Research Collection Doctoral Thesis Integration of myofibrils in the developing heart and challenges on the intercalated disc stability Author(s): Hirschy, Alain Publication Date: 2004 Permanent Link: https://doi.org/10.3929/ethz-a-005001295 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library Dissertation ETH N°15790 Integration of myofibrils in the developing heart and challenges on the intercalated disc stability A dissertation submitted to the SWISS INSTITUTE OF TECHNOLOGY ZURICH (ETHZ) For the degree of Doctor of Natural Sciences presented by Alain Hirschy Biologiste diplômé (Université de Neuchâtel, Switzerland) Born April 15, 1975 Citizen of Neuchâtel Accepted on the recommendation of Prof. Dr Jean-Claude Perriard, examiner Prof. Dr Lukas Sommer, co-examiner Prof. Dr Thierry Pedrazzini, co-examiner November 2004 Table of contents Table of contents I Abbreviations IV Abstract 1 Résumé 3 1 Introduction 5 1.1 The heart 5 1.1.1 Morphological development of the heart 5 1.1.2 Molecular pathways controlling heart development 6 1.1.3 Development of ventricular cardiomyocytes 6 1.2 Myofibrillogenesis and development of cell-cell contacts in the ventricular myocardium 7 1.2.1 The contractile apparatus 7 1.2.2 Assembly of sarcomeric proteins 7 1.2.3 Development of cell-cell contacts 9 1.2.4 Three types of cell-cell contact form the intercalated disc 10 1.2.5 The adherens junction 11 1.2.6 The desmosome 15 17 1.2.7 The gap junction 1.2.8 The costameres: cell to extracellular matrix contacts in contractile cells 19 1.3 Role of ß-catenin in signalling 21 1.3.1 Wnt signalling 21 1.3.2 Other regulators of ß-catenin 24 1.4 The conditional knockout approach 24 1.4.1 Cre/lox technology 24 1.4.2 MLC2v-Cre knock-in mouse 26 1.4.3 ß-catenin floxed mouse 28 1.5 Cardiomyopathies 29 1.5.1 Presentation of cardiomyopathies 29 1.5.2 Mutations in contractile and structural proteins lead to cardiomyopathies 29 1.5.3 MLP KO model 31 1.5.4 DRAL KO model 32 1.6 A im of the study 32 1.6.1 ICD development and integration of myofibrils 32 1.6.2 Reconstruction of ICD in vitro 33 1.6.3 Déstabilisation of the ICD 33 1.6.4 Additional stress on heart cells 34 2 Material and Methods 35 2.1 Cloning methods 35 2.1.1 Plasmid manipulation 35 2.1.2 Transformation of competent cells and bacterial culture 36 2.1.3 Plasmid DNA isolation 37 2.1.4 Sequencing PCR 37 2.2 RNA quantification 38 2.2.1 Total RNA isolation 38 2.2.2 RT-PCR analysis 38 2.3 Immunoblotting 39 2.3.1 SDS-sample preparation 39 2.3.2 Electrophoresis and transfer 39 2.3.3 Antibodies for immunoblot 40 2.3.4 Blotting and immunodetection 41 2.3.5 Densitometrie analysis 41 2.4 Isolation of mouse heart cells and cryosections 41 2.4.1 Isolation of rodent heart 41 I 2.4.2 Dissociation and culture of neonatal rat cardiomyocytes (NRCs) 42 2.4.3 Dissociation and culture of neonatal mouse cardiomyocytes 42 2.4.4 Isolation of adult heart cells 42 2.4.5 Cryosections 43 2.5 Transfection of neonatal rat cardiomyocytes 43 2.6 Fixation, immunofluorescence staining and apoptosis detection 43 2.6.1 Antibodies used in immunofluorescence 43 2.6.2 Immunofluorescence of heart whole mount preparations 45 2.6.3 Immunofluorescence of isolated cells 45 2.6.4 Immunofluorescence of cryosections 45 2.6.5 In Situ apoptosis detection 45 2.7 Microscopy 46 2.7.1 Standard fluorescence microscopy 46 2.7.2 Confocal microscopy 46 2.7.3 Cell measurements and volume reconstruction 46 2.8 Generation of conditional knockout mice and double knockouts by breeding 47 2.8.1 Animal strains, genetic background and maintenance 47 2.8.2 Genotyping 47 2.9 Hypertrophy induction and echocardiography 48 2.9.1 Hypertension-induced hypertrophy (1K1C model) 49 2.9.2 ß-adrenergic stimulation of the heart 49 2.9.3 Echocardiography of the mouse heart 49 2.10 Magnetic resonance imaging (MRI) 49 2.11 Statistical analysis 50 3 Results 51 3.1 Development of the intercalated disc in the heart of mouse embryos 51 3.1.1 Overview of the results 51 3.1.2 Myofibrillar and morphological changes in cardiomyocytes from embryonic stage to adult 51 3.1.3 Appearance of cardiomyocytes during heart development 52 3.1.4 Expression of cell-cell contact and extracellular matrix components in the heart 53 3.1.5 Growth of myofibrils 53 3.1.6 Orientation of myofibrils 54 3.1.7 Change in cell shape during cardiomyocyte development 54 3.1.8 Distribution of the adherens junctions during development 55 3.1.9 Distribution of desmosomes 56 3.1.10 Gap junctions 56 3.1.11 Distribution of the extracellular matrix (ECM) 57 3.2 Labelling of the ICD and myofibrils in vitro 58 3.2.1 Outline of the project 58 3.2.2 Tagging of cDNAs and expression vectors 59 3.2.3 Localisation of transfected catenin