6 ABSTRAKT Kardiovaskulární onemocnění představují celosvětově nejčastější příčinu úmrtí. Arytmie neboli poruchy srdečního rytmu k této mortalitě přispívají nemalou měrou. Výzkum v oblasti morfologie převodního systému srdečního má více jak stoletou historii, ale oblast ontogeneze není ani v dnešní době dostatečně prozkoumána. Přitom pochopení mechanismů indukce pracovního myokardu ve specializovanou tkáň by mohl být jeden z klíčů k otevření dveří nových možností diagnostiky a terapie onemocnění v této oblasti. Cílem této práce je pochopení vlivu mechanické zátěže na vznik a vývoj převodního systému srdečního. Chci prokázat, že mechanické zatížení srdce krevním oběhem hraje v embryonálním vývoji nezastupitelnou roli v diferenciaci převodního systému srdečního (PSS). Jako model pozorování jsem si vybral kuřecí srdce. Při porovnání dat získaných na srdci ptáků a savců můžeme najít drobné odlišnosti v detailech, nicméně hlavní principy a mechanismy se zdají být stejné. Kuřecí embryo se vyvíjí 21 dní, přičemž toto časové údobí lze podle Hamburgera a Hamiltona rozdělit do 46 stádií. Zpočátku představuje srdce pouze primitivní trubici, která se stáčí v srdeční kličku. Posléze je přeměněna na zralý čtyřoddílový orgán charakteristický pro vyšší obratlovce. Tyto morfologické změny korelují i se změnami šíření...

The scope of cardiac pathophysiology in sepsis has not been fully defined. Accordingly, we evaluated the effects of sepsis on heart rate (HR), HR variability, and conduction parameters in a murine model of sepsis. Electrocardiograms were recorded noninvasively from conscious mice before and after cecal ligation and puncture (CLP) or sham surgery. Responses of isolated atria to tyramine and isoproterenol were quantified to assess the functional state of sympathetic nerves and postjunctional sensitivity to adrenergic stimulation. Cecal ligation and puncture mice had lower HR compared with sham at 16 to 18 h postsurgery (sham, 741 ± 7 beats/min; CLP, 557 ± 31 beats/min; n = 6/group; P < 0.001), and there was significant prolongation of the PR, QRS, and QTc intervals. Slowing of HR and conduction developed within 4 to 6 h after CLP and were preceded by a decrease in HR variability. Treatment of CLP mice with isoproterenol (5 mg/kg, intraperitoneally) at 25 h after surgery failed to increase HR or decrease conduction intervals. The lack of in vivo response to isoproterenol cannot be attributed to hypothermia because robust chronotropic and inotropic responses to isoproterenol were evoked from isolated atria at 25°C and 30°C. These findings demonstrate that impaired regulation of HR (i.e., reduced HR variability) develops before the onset of overt cardiac rate and conduction changes in septic mice. Subsequent time-dependent decreases in HR and cardiac conduction can be attributed to hypothermia and would contribute to decreased cardiac output and organ perfusion. Because isolated atria from septic mice showed normal responsiveness to adrenergic stimulation, we conclude that impaired effectiveness of isoproterenol in vivo can be attributed to reversible effects of systemic factors on adrenergic receptors and/or postreceptor signaling.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20792号 / 医博第4292号 / 新制||医||1025(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 川村 孝, 教授 湊谷 謙司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

There is scarce information on the fate of cardiac progenitor cells (CPC) in the embryonic heart after chamber specification. Furthermore, the role of acetylcholinesterase (AChE) during heart development is unknown, despite record of its presence in the myocardium. Although three molecular variants of AChE (R, H and T) exist due to alternate splicing, temporal and spatial distribution of these splice variants during cardiac ontogeny is not well characterized. We hypothesized that the AChE “R” splice variant (AChE-R) is involved in directing lineage commitment of mouse ventricular CPCs to the conduction cell phenotype. It is possible that AChE may promote the breakdown of ACh and block the effects of ligand-binding via M2 receptors present on the surface of CPCs. Our study has also provided a platform to suggest that AChE may play a role in the molecular mechanisms underlying functional diversification of myocardial cells into conduction system cells during ontogenesis.

