a blue precipitate when cleaved by the β-Gal enzyme. Evaluation of the location of these cells in the embryonic heart showed a preferential distribution at the atrioventricular sulcus at E10.5, and later at E18.5 a more widely distributed ventricular pattern was observed. In addition, the right atrium showed a cluster of XGAL positive cells (blue cells) near the region of the sinoatrial node, while the distribution of XGAL positive cells in the left atrium was quite diffuse. Interestingly, when the adult heart was examined, it was discovered that cells capable of synthesizing epinephrine (adrenergic-derived) are found predominately on the left side of the heart. This left-sided distribution appears to be non-random and non-uniform, since specific regions are consistently XGAL positive, but not every cell in each region. Whole mount and 3-dimensional reconstruction of the iv β-Gal staining showed that these cells traverse the depth of the heart at the midventricular and apical regions. This finding is quite interesting and may provide new knowledge about the functional and structural characteristics of the adult heart. One observation is that these cells may contribute to the cardiomyopathy known as TakoTsubo or "Broken Heart" syndrome. The syndrome is characterized by left ventricular dysfunction during bouts of stress. Also, of particular intrigue is the anatomical correlation of the adrenergic derived cells and the helical ventricular myocardial band (HVMB). Careful examination of the spatial and directional pattern of these cells within the myocardium suggests they contribute primarily to a specific section of the HVMB. The significance of this finding is yet to be uncovered. Taken collectively, this study has shown a novel connection between two crucial developmental signaling pathways. Adrenergic hormone and retinoic acid signaling can now be viewed as cooperative partners in the development of the embryonic heart. In addition, this study has also shown that adrenergic derived cells in the adult heart have a distinctive left-sided distribution, which is non-random, non-uniform, and shows interesting features suggesting an anatomical connection to the HVMB and a clinical association to Tako-Tsubo syndrome. These findings will appreciably contribute to the knowledge base of the scientific community

Smyd1 is the founder of a gene family whose members contain split SET and MYND Domains. Smyd1 has several SET dependent lysine methyl-transferase substrates, including multiple histone lysines and at least one non-histone protein, skNAC. The MYND domain of Smyd1 is required for protein interactions, such as that with skNAC. Conventional Smyd1 knockouts die at E10 due to cardiac defects, including an enrichment of cardiac jelly, a decrease in trabeculation, and the loss of ventricular septation. dHand, a transcription factor specific for right ventricular development, and Irx4, a ventricle specific gene, are down regulated. I have shown that an approximately one kb stretch of DNA sequence upstream of the muscle specific first exon of Smyd1 is sufficient to drive expression of a reporter in transgenic mice. Cardiac specific expression is mediated by a proximal Mef2 binding site whereas skeletal muscle expression is dependent on E-boxes. I have fully analyzed this stretch of sequence via computational methods and made predictions on other potential regulatory factors. Through the use of Cre mediated conditional knockouts, I have shown that the phenotype of the conventional knockout was not due to the introduction of the Neomycin cassette at the gene locus or due to cell non-autonomous effects on the heart. Smyd1 is not only essential for cardiac septation, but throughout embryonic cardiac development, during embryonic skeletal muscle development, and in adult cardiac tissue. Conditionally deficient Smyd1 embryonic hearts are less affected than conventional Smyd1 knockouts, but are embryonically lethal and show poor trabeculation, cardiac