Mutations in the gene encoding the nuclear lamina protein lamin A/C (LMNA) and the associated integral inner nuclear membrane protein emerin (EMD) give rise to similar disease phenotypes and are both classified as Emery-Dreifuss muscular dystrophy (EDMD). However, the connection between the function of these nuclear envelope proteins and disease phenotype remains elusive. Given the consistent manifestation of dilated cardiomyopathy in EDMD, my investigation focused on deciphering the roles of these nuclear envelope proteins in the heart. To better understand their functions, I generated a set of isogenic human induced pluripotent stem cell (iPSC) lines with either LMNA mutation causing lamin A/C haploinsufficiency or EMD mutation causing emerin deficiency. I differentiated these iPSCs into cardiomyocytes (iPSC-CMs) and obtained their RNA transcript and protein expression profiles. I found that both mutant lines exhibited significant overlap in transcriptome and proteome changes. Analyzing alterations at both RNA and protein levels shed light on the potential functional roles of lamin A/C and emerin in cardiomyocytes and pathogenic mechanisms. To better understand the cardiac defects caused by loss of lamin A/C. I generated mice lines with tissue-specific and temporally regulated knockout of Lmna in the heart. The mutant mice experienced lethality due to heart failure, regardless of whether Lmna was knocked out at the embryonic or mature adult heart. This demonstrates that lamin A/C has a vital role in the normal function of cardiomyocytes.

Calcium (Ca²⁺) influx through Caᵥ1.2 channels mediates cardiac excitation-contraction coupling, tunes cardiac action potential duration and excitability, and regulates cardiomyocytes’ (CM) gene expression. Mechanisms regulating the sub-cellular localization, trafficking, and dynamics of surface Caᵥ1.2 in ventricular CMs are poorly understood though these are critical determinants of cardiac function. To gain new insights into Caᵥ1.2 organization, dynamics, and regulation at the CM surface we generated transgenic mice expressing an αMHC controlled cardiac-specific, dihydropyridine (DHP)- resistant α₁_ᴄ construct, tagged at the N-terminus with FLAG and HA epitopes, at the C- terminus with YFP, a 13-residue bungarotoxin binding site (BBS) inserted into in the third extracellular loop of domain II, and mutations that prevent cleavage of the C-terminus. We found robust inducible expression of DHP-resistant FLAG-HA-BBS-α₁_ᴄ-YFP in the heart that targeted to dyadic junctions, generated nisoldipine-resistant Ca²⁺ currents, supported cardiac excitation-contraction coupling, and was normally up-regulated by β-adrenergic activation with isoproterenol. Incubating transgenic CMs with AlexaFluor₆₄₇-conjugated α- bungarotoxin (BTX₆₄₇) enabled selective labeling of surface BBS-tagged Caᵥ1.2 channels. We used total internal fluorescence (TIRF) microscopy to investigate the spatiotemporal organization and dynamics of surface Caᵥ1.2 channels. Similar to endogenous Caᵥ1.2, transgenic α1C-YFP forms clusters with exponentially distributed sizes at the cell surface. A flow cytometry-based optical pulse-chase assay revealed surface Caᵥ1.2 channels in adult cardiomyocytes fully turn over within two hours. Application of angiotensin II (Ang II) decreased transgenic Caᵥ1.2 surface density and this effect was blocked by the selective Ang II receptor type I (AT1R) blocker losartan. Application of losartan by itself increased Caᵥ1.2 surface density, suggesting the potential presence of constitutively active Ang II receptors in adult CMs. Our results provide new insights into spatiotemporal organization, dynamics, and regulation of Caᵥ1.2 channels in adult CMs and introduce an approach that can be widely applied to elucidate spatiotemporal dynamics of cardiac ion channels and membrane proteins.

No description available.

