The IKs current constitutes part of the repolarisation reserve in the human myocardium, and whilst it does not play a major role at resting heart rates, it becomes a crucial component of repolarisation in the setting of increased sympathetic tone and high heart rates. The formation of the IKs current requires the KCNQ1 α-subunit and the KCNE1 β-subunit. Mutations in either of these subunits can lead to long QT syndrome types 1 and 5, respectively. Loss-of-function mutations in the IKs channel can reduce the repolarisation reserve and lead to action potential prolongation, predisposing to lethal cardiac arrhythmias such as torsades de pointes and ventricular fibrillation. It is widely recognised that the IKs channel requires the minor membrane phospholipid PIP2 for its function, and previous work in this laboratory found that mutations in a PIP2-binding region in KCNQ1 led to retention of the channel in the endoplasmic reticulum, suggesting that PIP2 may play a role in anterograde trafficking. Here, the rapamycin-inducible dimerisation system was used to manipulate levels of PIP2 and/or PI4P at the plasma membrane or Golgi, and the effect of this on IKs channel trafficking and function was investigated using molecular biology, confocal microscopy and electrophysiology. Despite difficulties with optimising the rapamycin-induced dimerisation system, it was observed that the IKs channel does not require PIP2 for anterograde trafficking, but is heavily reliant on PIP2 for channel opening. In addition, activation of the β1-adrenergic receptor (β 1-AR) led to an increase in the IKs current amplitude. The potential interplay between β1-AR and PIP2 signalling was also explored by depleting PIP2 during β1-AR stimulation. PIP2 depletion was less effective at inhibiting the IKs current during β1-AR stimulation, but this requires further investigation. In conclusion, the results suggest that the IKs channel is reliant on PIP2 for function, but not anterograde trafficking.

Heart failure patients have dysfunction of the cardiac conduction system that contributes to a high burden of arrhythmias including atrial fibrillation and sudden cardiac death. Heart failure has been associated with the inflammatory response, but it is unknown if inflammation is playing a role in the remodelling of the cardiac conduction system in heart failure. Inflammation has been shown to be present in the myocardium from failing hearts and it is known to have detrimental effects on cardiac function, inducing fibrosis, remodelling of ion channels and even arrhythmias. However, the effect of inflammation on the cardiac conduction system has not been investigated. The aims of this study were to determine if there is an increase of pro-inflammatory cytokines and inflammatory cells in the cardiac conduction system in heart failure. In addition, to identify if there is possible inflammation-associated fibrosis and apoptosis in the cardiac conduction system in heart failure. To test these aims, three models of heart failure were used: a rat model of pulmonary arterial hypertension, a rabbit model of congestive heart failure and a rat model of myocardial infarction. In the rat model of pulmonary arterial hypertension there was a bradycardia, a prolongation of the QT interval, and an increase in the atrioventricular and ventricular refractory periods, suggesting electrical remodelling in these animals. The rats with pulmonary arterial hypertension displayed an increase in pro-inflammatory cytokines such as interleukins 1β and TGFβ in the right side of the heart, including the sinoatrial node and right Purkinje fibres of the cardiac conduction system. In addition, in these areas, there was an increase in components of the extracellular matrix, including fibronectin, collagen I and vimentin. Histology revealed regions of non-myocyte nuclei, only in the right ventricle of the rats with pulmonary arterial hypertension. Immunohistochemistry demonstrated patches of CD68 and vimentin expression (markers for macrophages and fibroblasts, respectively) in the right side of the heart in these animals. TUNEL staining also revealed an increase in apoptosis in the right side of the heart. In the rabbit model of congestive heart failure, the region most affected by inflammation was the right atrium, while few changes were measured in the ventricles or cardiac conduction system. Although these results are surprising, it is suggested that the atria could be more sensitive to the physical stretch produced in this model. In the rat model of myocardial infarction, there were regions of non-myocyte nuclei in the border zone. This region also had increases in pro-inflammatory and fibrosis markers. In conclusion, this work has presented the novel finding that there can be inflammation in the cardiac conduction system in heart failure. This could be contributing to the arrhythmias seen in heart failure patients. This could possibly lead the way to anti-inflammatories as a possible novel therapeutic for heart failure patients.

