Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 5 uppsatser.

Thesis (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Progressive familial heart block I (PFHBI) is an inherited autosomal dominant cardiac conduction disorder which segregates in a large South African (SA) pedigree, two smaller SA families and a Lebanese family. It specifically affects conduction in the ventricles and is of unknown cause. Clinically, PFHBI is detected on electrocardiogram (ECG) by evidence of bundle-branch disease, i.e., as right bundle branch block, left anterior or posterior hemiblock, or complete heart block with broad QRS complexes. The PFHBI-causative gene was mapped to a lOcM region on chromosome 19ql3.3 using linkage analysis, and the locus was subsequently reduced to 7cM by genetic fine mapping. The present study involved a multi-strategy approach to search for the PFHBI gene. The objectives were the further reduction of the PFHBI locus by genetic fine mapping using published and novel markers, searching for short gene transcripts from publicly available databases and the generation of an integrated map of the locus to which genes were mapped. Prioritised genes were screened for PFHBI-causing mutations and, in addition, the PFHBI locus was searched for the presence of a G protein-encoding gene (PI 15- RhoGEF), a connexin (Cx) gene and any genes containing a CTG repeat expansion motif, since these genes are plausible PFHBI candidate genes. Genotyping and fine genetic mapping using known and novel polymorphic dinucleotide (CA)n and novel tetranucleotide (A3G)n repeat markers across the PFHBI locus were performed. Publicly available databases, such as LLNL (Livermore, USA), and GENEMAP (NCBI) were searched for ESTs which, in turn, were extended using clustering programmes, such as UNIGENE (NCBI) and STACK (SANBI), and the resulting consensus sequences were subsequently BLAST-searched against the protein databases. Using the available data, an integrated physical and genetic map of the PFHBI locus was generated and, as the HGP progressed, a number of novel genes were placed thereon. Subsequently, genes were prioritised on the basis of position, function and expression profile. Genetic fine mapping reduced the PFHBI locus from 7cM to 4cM. The EST approach yielded 38 ESTs, of which 24 ESTs matched proteins, such as activating transcription factor 5 (ATF5), actin-binding protein (KPTN) and zinc finger protein 473 (ZFP473) (May 2003). All the map data generated experimentally and computationally were placed on the PFHBI map. The PI 15-RhoGEF was excluded as a PFHBI candidate gene and although homologous sequences to connexin 37 (Cx37) was located on both chromosome 19 radiation hybrid clones (RHG12 and ORIM-7), it was not identified on the DNA clones spanning the PFHBI locus. No evidence of an expansion of a CTG repeat motif sequence in PFHBI-affected individuals was found. Five highly prioritised candidate genes, namely, 5CZ2-associated X protein (BAX), potassium voltage-gated channel Shaker-related subfamily member 7 (KCNA7’), potassium inwardly-rectifying channel, subfamily J, member 14 (KIR2.4), lin-7 homolog B {LIN-7B) and glycogen synthase 1 (GSYI) were selected for mutation screening. No disease associated mutations were identified in the exonic and flanking intronic regions of these genes. In summary, this study reduced the PFHBI locus substantially and generated a detailed map of the region. A number of attractive candidate genes were excluded from causing PFHBI; however, several plausible candidate genes are still present at this gene-rich locus and remain to be screened. Identifying the PFHBI-causative gene and associated mutation will provide a platform for further studies to understand the pathophysiology, not only of PFHBI, but also of other more commonly occurring conduction disturbances. / AFRIKAANSE