hemorrhaging, and a pericardial edema. I detail that the Nkx2.5-Cre mediated Smyd1 deletion phenocopies the skNAC conventional knockout and that both knockouts have similar changes in the expression levels of several genes. Furthermore, when Smyd1 is conditionally removed from adult cardiac tissue, survival rates are diminished. Surprisingly a skeletal muscle specific CKO of Smyd1 mediated by Myogenin-Cre has resulted in perinatal lethality, with a visible phenotype as early as E15. Evident in the phenotype is a large edema between the epithelium and skeletal muscle, fewer myoblasts, decreased muscle mass, increased degenerating cells, and a potentially defective differentiation process. / text

by Au Ka Wing. / "June 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 153-162). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Law Sui-Lin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 99-109). / Abstracts in English and Chinese. / CONTENTS --- p.i / ABSTRACT --- p.v / 撮要 --- p.vii / ACKNOWLEDGEMENTS --- p.ix / LIST OF FIGURES & TABLES --- p.xi / ABBREVIATIONS --- p.xv / Chapter Chapter 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- The Transition of Hyperplastic to Hypertrophic Growth During Heart Development --- p.1 / Chapter 1.2 --- The Controversial Capability of Heart Regeneration --- p.3 / Chapter 1.3 --- Challenges in Treating Heart Diseases --- p.5 / Chapter 1.4 --- A New Insight Behind Traditional Chinese Medicine (TCM) for Treating Heart Diseases --- p.7 / Chapter 1.5 --- The Potential Mitogenic TCMs on Cardiomyocytes --- p.10 / Chapter 1.5.1 --- Radix Astragali --- p.11 / Chapter 1.5.2 --- Radix Ophiopogonis --- p.12 / Chapter Chapter 2 --- MATERIALS & METHODS --- p.14 / Chapter 2.1 --- Materials --- p.14 / Chapter 2.2 --- Cell Culture --- p.16 / Chapter 2.2.1 --- Primary neonatal rat cardiomyocytes cell culture --- p.16 / Chapter 2.2.1.1 --- Mayer's hemalum-eosin staining --- p.17 / Chapter 2.2.2 --- Primary rat fibroblasts cell culture --- p.18 / Chapter 2.2.3 --- H9C2 cardiac cell culture --- p.18 / Chapter 2.3 --- TCMs Preparation and Treatment --- p.19 / Chapter 2.3.1 --- Preparation of TCMs powder from aqueous extracts --- p.19 / Chapter 2.3.2 --- Preparation of culture medium with TCMs powder --- p.19 / Chapter 2.3.3 --- Pre-treatment of undifferentiated and differentiated H9C2 cardiac cells with TCMs --- p.20 / Chapter 2.3.4 --- Post-treatment of differentiated H9C2 cardiac cells with TCMs --- p.20 / Chapter 2.4 --- Assessment of DNA Synthesis and Proliferation --- p.21 / Chapter 2.4.1 --- Tritiated thymidine incorporation assay --- p.21 / Chapter 2.4.2 --- 5-Bromo-2'-deoxy-uridine (BrdU) assay --- p.22 / Chapter 2.4.3 --- Cell counting --- p.23 / Chapter 2.4.4 --- Statistical analysis --- p.23 / Chapter 2.5 --- Screening of Differentially Expressed Genes in H9C2 Cells after TCM Treatment by cDNA Microarray --- p.25 / Chapter 2.5.1 --- Total RNA extraction --- p.25 / Chapter 2.5.2 --- RNA labeling --- p.26 / Chapter 2.5.2.1 --- Synthesis of fluorescence labeled probe --- p.26 / Chapter 2.5.2.2 --- Purification of fluorescence labeled probe --- p.27 / Chapter 2.5.3 --- Microarray hybridization --- p.28 / Chapter 2.5.3.1 --- Concentration of fluorescence labeled probe --- p.28 / Chapter 2.5.3.2 --- Hybridization --- p.28 / Chapter 2.5.3.3 --- Post-hybridization treatment --- p.29 / Chapter 2.5.4 --- Data collection --- p.29 / Chapter 2.5.4.1 --- Scanning of slide --- p.29 / Chapter 2.5.4.2 --- Image processing: spots finding and quantification --- p.30 / Chapter 2.5.5 --- Data normalization and analysis --- p.30 / Chapter 2.6 --- Confirmation of Differentially Expressed Genes in H9C2 Cells after TCM Treatment by RT-PCR --- p.32 / Chapter 2.6.1 --- DNase I digestion of total RNA sample --- p.32 / Chapter 2.6.2 --- First-strand cDNA synthesis --- p.32 / Chapter 2.6.3 --- RT-PCR of the candidate genes --- p.33 / Chapter Chapter 3 --- RESULTS --- p.36 / Chapter 3.1 --- Neonatal Primary Rat Cardiomyocytes --- p.36 / Chapter 