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

Stem and progenitor cells are an attractive cell source for the treatment of degenerative diseases due to their potential to differentiate into multiple cell types and provide large cell yields. Thus far, however, clinical applications have been limited due to inefficient differentiation into desired cell types with sufficient yields for adequate tissue repair and regeneration. The ability to spontaneously aggregate in suspension makes embryonic stem cells (ESCs) amenable to large-scale culture techniques for the production of large yields of differentiating cell spheroids (termed embryoid bodies or EBs); however, the introduction of hydrodynamic conditions may alter differentiation profiles within EBs and should be methodically examined. The work presented here employs a novel, laboratory-scale hydrodynamic culture model to systematically interrogate the effects of ESC culture hydrodynamics on cardiomyocyte differentiation through the modulation of a developmentally-relevant signaling pathway. The fluidic environment was defined using computational fluid dynamic modeling, and the effects of hydrodynamic conditions on EB formation, morphology and structure were assessed. Additionally, EB differentiation was examined through gene and protein expression, and indicated that hydrodynamic conditions modulate differentiation patterns, particularly cardiogenic lineage development. This work illustrates that mixing conditions can modulate common signaling pathways active in ESC differentiation and suggests that differentiation may be regulated via bioprocessing parameters and bioreactor design.

Herbs have been used for many centuries in diverse civilizations for the treatment of heart disease. Only a few natural supplements claim to have direct cardiovascular actions including hawthorn (Crataegus spp.) and berberine derived from the Berberidaceae family. Several different studies indicate important cardiovascular effects of hawthorn and berberine. For example, both exert positive inotropic effects and have been used in the treatment of congestive heart failure. Recently, it was shown that hawthorn extract preparations cause negative chronotropic effects in a cultured neonatal murine cardiomyocyte assay independent of beta-adrenergic receptor blockade. The aim of this study was to further characterize the effect of hawthorn extract to decrease the contraction rate of cultured cardiomyocytes. We hypothesized that hawthorn extract may be acting through muscarinic receptors to decrease contraction rate of cardiomyocytes. Atrial and ventricular cardiomyocytes were treated with hawthorn extract in the presence of atropine or himbacine. Changes in the contraction rate of cultured cardiomyocytes revealed that both muscarinic antagonists significantly attenuated the negative chronotropic activity of hawthorn extract. Using quinuclidinyl benzilate, L-[benzylic-4,4'-3H] ([³H]-QNB) as a radioligand antagonist, the effect of a partially purified hawthorn extract fraction to inhibit muscarinic receptor binding was quantified. Hawthorn extract fraction 3 dose-dependently inhibited [³H]-QNB binding to mouse heart membranes. These findings suggest that muscarinic receptors may be involved in the negative chronotropic effect of hawthorn extracts in neonatal murine cardiomyocytes. Berberine exhibits variable positive and negative chronotropic effects in different species. Our first aim was to examine the effect of