OPSOMMING: Progressiewe familiele hartblok I (PFHBI) is ‘n autosomaal dominant oorerflike kardiale geleidingstoomis wat in ‘n groot Suid-Afrikaanse (SA) familie, twee kleiner SA families en ‘n Lebanese familie segregeer. Dit affekteer hoofsaaklik die geleiding in die ventrikels en die oorsaak daarvan is onbekend. Klinies word PFHBI op elektrokardiogram (EKG) geidentifiseer as a bondeltak-siekte, naamlik, as regter bondeltakblok, linker anterior of posterior hemiblok, of volledige hartblok met wye QRS komplekse. Die PFHBI-veroorsakende geen is voorheen deur koppelingsanalise tot ‘n lOcM gebied op chromosoom 19ql3.3 gekarteer, en daaropvolgens is die lokus verklein tot 7cM deur genetiese fyn kartering. Die huidige studie behels ‘n veelvuldige-strategie benadering in die soektog na die PFHBI geen. Die doel van die studie was die verdere verkleining van die PFHBI lokus deur gebruik te maak van beide gepubliseerde en nuwe genetiese merkers, die identifisering van kort geentranskripte (ESTs) uit publieke databanke en die generasie van ‘n geintegreerde kaart van die lokus. Geprioritiseerde gene is geanaliseer vir die PFHBI-veroorsakende mutasie en, daarby, is die PFHBI lokus deursoek vir die teenwoordigheid van ‘n G proteien-enkodeeringsgeen (PIJ5-RhoGEF), ‘n konneksien (Kx) geen en enige gene wat ‘n uitgebreide CTG-herhalingsmotief bevat, aangesien hierdie gene as sterk PFHBI kandidaatgene geag is. Genotipering en fynkartering deur die gebruik van bekende asook nuwe polimorfiese dinukleotied- [(CA)n] en nuwe tertranukleotied- [(A3G)n] herhalingsmerkers wat die PFHBI lokus oorbrug, is uitgevoer. Publieke databanke, soos LLNL (Livermore, USA), en GENEMAP (NCBI) is ondersoek vir ESTs wat vervolgens verleng is deur gebruik te maak van groeperende programme soos UNIGENE (NCBI) en STACK (SANBI) en die gevolglike konsensus volgordes is daama met behulp van BLAST geanaliseer teen die protei'endatabanke. Die bekomde data is vervolgens gebruik om ‘n geintegreerde fisiese en genetiese kaart van die PFHBI lokus te produseer en, soos die mens genoomprojek gevorder het, is nuwe gene daarop geplaas. Daarna is gene geprioritiseer vir mutasie analise gebaseer op posisie, funksie en uitdrukkingsprofiele. Genetiese fynkartering het die PFHBI lokus van 7cM tot 4cM verklein. Die EST benadering het 38 ESTs gei'dentifiseer, waarvan 24 ESTs proteien gelyke gehad het, bv aktiverende transkripsie faktor 5 (ATF5), aktien-verbindingsprotei'en (KPTN) en sink-vingerproteien 473 (ZFP473) (Mei 2003). A1 die karterings data wat eksperimenteel en rekenaar-gewys gegenereer is, is op die PFHBI kaart geposisioneer. Die P115-RhoGEF is uitgeskakel as ‘n PFHBI kandidaatgeen en alhoewel ’n volgorde met homologie aan konneksien37 (Kx37) gevind is op albei chromosoom 19 radiasiehibried klone (RGH12 and ORIM-7), is dit nie gei'dentifiseer in die DNS klone wat die PFHBI lokus oorbrug nie. Geen bewyse van uitbreiding van CTG herhalingsmotiewe is gevind in PFHBIaangetasde persone nie. Vyf hoogs-geprioritiseerde kandidaat gene, naamlik, BCL2-geassosieerde X proteien (BAX), kalium spanningsbeheerde kanaal, subfamilie J, lid 14 (KIR2.4), lin-7 homoloog B (LIN-7b) en glikogeen sintase 1 (GYS1), is geselekteer vir mutasie-analise. Geen siekteveroorsakende mutasie is egter gei'dentifiseer in die eksoniese of die naasliggende introniese gebiede van hierdie gene nie. Ter opsomming, hierdie studie het die PFHBI lokus verklein en het ‘n omvattende kaart van die gebied gegenereer. Verskillende kandidaat gene is uitgesluit as die oorsaak van PFHBI, alhoewel daar nog heelwat goeie kandidaat gene in hierdie geen-ryke lokus is wat geanaliseer behoort te word. Die identifiseering van die PFHBI-veroorsakende mutasie sal ‘n platform bied vir verdere studies om die patofisiologie van nie alleen PFHBI nie, maar ook meer algemene geleidingstoomisse, te verstaan.