proteins 59 3.2.4 Localisation of transfected gap junction proteins 60 3.2.5 Localisation of transfected focal adhesion proteins 60 3.2.6 Expression of red fluorescent constructs 61 3.2.7 Localisation of transfected bicistronic constructs 61 3.3 Analysis of the ß-catenin conditional knockout 62 3.3.1 Outline of the project 62 3.3.2 Generation of a heart spécifie deletion of ß-catenin 62 3.3.3 Specificity and efficiency of the Cre mediated recombination 63 3.3.4 Deletion of ß-catenin through postnatal development 63 3.3.5 Deletion of ß-catenin at neonatal stage inculture 64 3.3.6 Regulation of other intercalated disc proteins in the absence of ß-catenin 65 3.3.7 Possible reasons for the long survival of ß-catenin in ICD 67 3.3.8 Is there a hypertrophic response in conditional ß-catenin KO hearts? 68 II 3.3.9 There is no significant increase of myocyte death in conditional ß-catenin KO hearts 69 3.3.10 Physiological parameters are not significantly altered in basal conditions 69 3.3.11 ß-catenin deletion improves fractional shortening in ß-adrenergic-induced hypertrophy 70 3.4 Importance of ß-catenin in DCM heart 71 3.4.1 ß-catenin knockout in MLP knockout mice 71 3.4.2.Early postnatal lethality associated with double KO mice 71 3.4.3 Hypertrophic response at RNA level 72 3.4.4 ANF and ot/ß-catenin protein expression 73 3.4.5 Sarcomeric organisation 73 3.5 Importance of ß-catenin in DRAL KO heart 74 3.5.1 ß-catenin knockout in DRAL knock out mice 74 3.5.2 Early postnatal lethality associated with double KO mice 74 3.5.3 Intraventricular septum defect in the double KO mice 74 4.1 Development of the intercalated disc: a long process of maturation 76 4.1.1 Critical analysis of the method: 76 4.1.2 How do myofibrils grow ? 76 4.1.3 What is the driving force for the alignment of myofibrils? 77 4.1.4 Embryonic developmental hypertrophy ? 77 4.1.5 Changes in nuclear morphology 78 4.1.6 What does the polarisation of AJ and DJ, compared with GJ mean? 78 4.1.7 The sorting out of cell-cell contacts and ECM contacts in postnatal heart 79 4.2 ICD-myofibril reconstruction in vitro 79 4.2.1 Criticisms of the method 79 4.2.2 a-catenin as a specific marker of cell-cell contact 80 4.2.3 RFP, a promising complement to GFP in dual labelling 80 4.2.4 Labelling of the ICD-myofibril interface in 3D and during myofibrillogenesis 81 4.3 Importance of ß-catenin under physiological and under stress conditions 81 4.3.1 Reasons for the slow disappearance of ß-catenin 81 4.3.2 Adaptation of adherens, desmosomal and gap junction to the ß-catenin deletion 82 4.3.3 No cardiac hypertrophy but cellular hypertrophy. Is this possible? 82 4.3.4 ß-catenin as a regulator of hypertrophy? 83 4.3.5 The sarcomeric structure is preserved 83 4.3.6 Deletion of ß-catenin in hypertrophic conditions: consequences on contractility 84 4.3.7 Early lethality of female cKO, dream or reality? 84 4.3.8 Regulation of ß-catenin in the healthy and hypertrophic heart 85 4.4 Deletion of ß-catenin on the top of LIM protein deletion 85 4.4.1 Critics of the mix in genetic backgound 85 4.4.2 Are there possible explanations for the early lethality of MLP-ß-catenin KOs? 86 4.4.3 Is there a possible explanation for the early lethality of ß-catenin cKO DRAL KO? 86 4.4.4 Ventricular septum defects as a cause of postnatal lethality? 87 4.4.5 Postnatal lethality in relationship with the initiation of developmental hypertrophy? 88 5 References 89 6 Appendix 114 6.1 Database of plasmids expressing Intercalated disc proteins and ES cells selection system 114 Acknowledgements 120 Curriculum Vitae 121 III Abbreviations ANF: Atrial natriuretic peptide APC: Adenomatous polyposis coli (tumour supressor protein) APS: Ammonium persulfate AR: Androgen receptor ARC: Adult rat cardiomyocyte aSK: Alpha skeletal actin BNP: Brain natriuretic peptide BSA: Bovine Serum Albumin ßMHC: Beta-myosin heavy chain cAMP: Cyclic Adenosine monophosphate cKO: Conditional knockout COS: African Green Monkey Kidney (epithelial cells) Cy2: Cyanine Cy3: Indocarbocyanine Cy5: Indodicarbocyanine DAPI: 4,5 diamino-2- phenylindoldihydrochlorid DCM: Dilated cardiomyopathy DNA: Deoxyribonucleic Acid dNTP's: Deoxynucleosid triphosphate DRAL: Down regulated in Rhabdomyosarcoma LIM protein DTT: Dithiothreitol Ex.x: Embryonic day x.x E.coli: Escherichia coli EDTA: Ethylene diamine tetraacetic acid EGF: Epidermal growth factor EGTA: Ethylene glycol-bis[ß-aminoethyl ether] N, N, N', N' tetraacetic acid ES cell: embryonic stem cell EtBr: Ethidium bromide FCS: Fetal calf serum FITC: Fluorescein isothiocyanate GAPDH: Glyceraldehyde 3-phosphate dehydrogenase GFP: Green Fluorescent Protein GSK3ß: Glycogen synthase kinase 3ß HCM: Hypertrophic cardiomyopathy