berberine in a cultured neonatal murine cardiomyocyte assay. Our study demonstrates that berberine has significant negative chronotropic actions on cardiomyocytes which is not an effect of beta-adrenergic receptor blockade. Pertussis toxin (PTX), a Gi/o protein inhibitor, blocked the negative chronotropic activity of berberine. Muscarinic, adenosine, opioid, and α₂ receptors are coupled through a G-protein (Gi/o) to adenylyl cyclase in an inhibitory fashion. Activation of these receptors are primarily responsible for PTX-sensitive negative chronotropic effects in heart. We hypothesized that berberine may be acting through one of these receptor type to decrease contraction rate of cardiomyocytes. For this purpose, we studied the effects of the muscarinic-receptor antagonists, atropine, himbacine, or AF- DX 116 on the negative chronotropic activity of berberine. Muscarinic antagonists completely blocked the effect of berberine on contraction rate of cardiomyocytes, whereas the bradycardic effect of berberine was not inhibited by the opioid, adenosine, or α2 receptor antagonists naloxone, CGS 15943, or phentolamine, respectively. Using [³H]QNB as a radioligand, we demonstrated that berberine bound to muscarinic receptors of adult mouse heart membranes with relatively high affinity. Furthermore, berberine dose-dependently inhibited [³H]QNB binding to muscarinic M2 receptors exogenously expressed in HEK 293 cells. Therefore, the findings of the present study suggest that berberine has muscarinic agonist effects in cultured neonatal murine cardiomyocytes, potentially explaining reported physiological effects of berberine. Cardiac hypertrophy represents the most important factor in the development of congestive heart failure. We investigated the inhibitory effect of berberine on hypertrophy of H9c2 cells. In rat heart-derived H9c2 myoblast cells treated with different hypertrophic agonists such as insulin growth factor II (IGF-II), arginine vasopressin (AVP), phenylephrine, and isoproterenol, protein content and size of cells were significantly increased compared to control group. However, the number of H9c2 cells after treatment with hypertrophic agonists did not differ significantly compared to control. The increases in area of cells and protein content induced by the hypertrophic agonists were inhibited by treatment with berberine in a concentration-dependent manner. Our findings have provided the first scientific evidence that berberine may have an inhibitory effect on hypertrophy of heart-derived cells, and provide a rationale for further studies to evaluate berberine's cardiac activity. / Graduation date: 2010

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

Ng, Sze Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 110-124). / Abstract also in Chinese. / Thesis Committee --- p.i / Acknowledgements --- p.ii / Contents --- p.iii / Abstract --- p.vii / 論文摘要 --- p.x / Abbreviations --- p.xi / List of Figures --- p.xiii / List of Tables --- p.xvii / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1 --- Embryonic Stem Cells (ESCs) --- p.1 / Chapter 1.1.1 --- What are ESCs and the characteristics of ESCs --- p.1 / Chapter 1.1.1.1 --- Pluripotent markers --- p.2 / Chapter 1.1.1.2 --- Germ layers' markers --- p.3 / Chapter 1.1.2 --- Mouse ESCs (mESCs) --- p.4 / Chapter 1.1.2.1 --- mESCs co-culture with mitotically inactivated mouse embryonic fibroblast (MEF) feeder layers --- p.4 / Chapter 1.1.2.2 --- Feeder free mESCs --- p.4 / Chapter 