Utilizou-se 30 corações de gatos sem raça definida, machos e fêmeas, adultos de idades variadas que não portavam nenhuma afecção cardíaca. Os corações foram injetados pela aorta torácica com Neoprene Latex 450, corados com pigmento vermelho. Verificou-se quando do predomínio da vascularização ventricular do tipo esquerda (63,34%) que a região irrigada pelo nó sinoatrial fica predominantemente na dependência do ramo proximal atrial direito (78,9%) ou com menor freqüência pelo ramo proximal atrial esquerdo (21,1%). Quando ocorreu a vascularização ventricular do tipo equilibrada (33,34%), a região ocupada pelo nó sinoatrial ficou na dependência mais frequentemente do ramo proximal atrial direito (80%), ou com menor freqüência a nutrição do nó se deu pelo ramo proximal atrial esquerdo (20%). Em um caso isolado, onde ocorreu a vascularização ventricular do tipo direita (3,34%), a nutrição da área ocupada pelo nó sinoatrial, ficou na dependência exclusiva do ramo intermédio atrial direito. Estes resultados indicam que nesta espécie não existe nenhuma relação entre a irrigação do nó sinoatrial e o tipo de vascularização ventricular. / We analyzed 30 hearts of cats without pedigree, males and females, adults of several ages. They were not carrying any heart problems. The hearts were injected by the thoracic aorta with Neoprene Latex 450 and stained with red pigment. We analyzed the prevalence of ventricular vascularization of the left type (63.34%) the irrigated region by the sinoatrial node was predominantly in the dependency of the Ramus proximalis atrii dextri (78.9%) or with less frequency by Ramus proximalis atrii sinister (21.1%). When occurred the ventricular vascularization of the type balanced (33.34%), the region occupied by the sinoatrial node was in the responsibility more often of the Ramus proximalis atrii dextri (80%) or with less frequency the nutrition of the sinoatrial node occurred by Ramus proximalis atril sinister (20%). In a singlecase, where we observed the ventricular vascularization of the right type (3.34%), the nutrition of the area occupied by the sinoatrial node, was the exclusive dependence of the Ramus intermedius atril dextri. These results showed that this specie there are no relationship between both the sinoatrial node irrigation and the type of ventricular vascularization, therefore, the nutrition of the sinoatrial node does not depend on the type of the ventricles vascularization.

Tvarkingą širdies darbą užtikrina širdies laidžioji sistema (ŠLS). Ją pažeidus sutrinka širdies darbas. Pažeidimo rizika atsiranda operacijos metu, kadangi ŠLS yra raumeninės kilmės audinys ir vizualiai neatskiriamas nuo aplinkinių audinių. ŠLS pažeidimo galima būtų išvengti, jei būtų žinomas tikslus ŠLS išsidėstymas arba egzistuotų ŠLS vaizdinimo metodika. Deja, bet atskirų ŠLS dalių tikslus išsidėstymas vis dar tikslinamas, o patologijų atvejais apskritai nėra žinomas. ŠLS vaizdinimo metodikos, tinkančios in vivo taikymams, taipogi nėra. Atlikus širdies audinių tyrimus nustoviąja fluorescencine spektroskopija nustatyti charakteringi intensyvumų skirtumai. Remiantis šiais skirtumais sukurta ŠLS atskyrimo metodika, paremta intensyvumų santykių skaičiavimu. Suskaičiuota vertė R = I(330)/I(380) yra skirtinga ŠLS, miokardui (MK) ir jungiamąjam audiniui (JA).Tokia metodika yra nejautri tyrimo sąlygoms ir gali būti naudojama ŠLS nustatymui. Ištyrus širdies audinius laikinės skyros spektroskopija nustatyta, kad ŠLS ir MK fluorescencijos gyvavimo trukmė bei santykinė sudėtis reikšmingai nesiskiria, tuo tarpu JA ir ŠLS tiek fluorescencijos gyvavimo trukmės, tiek santykinė komponentinė sudėtis yra skirtinga. Ištyrus širdies audinius konfokaliniu atspindžio mikroskopu nustatyta, jog dėl skirtingų šviesą atspindinčių komponentų bei skirtingo jų išsidėstymo galima identifikuoti MK, JA, Purkinje ląsteles ir ŠLS pluoštus. Atlikus tyrimus in vivo nustatyta, jog operacijos metu... [toliau žr. visą tekstą] / Rhythmical contraction of the heart is controlled by the cardiac conduction system (CCS). However, this highly important system visually could not be distinguished from the surrounding heart tissues – myocardium (MC) and connective tissue (CT); therefore during surgical procedures CCS could be damaged. The reliable method for CCS identification either in vivo or ex vivo does not exist therefore there is a definite need for developing a CCS imaging method. Fluorescence spectroscopy studies of cardiac tissues revealed, that most distinct spectral differences between CCS and the surrounding tissues were observed in 400 nm – 550 nm region under excitation from 330 nm – 380 nm region. The visualization method, based on the intensity ratios calculated for two excitation wavelengths, has been established. The calculated ratio R = I(330)/I(380) is different for CCS, CT and MC tissues, therefore the method may be used for identification of CCS. Time resolved fluorescence spectroscopy revealed no significant difference in composition and lifetimes between CCS and MC. On the other hand, the lifetimes and the relative spectral composition of CT differed significantly from those of CCS. Reflection confocal microscopy allows visualizing MC, CT, Purkinje cells and CCS bundles because of different reflection properties of tissue components and their specific distribution inside the tissue. The results of in vivo performed procedure revealed, that the distribution of fluorescence intensities... [to full text]