3.1.1 --- Preparation of high-purity neonatal primary rat cardiomyocytes --- p.36 / Chapter 3.1.2 --- Neonatal primary rat cardiomyocytes ceased to undergo DNA replication after 6-day in vitro culturing --- p.38 / Chapter 3.1.3 --- Both MD and HQ promoted the growth of day 1 primary rat cardiomyocytes in dose- and time-dependent manners --- p.40 / Chapter 3.1.4 --- HQ is more potent than MD in promoting the growth of day 7 primary rat cardiomyocytes --- p.43 / Chapter 3.2 --- H9C2 Cardiac cells --- p.45 / Chapter 3.2.1 --- Proliferative effect of MD and HQ on undifferentiated H9C2 cardiac cells --- p.45 / Chapter 3.2.2 --- Pre-treatment of HQ on H9C2 cardiac cells during differentiation --- p.50 / Chapter 3.2.3 --- Pre-treatment of MD and HQ on differentiated H9C2 cardiac cells --- p.52 / Chapter 3.2.4 --- Post-treatment of MD on differentiated H9C2 cardiac cells…… --- p.55 / Chapter 3.3 --- Primary Rat Fibroblasts --- p.57 / Chapter 3.3.1 --- Proliferative effect of MD and HQ on primary rat fibroblasts --- p.58 / Chapter 3.4 --- Screening of Differentially Expressed Genes in H9C2 Cells after HQ Treatment by cDNA Microarray --- p.60 / Chapter 3.4.1 --- Differentially expressed genes in undifferentiated H9C2 cardiac cells after HQ treatment --- p.60 / Chapter 3.4.2 --- Differentially expressed genes in differentiated H9C2 cardiac cells after HQ treatment --- p.66 / Chapter 3.4.3 --- Comparison of differentially expressed genes in both undifferentiated and differentiated H9C2 cardiac cells after HQ treatment --- p.72 / Chapter 3.5 --- Confirmation of Differentially Expressed Genes in H9C2 Cells after HQ Treatment by RT-PCR --- p.73 / Chapter 3.5.1 --- "Preferential up-regulation of N-G, N-G-dimethylarginine dimethylaminohydrolase mRNA expression level in undifferentiated H9C2 cardiac cells after HQ treatment " --- p.74 / Chapter 3.5.2 --- Preferential up-regulation of heme oxygenase-3 mRNA expression level in undifferentiated H9C2 cardiac cells after HQ treatment --- p.75 / Chapter 3.5.3 --- Preferential up-regulation of cyclin B mRNA expression level in differentiated H9C2 cardiac cells after HQ treatment --- p.76 / Chapter Chapter 4 --- DISCUSSION --- p.77 / Chapter 4.1 --- HQ Being a More Effective Mitogenic TCM than MD on Cardiomyocytes Exerted its Effect in Dose- and Time Dependent --- p.79 / Chapter 4.2 --- Mitogenic Effect of Both MD and HQ might Possibly Due to the Regulation of Intrinsic Factors --- p.82 / Chapter 4.3 --- HQ Rather Than MD Showed a Higher Specificity in Promoting DNA Synthesis in Cardiomyocytes --- p.83 / Chapter 4.4 --- The Differentially Expressed Genes were Supported by The Clinical Functions of HQ --- p.85 / Chapter 4.5 --- Relating the Differentially Expressed Genes with Cardiac Growth and Development --- p.87 / Chapter 4.6 --- The Hypothetic Mechanisms of Action that HQ Exerted on Cardiac Growth and Development --- p.92 / Chapter 4.7 --- Future Prospect --- p.94 / Chapter 4.7.1 --- In vivo study of HQ on the proliferation of rat cardiomyocytes from neonatal to postnatal development --- p.94 / Chapter 4.7.2 --- The study of transgenic mice carrying the target gene regulated by HQ on cardiac growth and development --- p.96 / Chapter 4.7.3 --- The determination of active component of HQ on cardiac growth and development --- p.97 / REFERENCES --- p.99 / APPENDIX --- p.110

Kou Ying Chuck. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 186-199). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Lau Lai Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 125-141). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iii / Acknowledgement --- p.v / Table of Contents --- p.vii / List of Figures --- p.xii / List of Tables --- p.xiv / Abbreviations --- p.xvii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Research initiative and significance --- p.1 / Chapter 1.2 --- Terminal differentiation --- p.4 / Chapter 1.3 --- Controversial terminal differentiation in cardiomyocytes --- p.5 / Chapter 1.4 --- Molecular switch from hyperplasia to hypertrophy in neonatal myocardial development --- p.7 / Chapter 1.5 --- Interferons --- p.8 / Chapter 1.6 --- Functions induced by interferons --- p.9 / Chapter 1.7 --- Interferons in cardiomyocytes --- p.12 / Chapter 1.8 --- Interferon-inducible transmembrane gene family --- p.13 / Chapter 1.9 --- Our hypothesis and objective --- p.16 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Sequence analysis --- p.18 / Chapter 2.2 --- Cell culture --- p.18 / Chapter 2.3 --- Induction of differentiation of H9C2 cells --- p.19 / Chapter 2.4 --- In vitro induction of IFITMs by interferon treatments --- p.19 / Chapter 2.5 --- RNA isolation --- p.20 / Chapter 2.5.1 --- Experimental animals and sampling --- p.20 / Chapter 2.5.2 --- Total RNA Isolation --- p.20 / Chapter 2.5.3 --- RNA Quantification and Quality Check --- p.21 / Chapter 2.5.4 --- Purification by Qiagen-RNeasy Column and DNase I Digestion --- p.21 / Chapter 2.6 --- First-strand cDNA synthesis --- p.22 / Chapter 2.7 --- Quantitative real-time polymerase chain reaction --- p.22 / Chapter 2.8 --- Cloning protocol --- p.25 / Chapter 2.8.1 --- "Construction of pEGFP-IFITMl, pEGFP-IFITM2 and pEGFP-IFITM3 fusion proteins" --- p.25 / Chapter 2.8.1.1 --- Amplification of DNA fragments --- p.25 / Chapter 2.8.1.2 --- Purification of PCR product --- p.26 / Chapter 2.8.1.3 --- Restriction endonuclease digestion --- p.26 / Chapter 2.8.1.4 --- Insert/vector ligation --- p.27 / Chapter 2.8.1.5 --- Preparation of chemically competent bacterial cells --- p.27 / Chapter 2.8.1.6 --- Transformation of ligation product into chemically competent bacterial cells DH5a --- p.28 / Chapter 2.8.1.7 --- Recombinant clone screening by PCR --- p.29 / Chapter 2.8.1.8 --- Small-scale preparation of recombinant plasmid DNA --- p.29 / Chapter 2.8.1.9 --- Dideoxy DNA sequencing --- p.30 / Chapter 2.8.1.10 --- Large-scale preparation of recombinant plasmid DNA --- p.30 / Chapter 2.8.2 --- "Construction of IFITMl-pcDNA4, IFITM2-pcDNA4 and IFITM3- pcDNA4 constructs" --- p.33 / Chapter 2.8.2.1 --- Amplification of DNA fragments --- p.33 / Chapter 2.8.2.2 --- Insert/vector ligation --- p.33 / Chapter 2.8.2.3 --- Transformation of ligation product into one shot® TOP1 OF´ة chemically competent E. coli cells --- p.34 / Chapter 2.9 --- Transient transfection --- p.36 / Chapter 2.10 --- Subcellular fractionation --- p.37 / Chapter 2.11 --- Isolation of total protein cell lysate --- p.38 / Chapter 2.12 --- Protein concentration determination --- p.38 / Chapter 2.13 --- Protein gel electrophoresis and western blotting --- p.39 / Chapter 2.13.1 --- Preparation of SDS-polyacrylamide gel --- p.39 / Chapter 2.13.2 --- Preparation of protein samples --- p.39 / Chapter 2.13.3 --- SDS-polyacrylamide gel electrophoresis --- p.40 / Chapter 2.13.4 --- Protein transfer to nylon membrane --- p.40 / Chapter 2.13.5 --- Antibodies and detection --- p.40 / Chapter 2.13.6 --- Stripping membrane --- p.41 / Chapter 2.14 --- Bromodeoxyuridine proliferation assay --- p.42 / Chapter 2.14.1 --- Bromodeoxyuridine labeling and detection --- p.42 / Chapter 2.14.2 --- Cell number determination --- p.42 / Chapter 2.15 --- Fluorescence microscopy --- p.43 / Chapter 2.16 --- Confocal microscopy --- p.43 / Chapter 2.17 --- Statistical analysis --- p.44 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- Sequence analysis --- p.45 / Chapter 3.1.1 --- Primary structure analysis --- p.45 / Chapter 3.1.2 --- Transmembrane he lice prediction --- p.46 / Chapter 3.1.3 --- Conserved domain prediction --- p.51 / Chapter 3.1.4 --- Sequence alignments across different species --- p.52 / Chapter 3.2 --- Differential expression during rat myocardial development --- p.53 / Chapter 3.3 --- Altered mRNA levels during differentiation of H9C2 cells --- p.55 / Chapter 3.4 --- "Cloning of IFITMl, IFITM2 and IFITM3" --- p.60 / Chapter 3.5 --- Subcellular localization --- p.61 / Chapter 3.5.1 --- Fluorescence microscopy --- p.61 / Chapter 3.5.2 --- Subcellular