1.1.3 --- Promising uses of ESCs and their shortcomings --- p.5 / Chapter 1.1.4 --- Characteristics of ESC-derived cardiomyocytes (ESC-CMs) --- p.6 / Chapter 1.2 --- Cardiovascular diseases (CVD) --- p.7 / Chapter 1.2.1 --- Background --- p.7 / Chapter 1.2.2 --- Current treatments --- p.8 / Chapter 1.2.3 --- Potential uses of ESC-CMs for basic science research and therapeutic purposes --- p.9 / Chapter 1.2.4 --- Current hurdles in application of ESC-CMs for clinical uses --- p.10 / Chapter 1.3 --- Cardiac gene markers --- p.13 / Chapter 1.3.1 --- Atrial-specific --- p.13 / Chapter 1.3.2 --- Ventricular-specific --- p.19 / Chapter 1.4 --- Lentiviral vector-mediated gene transfer --- p.27 / Chapter 1.5 --- Cell cycle in ESCs --- p.29 / Chapter 1.5.1 --- Cell cycle --- p.29 / Chapter 1.5.2 --- Characteristics of cell cycle in ESCs --- p.30 / Chapter 1.6 --- Potassium (K+) channels --- p.31 / Chapter 1.6.1 --- Voltage gated potassium (Kv) channels --- p.32 / Chapter 1.6.2 --- Role of Kv channels in maintenance of membrane potential --- p.32 / Chapter 1.7 --- Objectives and significances --- p.33 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.35 / Chapter 2.1 --- Mouse embryonic fibroblast (MEF) culture --- p.35 / Chapter 2.1.1 --- Derivation of MEF --- p.3 5 / Chapter 2.1.2 --- MEF culture --- p.37 / Chapter 2.1.3 --- Irradiation of MEF --- p.37 / Chapter 2.2 --- mESC culture and their differentiation --- p.38 / Chapter 2.2.1 --- mESC culture --- p.38 / Chapter 2.2.2 --- Differentiation of mESCs --- p.39 / Chapter 2.3 --- Subcloning --- p.40 / Chapter 2.3.1 --- Amplification of Irx4 --- p.40 / Chapter 2.3.2 --- Purification of DNA products --- p.41 / Chapter 2.3.3 --- Restriction enzyme digestion --- p.42 / Chapter 2.3.4 --- Ligation of Irx4 with iDuet101A vector --- p.43 / Chapter 2.3.5 --- Transformation of ligation product into competent cells --- p.43 / Chapter 2.3.6 --- Small scale preparation of bacterial plasmid DNA --- p.44 / Chapter 2.3.7 --- Confirmation of positive clones by restriction enzyme digestion --- p.45 / Chapter 2.3.8 --- DNA sequencing of the cloned plasmid DNA --- p.45 / Chapter 2.3.9 --- Large scale preparation of target recombinant expression vector --- p.45 / Chapter 2.4 --- Lentiviral vector-mediated gene transfer to mESCs --- p.47 / Chapter 2.4.1 --- Lentivirus packaging --- p.47 / Chapter 2.4.2 --- Lentivirus titering --- p.48 / Chapter 2.4.3 --- Multiple transduction to mESCs --- p.48 / Chapter 2.4.4 --- Hygromycin selection on mESCs --- p.49 / Chapter 2.5 --- Selection of stable clone --- p.49 / Chapter 2.5.1 --- Monoclonal establishment and clone selection --- p.49 / Chapter 2.6 --- Differentiation of cell lines after selection --- p.50 / Chapter 2.7 --- Gene expression study on control and Irx4-overexpressed mESC lines --- p.50 / Chapter 2.8 --- Analysis of mESCs at different phases of the cell cycle --- p.55 / Chapter 2.8.1 --- Go/Gi and S phase synchronization --- p.55 / Chapter 2.8.2 --- Cell cycle analysis by propidium iodide (PI) staining followed by flow cytometric analysis --- p.55 / Chapter 2.8.3 --- Gene expression study by qPCR of Kv channel isoforms --- p.56 / Chapter 2.8.4 --- Membrane potential measurement by membrane potential-sensitive dye followed by flow cytometry --- p.57 / Chapter 2.9 --- Apoptotic study --- p.58 / Chapter 2.10 --- Determination of pluripotent characteristic of mESCs --- p.59 / Chapter 2.10.1 --- Expression of germ layers' markers by qPCR --- p.59 / Chapter 2.10.2 --- Differentiation by hanging drop method and suspension method --- p.61 / Chapter CHAPTER THREE --- RESULTS --- p.62 / Chapter 3.1 --- mESC culture --- p.62 / Chapter 3.1.1 --- Cell colony morphology of feeder free mESCs --- p.62 / Chapter 3.2 --- Subcloning --- p.63 / Chapter 3.2.1 --- PCR cloning of Irx4 --- p.63 / Chapter 3.2.2 --- Restriction digestion on iDuet101A --- p.64 / Chapter 3.2.3 --- Ligation of Irx4 to iDuet101A backbone --- p.66 / Chapter 3.2.4 --- Confirmation of successful ligation --- p.67 / Chapter 3.3 --- Lentivirus packaging --- p.68 / Chapter 3.3.1 --- Transfection --- p.68 / Chapter 3.4 --- Multiple transduction of mESCs and hygromycin selection of positively-transduced cells --- p.69 / Chapter 3.5 --- FACS --- p.70 / Chapter 3.6 --- Irx4 and iduet clone selection --- p.71 / Chapter 3.7 --- Characte rization of mESCs after clone selection --- p.74 / Chapter 3.7.1 --- Immunostaining of pluripotent and differentiation markers --- p.74 / Chapter 3.8 --- Differentiation of cell lines after selection --- p.77 / Chapter 3.8.1 --- Size of EBs of the cell lines during differentiation --- p.77 / Chapter 3.9 --- Gene expression study by qPCR --- p.79 / Chapter 3.10 --- Kv channel expression and membrane potential of mESCs at Go/Gi phase and S phases --- p.84 / Chapter 3.10.1 --- Expression of Kv channels subunits at G0/Gi phase and S phase --- p.86 / Chapter 3.10.2 --- Membrane potential at Go/Gi phase and S phase --- p.87 / Chapter 3.11 --- Effects of TEA+ on feeder free mESCs --- p.89 / Chapter 3.11.1 --- Apoptotic study --- p.89 / Chapter 3.11.2 --- Expression of germ layers´ة markers --- p.91 / Chapter 3.11.3 --- Embryo id bodies (EBs) measurement after differentiation --- p.92 / Chapter CHAPTER FOUR --- DISCUSSION --- p.95 / Chapter 4.1 --- Effect of overexpression of Irx4 on the cardiogenic potential of mESCs --- p.95 / Chapter 4.2 --- Role of Kv channels in maintaining the chacteristics of mESCs --- p.99 / Chapter 4.2.1 --- Inhibition of Kv channels led to a redistribution of the proportion of cells in different phases of the cell cycle: importance of Kv channels in cell cycle progression in native ESCs --- p.99 / Chapter 4.2.2 --- Inhibition of Kv channels led to a loss of pluripotency at molecular and functional levels: importance of Kv channels in the fate determination of mESCs --- p.102 / Chapter 4.3 --- Insights from the present investigation on the future uses of ESCs --- p.105 / Conclusions --- p.108 / References --- p.110

Law, Sau Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 111-117). / Abstract also in Chinese. / Thesis Committee --- p.i / Acknowledgements --- p.ii / Contents --- p.iii / Abstract --- p.vii / 論文摘要 --- p.x / Abbreviations --- p.xi / List of Figures --- p.xiii / List of Tables --- p.xxiii / Chapter CHAPTER ONE --- INTRODUCTION / Chapter 1.1 --- Embryonic Stem (ES) Cells / Chapter 1.1.1 --- Characteristics of ES Cells l / Chapter 1.1.2 --- Therapeutic Potential of ES Cells --- p.3 / Chapter 1.1.3 --- Myocardial Infarction and ES cells-derived Cardiomyocytes --- p.4 / Chapter 1.1.4 --- Current Hurdles of Using ES cells-derived Cardiomyocytes for Research and Therapeutic Purposes --- p.6 / Chapter 1.2 --- Transcription Factors for Cardiac Development / Chapter 1.2.1 --- GATA-binding Protein 4 (GATA-4) --- p.8 / Chapter 1.2.2 --- Myocyte Enhancer Factor 2C (MEF2C) --- p.10 / Chapter 1.2.3 --- "NK2 Transcription Factor Related, Locus 5 (Nkx2.5)" --- p.11 / Chapter 1.2.4 --- Heart and Neural Crest