Disertacijos objektas yra žmogaus širdies laidžiosios sistemos ir kitų širdies audinių morfologinių skirtumų tyrimas spektroskopiniais, histocheminiais ir proteominiais metodais. Disertacijoje pateikti žmogaus širdies laidžiosios sistemos ir kitų širdies audinių palyginamieji spektroskopiniai ir proteominiai tyrimai parodė aiškius struktūrinius šių audinių skirtumus. Baltymų elektroforezės metodu rasta baltymų grupė aptinkama tik širdies laidžiosios sistemos audinyje. Darbe konstatuojama, kad rasti fluorescenciniai ir baltyminės sudėties skirtumai tarp žmogaus širdies laidžiosios sistemos ir miokardo audinių leidžia manyti, kad bioelektrinio impulso sklidimo greičių skirtumus sąlygoja ne tik ląstelių išsidėstymas, skersmuo, bet ir specifiniai morfologiniai skirtumai. Pasinaudojus nustatytais morfologiniais skirtumais galima sukurti žmogaus širdies laidžiosios sistemos vaizdinimo metodiką, kuri leistų nustatyti širdies laidžiosios sistemos anatomines ypatybes. / The theme covered in the dissertation is about investigation of the morphological differences of the conduction system of the human heart and those of other heart tissues, applying spectroscopic, histochemical and proteomic methods. The described spectroscopical and proteomic investigations of the human heart conduction system and other heart tissues in the dissertation indicate clear structural differences between these tissues. Electrophoresis shows protein groups which may be detected only in a conduction system tissue. The dissertation concludes that estimated fluorescence and proteomic differences between His bundle and myocardium tissues may allow us to suggest that distinction of the bioelectrical impulse velocity in these tissues is determined by the specific morphological odds. According to these differences it is possible to create the visualization method of the conduction system.

The theme covered in the dissertation is about investigation of the morphological differences of the conduction system of the human heart and those of other heart tissues, applying spectroscopic, histochemical and proteomic methods. The described spectroscopical and proteomic investigations of the human heart conduction system and other heart tissues in the dissertation indicate clear structural differences between these tissues. Electrophoresis shows protein groups which may be detected only in a conduction system tissue. The dissertation concludes that estimated fluorescence and proteomic differences between His bundle and myocardium tissues may allow us to suggest that distinction of the bioelectrical impulse velocity in these tissues is determined by the specific morphological odds. According to these differences it is possible to create the visualization method of the conduction system. / Disertacijos objektas yra žmogaus širdies laidžiosios sistemos ir kitų širdies audinių morfologinių skirtumų tyrimas spektroskopiniais, histocheminiais ir proteominiais metodais. Disertacijoje pateikti žmogaus širdies laidžiosios sistemos ir kitų širdies audinių palyginamieji spektroskopiniai ir proteominiai tyrimai parodė aiškius struktūrinius šių audinių skirtumus. Baltymų elektroforezės metodu rasta baltymų grupė aptinkama tik širdies laidžiosios sistemos audinyje. Darbe konstatuojama, kad rasti fluorescenciniai ir baltyminės sudėties skirtumai tarp žmogaus širdies laidžiosios sistemos ir miokardo audinių leidžia manyti, kad bioelektrinio impulso sklidimo greičių skirtumus sąlygoja ne tik ląstelių išsidėstymas, skersmuo, bet ir specifiniai morfologiniai skirtumai. Pasinaudojus nustatytais morfologiniais skirtumais galima sukurti žmogaus širdies laidžiosios sistemos vaizdinimo metodiką, kuri leistų nustatyti širdies laidžiosios sistemos anatomines ypatybes.

Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Heart failure due to cardiomyopathy or cardiac conduction disease is a major cause of mortality and morbidity in both developed and developing countries. Although defined as separate clinical entities, inherited forms of cardiomyopathies and cardiac conduction disorders have been identified that present with overlapping clinical features and/or have common molecular aetiologies. The objective of the present study was to identify the molecular cause of progressive familial heart block type II (PFHBII), an inherited cardiac conduction disorder that segregates in a South African Caucasian Afrikaner family (Brink and Torrington, 1977). The availability of family data tracing the segregation of PFHBII meant that linkage analysis could be employed to identify the chromosomal location of the disease-causative gene. Human Genome Project (HGP) databases have provided additional resources to facilitate the identification of positional candidate genes. Clinical examinations were performed on individuals of the PFHBII-affected family, and, where available, clinical records of subjects examined in a previous study by Brink and Torrington (1977) were re-assessed. Retrospective data suggested redefining the classification of PFHBII. Subsequently, linkage analysis was used to test described dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM) and cardiac conduction-causative loci on chromosomes 1, 2, 3, 6, 7, 9, 11, 14, 15 and 19 for their involvement in the development of PFHBII. Once a locus was mapped, bioinformatics tools were applied to identify and prioritise positional candidate genes for mutation screening. The retrospective and prospective clinical study redefined PFHBII as a cardiac conduction and DCM-associated disorder and simultaneously allowed more family members to be traced.Fortuitously, candidate loci linkage analysis mapped the PFHBII locus to chromosome 1q32, to a region that overlapped a previously described DCM-associated disorder (CMD1D), by the generation of a maximum pairwise lod score of 3.13 at D1S3753 (theta [θ]=0.0) and a maximum multipoint lod score of 3.7 between D1S3753 and D1S414. However, genetic fine mapping and haplotype analysis placed the PFHBII-causative locus distal to the CMD1D locus, within a 3.9 centimorgan (cM) interval on chromosome 1q32.2-q32.3, telomeric of D1S70 and centromeric of D1S505. Bioinformatics analyses prioritised seven candidate genes for mutation analysis, namely, a gene encoding a potassium channel (KCNH1), an extracellular matrix protein (LAMB3), a protein phosphatase (PPP2R5A), an adapter protein that interacts with a cytoskeletal protein (T3JAM), a putative acyltransferase (KIAA0205) and two genes encoding proteins possibly involved in energy homeostasis (RAMP and VWS59). The PFHBII-causative mutation was not identified, although single sequence variations were identified in four of the seven candidate genes that were screened. Although the molecular aetiology was not established, the present study defined the underlying involvement of DCM in the pathogenesis of PFHBII. The new clinical classification of PFHBII has been published (Fernandez et al., 2004) and should lead to tracing more affected individuals in South Africa or elsewhere. The identification of a novel disease-causative locus may point toward the future identification of a new DCM-associated aetiology, which, in turn, might provide insights towards understanding the associated molecular pathophysiologies of heart failure. / AFRIKAANSE OPSOMMING: Hartversaking as gevolg van kardiomiopatie of kardiale geleidingsiekte is ‘n hoof-oorsaak van mortaliteit and morbiditeit in beide ontwikkelde en ontwikkelende lande. Alhoewel gedefinieer as verskillende kliniese entiteite is oorerflike vorms van kardiomiopatie en kardiale geleidingsstoornisse geïdentifiseer met oorvleuelende kliniese eienskappe en/of molukulêre oorsake. Die doelwit van hierdie studie was om die molukulêre oorsaak van progressiewe familiële hartblok tipe II (PFHBII), ‘n oorerflike kardiale geleidingsstoornis, wat in ‘n Suid-Afrikaanse Kaukasiër familie segregeer (Brink en Torrington, 1977), te identifiseer. Die beskikbaarheid van familie data, beteken dat koppelingsanalise gebruik kan word om die chromosomale posisie van die siekte-veroorsakende geen te identifiseer. Menslike Genoom Projek (MGP) databanke het addisionele hulpbronne beskikbaar gestel om die identifikasie van posisionele kandidaat gene te vergemaklik. Kliniese ondersoeke is uitgevoer op PFHBII-geaffekteerde familielede, en waar beskikbaar is kliniese rekords van persone, wat in ‘n vorige studie deur Brink en Torrington (1977) geassesseer was, herontleed. Retrospektiewe data-analise het