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Effect of temperature on arrhythmogenesis during heart development
Vostárek, František ; Sedmera, David (advisor) ; Tomek, Viktor (referee) ; Naňka, Ondřej (referee)
5 Abstract: Aims: The main objective of this work was to analyze in detail the effects of acute temperature changes on the function of isolated chick embryonic heart in vitro in comparison with natural conditions in ovo. Methods: The effects of temperature change (34 řC, 37 řC and 40 řC - hypo-, normo- and hyperthermia, respectively) on calcium dynamics in four days old isolated chick hearts in vitro were investigated by high-speed calcium optical imaging. For comparison and validation of in vitro measurements, experiments were also performed in ovo using videomicroscopy. Artificial electrical stimulation experiments were performed in vitro and in ovo to uncover conduction limits of different heart segments. Results: We observed almost linear dependence of sinus frequency on temperature in our temperature range. Sinus frequency during hypothermia and hyperthermia in vitro and in ovo changed about 20% in comparison with normothermia. We observed no significant changes in amplitude of calcium transients during temperature change to hypothermia but hyperthermia caused a significant decrease in amplitude of calcium transients (atria 35%, ventricles 38%). We observed a wide spectrum of arrhythmias, which occurred spontaneously even during normothermia in vitro. Occurrence of arrhythmias in vitro significantly...
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Role of endothelial and vascular smooth muscle cells in the origin, progression and therapy of vascular diseases
Chlupáč, Jaroslav ; Bačáková, Lucie (advisor) ; Piťha, Jan (referee) ; Sedmera, David (referee)
Introduction: Vascular surgery for atherosclerosis is confronted by the lack of a suitable bypass material. Synthetic vascular prostheses include polyethylene terephthalate (PET) and expanded polytetrafluoroethylene (ePTFE). However, these materials become thrombosed in small-caliber applications (<6 mm) because of the lack of an endothelium. The objectives of this study were to make modifications to clinically-used PET vascular prostheses with tissue-engineered surfaces to improve their bio-compatibility towards vascular smooth muscle cells (VSMC) and endothelial cells (EC). Methods: Blood coagulation protein fibrin (Fb) and extracellular matrix proteins collagen (Co), laminin (LM) and fibronectin (FN) were used. Cell adhesive assemblies were prepared: Co, Co/LM, Co/FN, Co/Fb, Co/Fb/FN. Cell culture experiments were performed: (1) planar static, (2) planar dynamic with simulation of blood flow, (3) tubular dynamic, and (4) animal porcine implantation. Results: The growth of EC and VSMC on commercial prostheses (ePTFE, PET and PET/Co) was low. The growth of both cell types was lower on PET/Co than on PET. After modification with protein assemblies, the highest numbers of EC were reached on PET/Co and on PET/Co +Co/Fb. There was no difference in the densities of VSMC among various assemblies. The...
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Role of Nkx2.5 in development and electrophysiology of the mouse heart
Hámor, Peter ; Sedmera, David (advisor) ; Neckář, Jan (referee)
Role of Nkx2.5 in development and electrophysiology of the mouse heart Prague 2016 Peter Hámor, B.S. ABSTRACT The objective of this thesis is to investigate the role of Nkx2.5 gene dosage on electrophysiology of the mouse heart in prenatal stage of its development. The main goal of this work is to search for differences in conduction of electric impulses through the embryonic mouse hearts of different genotype. Special method of capturing the conduction of electric impulse through myocardium, called optical mapping, was used to visualize the electrical activity. Thanks to this method I was able to construct images and videos capturing the spread of the impulse with identification of the beginning of the activation and its direction in the heart. These outputs, or optical maps, help to define anomalies and defects in mutants compared with a normal functioning heart. The thesis focuses on the expression of the transcription factor Nkx2.5 and regulatory components related with the correct formation and physiology of the heart until 9.5 days post coitum. Embryos at this developmental stage were optically mapped and analysed according to their genotype. While the wild type and heterozygote mouse embryos exhibited high degree of similarity, the homozygous mutants were dramatically different. Considering this work...
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Mechanisms of conduction system development in vertebrates
Šaňková, Barbora ; Sedmera, David (advisor) ; Neckář, Jan (referee) ; Melenovský, Vojtěch (referee)
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...