Derivatives Expressed 1 /2 (HANDI/2) --- p.11 / Chapter 1.2.5 --- T-box Protein 5 (Tbx5) --- p.13 / Chapter 1.2.6 --- Serum Response Factor (SRF) --- p.14 / Chapter 1.2.7 --- Specificity Protein 1 (Spl) --- p.15 / Chapter 1.2.8 --- Activator Protein 1 (AP-1) --- p.16 / Chapter 1.3 --- Reactive Oxygen Species (ROS) / Chapter 1.3.1 --- Cellular Production of ROS --- p.18 / Chapter 1.3.2 --- Maintenance of Redox balance --- p.18 / Chapter 1.3.3 --- Redox Signaling --- p.19 / Chapter 1.4 --- Nitric Oxide (NO) and NO Signaling --- p.20 / Chapter 1.5 --- Aims of the Study --- p.22 / Chapter CHAPTER TWO --- MATERIALS AND METHODS / Chapter 2.1 --- Mouse Embryonic Fibroblast (MEF) Culture / Chapter 2.1.1 --- Derivation of MEF --- p.23 / Chapter 2.1.2 --- Maintenance of MEF Culture --- p.24 / Chapter 2.1.3 --- Irradiation of MEF --- p.25 / Chapter 2.2 --- Mouse ES Cell Culture / Chapter 2.2.1 --- Maintenance of Undifferentiated Mouse ES Cell Culture --- p.26 / Chapter 2.2.2 --- Differentiation of Mouse ES Cells --- p.26 / Chapter 2.2.3 --- Exogenous addition of hydrogen peroxide (H2O2) and NO --- p.27 / Chapter 2.3 --- ROS Localization Study / Chapter 2.3.1 --- Frozen Sectioning --- p.28 / Chapter 2.3.2 --- Confocal microscopy for ROS detection --- p.28 / Chapter 2.4 --- Intracellular ROS Measurement / Chapter 2.4.1 --- "Chemistry of 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA)" --- p.29 / Chapter 2.4.2 --- Flow Cytometry for ROS Measurement --- p.29 / Chapter 2.5 --- Gene Expression Study / Chapter 2.5.1 --- Primer Design --- p.30 / Chapter 2.5.2 --- RNA Extraction --- p.31 / Chapter 2.5.3 --- DNase Treatment --- p.32 / Chapter 2.5.4 --- Reverse Transcription --- p.32 / Chapter 2.5.5 --- Quantitative Real Time PCR --- p.33 / Chapter 2.5.6 --- Quantification of mRNA Expression --- p.34 / Chapter 2.6 --- Protein Expression Study / Chapter 2.6.1 --- Total Protein Extraction --- p.34 / Chapter 2.6.2 --- Nuclear and Cytosolic Protein Extraction --- p.35 / Chapter 2.6.3 --- Measurement of Protein Concentration --- p.36 / Chapter 2.6.4 --- De-sumoylation Assay --- p.36 / Chapter 2.6.5 --- De-phosphorylation Assay --- p.37 / Chapter 2.6.6 --- De-glycosylation Assay --- p.38 / Chapter 2.6.7 --- Western Blot --- p.39 / Chapter 2.7 --- Statistical Analysis --- p.41 / Chapter CHAPTER THREE --- RESULTS / Chapter 3.1 --- Study of Endogenous ROS / Chapter 3.1.1 --- Level and Distribution of Endogenous ROS --- p.47 / Chapter 3.1.2 --- Quantification of intracellular ROS --- p.48 / Chapter 3.2 --- Effect of Exogenous Addition of Nitric Oxide (NO) on Cardiac Differentiation / Chapter 3.2.1 --- Beating Profile of NO-treated Embryoid Bodies (EBs) --- p.50 / Chapter 3.3 --- Effect of Exogenous Addition of H2O2 on Cardiac Differentiation / Chapter 3.3.1 --- Beating Profile of H2O2-treated EBs --- p.51 / Chapter 3.3.2 --- mRNA Expression of Cardiac Structural Genes --- p.52 / Chapter 3.3.3 --- Protein Expression of Cardiac Structural Genes --- p.54 / Chapter 3.3.4 --- mRNA Expression of Cardiac Transcription Factors --- p.58 / Chapter 3.3.5 --- Protein Expression of Cardiac Transcription Factors --- p.67 / Chapter 3.3.6 --- Post-translational Modifications of Cardiac Transcription Factors --- p.74 / Chapter 3.3.7 --- Translocation of Cardiac Transcription Factors --- p.89 / Chapter CHAPTER FOUR --- DISCUSSION / Chapter 4.1 --- Changes in the Level of Endogenous ROS During Cardiac Differentiation of Mouse ES Cells --- p.96 / Chapter 4.2 --- H2O2 and NO Have Opposite Effects Towards Cardiac Differentiation --- p.97 / Chapter 4.3 --- Exogenous Addition of H2O2 Advances Differentiation of Mouse ES Cells into Cardiac Lineage --- p.99 / Chapter 4.4 --- Possible Role of H2O2 in Mediating Cardiac Differentiation of Mouse ES Cells --- p.103 / Chapter 4.5 --- Future Directions --- p.108 / Conclusions --- p.110 / References --- p.111