Le système de conduction ventriculaire (SCV) est responsable de la propagation rapide de l’activité électrique dans le cœur. Il est composé du faisceau de His, des branches droite et gauche, et des fibres de Purkinje. 1) Le développement du SCV a été étudié par l’utilisation de souris transgéniques exprimant la recombinase Cre inductible, insérée par recombinaison homologue au locus du gène de la connexine-40 (Cx40). Ce gène code pour une protéine exprimée dans les trabécules ventriculaires et le SCV. La recombinaison est observée uniquement dans les cellules exprimant la Cx40 et leur descendance. Mes résultats révèlent une restriction progressive du destin des trabécules Cx40-positives en cardiomyocytes conducteurs. Les progéniteurs Cx40-positifs participent à la formation du myocarde contractile et du SCV de E10.5 à E14.5 ; alors qu’ils ne donnent que des cardiomyocytes conducteurs après E16.5. 2) L’analyse du SCV a été étudiée dans un modèle de malformation cardiaque congénitale. Le mutant Tbx1-/-, modèle du syndrome de DiGeorge humain, présente une communication inter-ventriculaire. Des défauts morphologiques du SCV sont détectés chez ces mutants : absence de branche droite et un faisceau de His moins compacté. Ceux-ci sont corrélés avec des défauts de conduction. Le phénotype observé ne résulte pas d’un défaut d’activation du programme génique à l’origine de l’établissement du SCV ; mais semble dû à la présence de la communication inter-ventriculaire qui empêcherait les cellules progénitrices du SCV de rejoindre le sommet du septum inter-ventriculaire. / The ventricular conduction system (VCS) is responsible for the rapid propagation of electrical activity in the heart. The VCS is composed of the His bundle, the left and right bundle branches, and the peripheral Purkinje fibers. 1) The development of the VCS has been studied by using transgenic mice expressing the inducible Cre recombinase, introduced by homologous recombination at the locus of gene of connexin-40 (Cx40). Cx40 encodes for a protein expressing in the ventricular trabeculae and the VCS. The recombination is observed in the cells expressing Cx40 and in their descendants. My results suggest a progressive restriction of the fate of Cx40-positive trabeculae in conductive cardiomyocytes. Cx40-positive progenitors give rise to the formation of the compact myocardium and of the VCS when they are induced between E10.5-E14.5; while they participate only in the cardiomyocytes of VCS after E16.5. 2) The analysis of the VCS has been studied in a model of congenital heart malformation. The mutant Tbx1-/-, model of the DiGeorge syndrome, present a ventricular septal defect. Morphological defects of the VCS are found in Tbx1-/- hearts: an absence of right bundle branch and a non-compacted His bundle; which are correlated with functional defect. The phenotype observed in these mutants does not result from a defect of the activation in the genetic program being at the origin of the establishment in the VCS, but seems to be explain by the presence of a large ventricular septal defect, because it could block the progression of the progenitors of the VCS along the crest of the inter-ventricular septum.

It was shown almost 50 years ago that hypothermic incubation of chicken embryos results in a reduction in the size of embryos and an increase in the heart weight, presumably by hypertrophy (increase in cell volume). The chicken embryos were incubated in normothermia (37.5 ř C) and hypothermia (33.5 ř C) from the eleventh embryonic day. On the 17th day, the embryos were weighed and then their hearts were weighed. In agreement with the previous results, hypothermic embryos were 29% smaller and their hearts 18% heavier. The heart-to-body weight ratio was 67% higher in the hypothermic group. The measured cell size was very similar in the target areas and it was also between the two groups. The left ventricle width was twofold that the right one and the difference was not significantly higher in the hypothermia model. Purkinje fibers, the terminal part of the conduction system, were smaller than the working cardiomyocytes. Purkinje fibers were slightly enlarged after hypothermic incubation. The proliferation rate was measured by immunohistochemical labeling of anti-phospho histone H3. The experimental group showed much higher proliferation rate; it reached statistical significance in the right ventricle. Thus, hypothermic incubation resulted in increased growth of embryonic heart based on hyperplasia...

No description available.

Stress hormones, adrenaline and noradrenaline, have been shown to be critical for heart development. Mice lacking dopamine greek lower case letter beta]-hydroxylase (Dbh), an enzyme responsible for synthesis of these adrenergic hormones, die during mid-gestation due to cardiac failure. Prior research showed that adrenergic cells are found within the electrical conduction system of the heart, and adrenergic deficiency leads to slowed cardiac conduction during embryogenesis. Microarray analysis of wild-type (Dbh+/+) and knockout (Dbh-/-) mouse hearts revealed significant differences in expression of retinoic acid (RA) signaling genes. RA signaling has also been shown to be critical for heart development. These data suggest that heart failure due to adrenergic deficiency may be dependent upon RA signaling. This led to the hypothesis that adrenergic hormones promote the development of the electrical conduction system through modulation of RA signaling. To test this, embryonic mouse hearts were cultured with LE 135, a RA receptor blocker. Heart rate, arrhythmic index (AI) and conduction time were measured. Under these conditions there was a marked increase in arrhythmias. Hearts treated with LE 135 showed a mean AI of 0.232±0.057 after 24 hours of treatment while when untreated had an AI of 0.083±0.028 (p<0.05;n=15). In contrast, there was no significant change in heart rate or conduction speed after 24 hours with or without the retinoic acid receptor blocker. To determine if adrenergic stimulus influences retinoic acid response, an established RA-sensitive reporter cell line was employed. These F9-RARE-LacZ cells were treated with forskolin (cAMP regulator) and isoproterenol (greek lower case letter beta]-agonist) to measure changes in RA signaling. Evaluation of RA signaling showed an increase in retinoic acid responsiveness when treated with an adrenergic signaling agonist.; These results suggest that proper retinoic acid signaling is essential for maintaining cardiac rhythmicity during embryonic development and adrenergic stimulation can influence this response.

Thesis (PhD)--Stellenbosch University, 2004. / Bibliography / 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 opsporing van 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.

Group of specialized cells that form cardiac conduction system is responsible for generation and coordinated propagation of the electrical impulse in the heart. Changes in its development can be connected with arrhythmias; therefore, a good level of knowledge is necessary and relevant for basic science and clinical practice. For correct development of the conduction system are important genes coding gap junctions proteins, ion channels, transcription factors and other molecules involved in signaling cascades (endothelin, neuregulin). Development of conduction system is determined in addition to genetic factors also by epigenetics and environmental factors. This thesis with its individual papers on which it is based is addressing different aspects of conduction system development, which appears to be a complex process. Another feature which is linking all papers together, is the methodological approach enabling us to study function of the conduction system - optical mapping. In the first publication we studied by the means of in vitro organ culture the impact of work load without interfering hemodynamics on the conduction system maturation in the chick embryonic heart. The phenotype observed during experiments was developmental regression of conduction system maturation together with changes in...