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Developmental mechanisms of arrhythmias - role of connexins in arrhythmogenesis
Beneš, Jiří ; Sedmera, David (advisor) ; Kolář, František (referee) ; Král, Jiří (referee)
Developmental mechanisms of arrhythmias - role of connexins in arrhythmogenesis MUDr.Jiří Beneš Abstract: Objective: The aim of this study is an improvement of our knowledge concerning the role of connexins in arrhythmogenesis. The main focus is on the role of connexin40 (Cx40) in heart development in mice and changes in connexin43 expression in volume overload heart failure rat model. Methods: The infuence of Cx40 on heart develoment was studied on transgenic mouse Cx40:GFP model using the method of optical mapping. Volume overload heart failure was examined in the rats with aortocaval shunt. Morphological changes in hearts were examined using imunofluorescence microscopical techniques. Results: In the atria, Cx40 is important especially during the early stages. Cx43 can partially substitute its function from 12.5 embryonic day on. Cx40 deficiency leads to decreased conduction velocity and ectopic sites of activation. Absence of Cx40 in ventricular conduction system leads to the development of right bunde branch block. Volume overload in rats leads to excentric hypertrofy and later to heart failure. We described morphological as well as microscopical changes in failing hearts. Without the presence of fibrosis, the main arrhythmogenic substrate we found was reduced amount of Cx43 and its dephosphorylation....
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Multipotent mesenchymal stromal cells in orthopedic: Potentiation of bone healing
Stehlík, David ; Trč, Tomáš (advisor) ; Janíček, Pavel (referee) ; Sedmera, David (referee)
The aim of the thesis was development of an innovative treatment of bone defects. Human multipotent mesenchymal stromal cells (MSC) play a crucial role in bone healing. Clinical applications of MSC require large amount of cells, which could be obtained by autologous expansion of MSC harvested from bone marrow. As a first step, the standard protocol of MSC expansion based on αMEM medium and fetal bovine serum (FBS) was used. Experiments replacing FBS by pooled human serum (HS) in the culture medium concluded in patenting of a new MSC cultivation protocol (EU 1999250, CR 301141). This one-step cultivation protocol and xenogeneic protein-free cultivation medium is based on CellGro® for Hematopoietic Cells' Medium, HS, human recombinant growth factors, dexamethasone, insulin and ascorbic acid. The preclinical in vitro and in vivo experiments with MSC from both expansion protocols were carried out. Fibrillar polylactic scaffolds were seeded with MSC, cultured, differentiated and implanted in immunodeficient mice (NOD/LtSz-Rag1-). Bone-like mineralized tissue containing vessels was observed. The MSC cultured according to patented method were classified as Advanced-therapy Medicinal Product and has to fulfil the European Medicines Agency regulations to enter the clinical trials. Nevertheless the use of MSC seems...
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Effect of incubation temperature on growth of the working and conducting myocardium in the embryonic chick
Skuhrová, Kristýna ; Sedmera, David (advisor) ; Gregorovičová, Martina (referee)
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...
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Effect of temperature on arrhythmogenesis during heart development
Vostárek, František ; Sedmera, David (advisor) ; Tomek, Viktor (referee) ; Naňka, Ondřej (referee)
5 Abstract: Aims: The main objective of this work was to analyze in detail the effects of acute temperature changes on the function of isolated chick embryonic heart in vitro in comparison with natural conditions in ovo. Methods: The effects of temperature change (34 řC, 37 řC and 40 řC - hypo-, normo- and hyperthermia, respectively) on calcium dynamics in four days old isolated chick hearts in vitro were investigated by high-speed calcium optical imaging. For comparison and validation of in vitro measurements, experiments were also performed in ovo using videomicroscopy. Artificial electrical stimulation experiments were performed in vitro and in ovo to uncover conduction limits of different heart segments. Results: We observed almost linear dependence of sinus frequency on temperature in our temperature range. Sinus frequency during hypothermia and hyperthermia in vitro and in ovo changed about 20% in comparison with normothermia. We observed no significant changes in amplitude of calcium transients during temperature change to hypothermia but hyperthermia caused a significant decrease in amplitude of calcium transients (atria 35%, ventricles 38%). We observed a wide spectrum of arrhythmias, which occurred spontaneously even during normothermia in vitro. Occurrence of arrhythmias in vitro significantly...
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Mechanisms of conduction system development in vertebrates
Šaňková, Barbora ; Sedmera, David (advisor) ; Neckář, Jan (referee) ; Melenovský, Vojtěch (referee)
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...
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