Thesis (MScMedSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Introduction: Primary culture of isolated adult rat cardiomyocytes (ARCMs) is an important model for cardiovascular research, but successful maintenance of these cells in culture for their use in experiments remains challenging (Xu et al, 2009; Louch et al, 2011). Most studies are done on acutely isolated cardiomyocytes immediately after isolation, which is due to low survival of these cells in culture. Obstacles in culture are due to the type of medium and attachment factors (tissue culture adhesives) used to culture and grow these cells. Although we previously identified an optimum medium and adhesive for culture, an adhesive that permits cells to remain attached to the culture surface until after an ischemia/reperfusion insult was elusive. Aims: We therefore aimed to identify the best attachment factor and concentration that will allow adult rat cardiomyocytes to remain attached to the culture surfaces after ischemia/reperfusion experiments. Methods: Cardiomyocytes were isolated from adult Wistar rat hearts and cultured overnight on different concentrations (25 -200 μg/ml) of collagen 1, collagen 4, extracellular matrix (ECM), laminin/entactin (L/E) and laminin. Following overnight cultures, experiments were done in PBS and in PBS versus MMXCB to compare ARCM attachment and viability. Cardiomyocytes cultured on ECM, L/E and L (25−200μg/ml) were subjected to 1 hour of simulated ischemia using MMXCB that contained 3mM SDT and 10mM 2DG, followed by 15 minutes reperfusion. Cell viability was determined by staining cells with JC-1 and images of cells in a field view of 1.17μm/mm2 were captured using fluorescence microscopy. The cells were analysed according to morphology and fluorescence intensity. Results: Total and rod-shaped ARCMs attachment was improved when MMXCB was used as an experimental buffer instead of PBS. Regardless of the buffer used, morphological viability was poor on substrates of Col 1 and Col 4. In contrast to collagens, ARCMs attached efficiently and morphological viability was high on substrates of ECM, L/E and L in MMXCB, but this was greatly reduced in PBS. Mitochondrial viability was high in MMXCB compared to PBS on Col 1 and Col 4 at 75−175μg/ml and on ECM, L/E and L at all concentrations, except at 50 and 150μg/ml ECM, 175μg/ml L/E and 25μg/ml L. When cardiomyocytes cultured on ECM, L/E and L were subjected to simulated ischemia, total ARCMs, rod-shaped and R/G fluorescence (mitochondrial viability) was reduced at all concentrations compared to the control group. Hypercontracted cells were higher in the ischemic treated cells compared to the controls on ECM at 75−150μg/ml and 200μg/ml, L/E at 50,100μg/ml and 175μg/ml and on L at 125μg/ml. Total numbers of ARCMs attached on ECM, L/E and L in the ischemic group consisted of similar numbers of non-viable hypercontracted and viable rod-shaped cells. Conclusion: Cardiomyocytes should be cultured on ECM or L/E or L at concentrations from 25−200μg/ml in MMXCB. PBS is harmful to cultured ARCMs and should thus not be used as an experimental buffer. Ischemia/reperfusion