Rhythmical contraction of the heart is controlled by the cardiac conduction system (CCS). However, this highly important system visually could not be distinguished from the surrounding heart tissues – myocardium (MC) and connective tissue (CT); therefore during surgical procedures CCS could be damaged. The reliable method for CCS identification either in vivo or ex vivo does not exist therefore there is a definite need for developing a CCS imaging method. Fluorescence spectroscopy studies of cardiac tissues revealed, that most distinct spectral differences between CCS and the surrounding tissues were observed in 400 nm – 550 nm region under excitation from 330 nm – 380 nm region. The visualization method, based on the intensity ratios calculated for two excitation wavelengths, has been established. The calculated ratio R = I(330)/I(380) is different for CCS, CT and MC tissues, therefore the method may be used for identification of CCS. Time resolved fluorescence spectroscopy revealed no significant difference in composition and lifetimes between CCS and MC. On the other hand, the lifetimes and the relative spectral composition of CT differed significantly from those of CCS. Reflection confocal microscopy allows visualizing MC, CT, Purkinje cells and CCS bundles because of different reflection properties of tissue components and their specific distribution inside the tissue. The results of in vivo performed procedure revealed, that the distribution of fluorescence intensities... [to full text] / Tvarkingą širdies darbą užtikrina širdies laidžioji sistema (ŠLS). Ją pažeidus sutrinka širdies darbas. Pažeidimo rizika atsiranda operacijos metu, kadangi ŠLS yra raumeninės kilmės audinys ir vizualiai neatskiriamas nuo aplinkinių audinių. ŠLS pažeidimo galima būtų išvengti, jei būtų žinomas tikslus ŠLS išsidėstymas arba egzistuotų ŠLS vaizdinimo metodika. Deja, bet atskirų ŠLS dalių tikslus išsidėstymas vis dar tikslinamas, o patologijų atvejais apskritai nėra žinomas. ŠLS vaizdinimo metodikos, tinkančios in vivo taikymams, taipogi nėra. Atlikus širdies audinių tyrimus nustoviąja fluorescencine spektroskopija nustatyti charakteringi intensyvumų skirtumai. Remiantis šiais skirtumais sukurta ŠLS atskyrimo metodika, paremta intensyvumų santykių skaičiavimu. Suskaičiuota vertė R = I(330)/I(380) yra skirtinga ŠLS, miokardui (MK) ir jungiamąjam audiniui (JA).Tokia metodika yra nejautri tyrimo sąlygoms ir gali būti naudojama ŠLS nustatymui. Ištyrus širdies audinius laikinės skyros spektroskopija nustatyta, kad ŠLS ir MK fluorescencijos gyvavimo trukmė bei santykinė sudėtis reikšmingai nesiskiria, tuo tarpu JA ir ŠLS tiek fluorescencijos gyvavimo trukmės, tiek santykinė komponentinė sudėtis yra skirtinga. Ištyrus širdies audinius konfokaliniu atspindžio mikroskopu nustatyta, jog dėl skirtingų šviesą atspindinčių komponentų bei skirtingo jų išsidėstymo galima identifikuoti MK, JA, Purkinje ląsteles ir ŠLS pluoštus. Atlikus tyrimus in vivo nustatyta, jog operacijos metu... [toliau žr. visą tekstą]

Utilizou-se 30 corações de gatos sem raça definida, machos e fêmeas, adultos de idades variadas que não portavam nenhuma afecção cardíaca. Os corações foram injetados pela aorta torácica com Neoprene Latex 450, corados com pigmento vermelho. Verificou-se quando do predomínio da vascularização ventricular do tipo esquerda (63,34%) que a região irrigada pelo nó sinoatrial fica predominantemente na dependência do ramo proximal atrial direito (78,9%) ou com menor freqüência pelo ramo proximal atrial esquerdo (21,1%). Quando ocorreu a vascularização ventricular do tipo equilibrada (33,34%), a região ocupada pelo nó sinoatrial ficou na dependência mais frequentemente do ramo proximal atrial direito (80%), ou com menor freqüência a nutrição do nó se deu pelo ramo proximal atrial esquerdo (20%). Em um caso isolado, onde ocorreu a vascularização ventricular do tipo direita (3,34%), a nutrição da área ocupada pelo nó sinoatrial, ficou na dependência exclusiva do ramo intermédio atrial direito. Estes resultados indicam que nesta espécie não existe nenhuma relação entre a irrigação do nó sinoatrial e o tipo de vascularização ventricular. / We analyzed 30 hearts of cats without pedigree, males and females, adults of several ages. They were not carrying any heart problems. The hearts were injected by the thoracic aorta with Neoprene Latex 450 and stained with red pigment. We analyzed the prevalence of ventricular vascularization of the left type (63.34%) the irrigated region by the sinoatrial node was predominantly in the dependency of the Ramus proximalis atrii dextri (78.9%) or with less frequency by Ramus proximalis atrii sinister (21.1%). When occurred the ventricular vascularization of the type balanced (33.34%), the region occupied by the sinoatrial node was in the responsibility more often of the Ramus proximalis atrii dextri (80%) or with less frequency the nutrition of the sinoatrial node occurred by Ramus proximalis atril sinister (20%). In a singlecase, where we observed the ventricular vascularization of the right type (3.34%), the nutrition of the area occupied by the sinoatrial node, was the exclusive dependence of the Ramus intermedius atril dextri. These results showed that this specie there are no relationship between both the sinoatrial node irrigation and the type of ventricular vascularization, therefore, the nutrition of the sinoatrial node does not depend on the type of the ventricles vascularization.

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...

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.

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...

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...