die kliniese herdefinisie van PFHBII voorgestel. Daarna is koppelingsanalise gebruik om dilateerde kardiomiopatie (DKM), hipertrofiese kardiomiopatie (HKM) en kardiale geleidingssiekte-veroorsakende loki op chromosoom 1, 2, 3, 6, 7, 9, 11, 14, 15 en 19 te ondersoek vir hul moontlike bydrae tot die ontwikkeling van PFHBII. Toe die lokus gekarteer was, is bioinformatiese ondersoeke gebruik om posisionele kandidaat gene te identifiseer en prioritiseer vir mutasie analise. Die retrospektiewe en prospektiewe kliniese ondersoek het PFHBII herdefinieer as ‘n geleidingsstoornis en DKM-verbonde siekte, en terselfde tyd het dit gelei tot die opsporingvan nog familielede. Toevallig het kandidaat loki-analise die PFHBII lokus op chromosoom 1q32 gekarteer, na ‘n gebied wat met ‘n voorheen-beskyfde DKM-verbonde stoornis (CMD1D) oorvleuel, met die opwekking van ‘n makisimum paargewyse lod-getal van 3.13 by D1S3753 (theta [θ] = 0.0) en ‘n maksimum multipunt lod-getal van 3.7 tussen D1S3753 en D1S414. Genetiese fynkartering en haplotipe-analise het die PFHBII-veroorsakende lokus afwaards van die CMD1D lokus geplaas, in ‘n 3.9 centimorgan (cM) gebied op chromosoom 1q32.2-q32.3, telomeries van D1S70 en sentromeries van D1S505. Bioinformatiese analise het daarnatoe gelei dat sewe kandidaat gene vir mutasie analise geprioritiseerd is, naamlik, gene wat onderskeidelik ‘n kalium kanaal (KCNH1), ‘n ekstrasellulêre matriksproteïen (LAMB3), ‘n proteïen fosfatase (PPP2R5A), ‘n aansluiter proteïen wat met ‘n sitoskilet proteïen bind (T3JAM), ‘n asieltansferase (KIAA0205) en twee gene moontlik betrokke in energie homeostase (RAMP en VWS59) enkodeer. Die PFHBII-veroorsakende geen is nie geïdentifiseer nie, alhoewel enkele volgorde-wisselings geïdentifiseer is in vier van die sewe geanaliseerde kandidaat gene. Alhowel die molekulêre oorsaak van die siekte nie vasgestel is nie, het die huidige studie die onderliggende betrokkenheid van DKM in die pathogenese van PFHBII gedefinieer. Die nuwe kliniese klassifikasie van PFHBII is gepubiliseer (Fernandez et al., 2004) en sal lei tot die identifisering van nog geaffekteerde persone in Suid Afrika of in ander lande. Die identifikasie van ‘n nuwe siekte-verbonde lokus mag lei tot die toekomstige identifikasie van ‘n nuwe DKM-verbonde genetiese oorsaak wat, opsig self, dalk insig kan gee in die molekulêre patofisiologie van hartversaking.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2009. / Härtill 4 uppsatser.

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.

Indiana University-Purdue University Indianapolis (IUPUI) / The heart is a complex organ that is composed of numerous cell types, which must integrate their programs for proper specification, differentiation, and cardiac morphogenesis. During cardiac development the basic helix-loop-helix transcription factor Hand2 is dynamically expressed within the endocardium and extra-cardiac lineages such as the epicardium, cardiac neural crest cells (cNCCs), and NCC derived components of the autonomic nervous system. To investigate Hand2 function within these populations we utilized multiple murine Hand2 Conditional Knockout (H2CKO) genetic models. These studies establish for the first time a functional requirement for Hand2 within the endocardium, as several distinct phenotypes including hypotrabeculation, tricuspid atresia, aberrant septation, and precocious coronary development are observed in endocardial H2CKOs. Molecular analyses reveal that endocardial Hand2 functions within the Notch signaling pathway to regulate expression of Nrg1, which encodes a crucial secreted growth factor. Furthermore, we demonstrate that Notch signaling regulates coronary angiogenesis via Hand2 mediated modulation of Vegf signaling. Hand2 is strongly expressed within midgestation NCC and endocardium derived cardiac cushion mesenchyme. To ascertain the function of Hand2 within these cells we employed the Periostin Cre (Postn-Cre), which marks cushion mesenchyme, a small subset of the epicardium, and components of the autonomic nervous system, to conditionally ablate Hand2. We find that Postn-Cre H2CKOs die shortly after birth despite a lack of cardiac structural defects. Gene expression analyses demonstrate that Postn-Cre ablates Hand2 from the adrenal medulla, causing downregulation of Dopamine Beta Hydroxylase (Dbh), a gene encoding a crucial catecholaminergic biosynthetic enzyme. Electrocardiograms demonstrate that 3-day postnatal Postn-Cre H2CKO pups exhibit significantly slower heart rates than control littermates. In conjunction with the aforementioned gene expression analyses, these results indicate that loss of Hand2 function within the adrenal medulla results in a catecholamine deficiency and subsequent heart failure.