can be simulated on ARCMs cultured on ECM, L/E or L from 25−200μg/ml, provided that a modified culture buffer is used as experimental buffer. / AFRIKAANSE OPSOMMING: Inleiding: Primêre selkulture van geïsoleerde volwasse rot kardiomiosiete (VRKMe) is ‘n belangrike model vir kardiovaskulêre navorsing, maar om hierdie selle suksesvol in kultuur te onderhou is ‘n groot uitdaging (Xu et al, 2009; Louch et al, 2011). Die meeste navorsingstudies maak gebruik van akuut geïsoleerde kardiomiosiete onmiddelik na isolasie omdat oorlewing van hierdie selle in kultuur baie laag is. Die struikelblokke in kultuur is as gevolg van die tipe medium en weefselkultuurgom wat gebruik word. Ons het voorheen 'n optimale medium en weefselkultuurgom geïdentifiseer vir VRKM kultuur oorlewing, maar die weefselkultuurgom was nie effektief genoeg om die selle aan die kultuuroppervlak te laat bly vaskleef, tot na die einde van 'n isgemie/herperfusie eksperiment nie. Doel: Die doel was dus om die beste weefselkultuurgom en konsentrasie te identifiseer, wat sal toelaat dat VRKMe verbonde bly aan die kultuuroppervlaktes tot na die einde van isgemie/herperfusie eksperimente. Metodes: Kardiomiosiete was geïsoleer vanaf volwasse Wistar rotharte en oornag in kultuur op verskillende konsentrasies (25 -200 μg/ml) van kollageen 1, kollageen 4, ekstrasellulêre matriks (ESM), laminin/entactin (L/E) en laminin onderhou. Die volgende dag was die VRKMe vir eksperimentasie in PBS en in PBS teenoor MMXCB gebruik, om selbehoud en oorlewing te vergelyk. Kardiomiosiete op ESM, L/E en L (25−200μg/ml) was aan 1 uur van gesimuleerde isgemie blootgestel, in MMXCB wat 3mM SDT en 10mM 2DG bevat het, gevolg deur 15 minute herperfusie. Sel oorlewing was bepaal deur selle te kleur met JC-1 en daarna was fluoressensiebeelde van die selle in ‘n veldgebied van 1.17μm/mm2 geneem. Die selle was volgens selmorfologie en fluoressensie intensiteit ontleed. Resultate: Met die gebruik van MMXCB as eksperimentele buffer in plaas van PBS, het die aantal totale en staafvormige VRKMe verbinding verbeter. Morfologiese onderhoud was sleg op kollageen 1 en 4, ongeag van watter buffer gebruik was. In kontras met die kollagene was die VRKM verbinding en morfologiese onderhoud op ESM, L/E en L in MMXCB effektief verbeter, maar in PBS aansienlik verminder. Mitochondriale lewensvatbaarheid in MMXCB teenoor PBS op kollageen 1 en 4 by 75−175μg/ml, sowel as op ECM, L/E en L by alle konsentrasies, was hoog, behalwe by 50 en 150μg/ml ESM, 175μg/ml L/E en 25μg/ml L. Isgemie blootstelling van kardiomiosiete gekultuur op alle konsentrasies van ESM, L/E en L, het ‘n afname in die totale, staafvormige en R/G fluoressensie (mitochondriale lewensvatbaarheid) teweeggebring. Meer hiperkontrakteerde kardiomiosiete was in die isgemie behandelde groepe as in die kontrole groepe teenwoordig, spesifiek op ESM by 75−150μg/ml en 200μg/ml, op L/E by 50,100μg/ml en 175μg/ml asook op L by 125μg/ml. In die isgemie groepe het die totale aantal VRKMe op ESM, L/E en L meestal uit ‘n gelyke hoeveelheid hiperkontrakteerde en staafvormige selle bestaan. Gevolgtrekking: Kardiomiosiete moet op ESM of L/E of L by konsentrasises van 25−200μg/ml in MMXCB gekultuur word. PBS is nadelig vir VRKMe in kultuur en moet dus nie gebruik word as eksperimentele buffer nie. Isgemie/herperfusie eksperimente kan gesimuleer word op VRKMe wat op 25−200μg/ml ESM, L/E of L gekultuur is, mits ‘n gemodifiseerde kultuur buffer gebruik word as eksperimentele buffer.