fractionation --- p.70 / Chapter 3.6 --- "In vitro induction by interferons-α, β and γ" --- p.72 / Chapter 3.7 --- "DNA synthesis after in vitro induction of interferons-α, β and γ" --- p.79 / Chapter 3.8 --- "Proliferating cell nuclear antigen expression after in vitro induction of interferons-α, β and γ" --- p.87 / Chapter 3.9 --- "DNA synthesis after overexpression of IFITM1, IFITM2 and IFITM3" --- p.93 / Chapter 3.10 --- "Proliferating cell nuclear antigen expression after overexpression of IFITM1, IFITM2 and IFITM3" --- p.95 / Chapter 3.11 --- "β-catenin and cyclin D1 expression after in vitro induction of interferons-α, β and γ" --- p.97 / Chapter 3.12 --- "β-catenin and cyclin D1 expression after overexpression of IFITMl, IFITM2 and IFITM3" --- p.101 / Chapter CHAPTER 4 --- DISCUSSION / Chapter 4.1 --- "Upregulation of IlFITMl, IFITM2 and IFITM3 during myocardial development" --- p.103 / Chapter 4.2 --- "Subcellular localization of IFITMl, IFITM2 and IFITM3" --- p.105 / Chapter 4.3 --- "Induction by interferons-α, β and γ" --- p.107 / Chapter 4.4 --- Inhibition of DNA synthesis by interferons-α and β and IFITM1 --- p.109 / Chapter 4.5 --- Involvement of IFITM family in canonical Wnt pathway --- p.112 / Chapter 4.6 --- Other possible pathways involved --- p.117 / Chapter CHAPTER 5 --- FUTURE PROSPECTS / Chapter 5.1 --- Production of antibodies --- p.118 / Chapter 5.2 --- Silencing or knockout approach --- p.118 / Chapter 5.3 --- Target genes of Wnt/β-catenin signaling --- p.119 / Chapter 5.4 --- Other signaling pathways involved --- p.119 / Chapter 5.5 --- Use of primary cardiomyocytes --- p.120 / APPENDIX --- p.121 / REFERENCES --- p.124

Indiana University-Purdue University Indianapolis (IUPUI) / Autonomic innervation of the heart begins in utero and continues during the neonatal phase of life. A balance between the sympathetic and parasympathetic arms of the autonomic nervous system is required to regulate heart rate as well as the force of each contraction. Our lab studies the development of sympathetic innervation of the early postnatal heart in a conditional knockout (cKO) of Src homology protein tyrosine phosphatase 2 (Shp2). Shp2 is a ubiquitously expressed non-receptor phosphatase involved in a variety of cellular functions including survival, proliferation, and differentiation. We targeted Shp2 in post-migratory neural crest (NC) lineages using our novel Periostin-Cre. This resulted in a fully penetrant mouse model of diminished cardiac sympathetic innervation and concomitant bradycardia that progressively worsen. Shp2 is thought to mediate its basic cellular functions through a plethora of signaling cascades including extracellular signal-regulated kinases (ERK) 1 and 2. We hypothesize that abrogation of downstream ERK1/2 signaling in NC lineages is primarily responsible for the failed sympathetic innervation phenotype observed in our mouse model. Shp2 cKOs are indistinguishable from control littermates at birth and exhibit no gross structural cardiac anomalies; however, in vivo electrocardiogram (ECG) characterization revealed sinus bradycardia that develops as the Shp2 cKO ages. Significantly, 100% of Shp2 cKOs die within 3 weeks after birth. Characterization of the expression pattern of the sympathetic nerve marker tyrosine hydroxylase (TH) revealed a loss of functional sympathetic ganglionic neurons and reduction of cardiac sympathetic axon density in Shp2 cKOs. Shp2 cKOs exhibit lineage-specific suppression of activated pERK1/2 signaling, but not of other downstream targets of Shp2 such as pAKT (phosphorylated-Protein kinase B). Interestingly, restoration of pERK signaling via lineage-specific expression of constitutively active MEK1 (Mitogen-activated protein kinase kinase1) rescued TH-positive cardiac innervation as well as heart rate. These data suggest that the diminished sympathetic cardiac innervation and the resulting ECG abnormalities are a result of decreased pERK signaling in post-migratory NC lineages.