Aldolase from human heart has been purified 128-fold to a final specific activity of 11.52 units per mg. The purification procedure employed column chromatography on phosphocellulose.

No description available.

The pathological consequences of even mild increases in blood pressure warrant treatment for hypertension in its early stages. However, chronic drug treatment programmes are generally not advantageous during the early stages of hypertension. Augmented sympathetic outflow to the heart plays a role in the early stages of hypertension, and perhaps the development of hypertension. Environmental factors are often responsible for increases in sympathetic outflow to the heart. Therefore, an alternative hypertensive treatment involves behavioural control over increases in sympathetic activity. This treatment includes biofeedback training. The literature indicates that the R-wave to ear pulse wave interval (RPI) is the most appropriate index of sympathetic influences for biofeedback training. An experiment is reported in which unconstrained normotensive subjects were asked to produce changes in RPI with and without the aid of analog feedback. Five subjects learned to produce bidirectional changes in RPI. These subjects generally showed more RPI shortening than lengthening. The data indicate that moderately heavy levels of exercise were employed to shorten RPI. This is consistent with increased sympathetic activity. Some subjects were consistently able to lengthen RPI. However, this study produced converging evidence indicating that RPI lengthening was often a product of reduced left ventricular preload. Preload influences on RPI appear to have led subjects to adopt behavioural strategies which were inconsistent with reduced sympathetic activity during attempts to lengthen RPI. Therefore, caution must be employed when using RPI to index and teach control over sympathetic activity. It is suggested that incorporating information about left ventricular ejection time or cardiac interbeat interval will improve RPI as a measure of sympathetic influences on the human heart. / Thesis / Master of Arts (MA)

No description available.

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.

Cardiac accessory β-subunits are part of macromolecular ion channel complexes. They can modulate electrophysiological properties of resulting ion currents and action potentials and are supposed to contribute to cardiac disease e.g. arrhythmias or Brugada syndrome. In my thesis, we characterized the functions of dipeptidyl peptidase-like protein 10 (DPP10), a transmembrane β-subunit of cardiac Na+ and K+ channels. Previous studies revealed that DPP10 is expressed in human heart and acts as regulator of Kv channel kinetics. In electrophysiological experiments, we found that DPP10 modulates Ito through Kv4.3 channel complexes by accelerating current densities and the time course of activation, inactivation and recovery from inactivation. Interestingly, co-expression of DPP10 with Kv4.3 and KChIP2 in CHO cells induced a slowly inactivating fraction of Ito, providing evidence for a contribution of Ito on the sustained outward K+ current in cardiomyoctes. Until then, the sustained fraction of K+ currents was thought to be due to IKur. We further studied the contribution of Kv4-mediated Ito to total K+ currents in human atrial myocytes using 4-Aminopyridine to block IKur in combination with Heteropoda toxin 2 to block Kv4 channels. Using this approach, it was possible to separate an Ito fraction of about 19% contributing to the late current component. These data suggest that the generation of a sustained current component of Ito induced by DPP10 may affect the late repolarization phase of an atrial action potential. To further explore the functions of DPP10, we investigated a potential interaction with Nav channels in cardiomyocytes. It was possible to detect DPP10 in human ventricles, with higher expression levels in patients with heart failure. We demonstrated that DPP10 affects cellular action potentials in isolated rat cardiomyocytes after adenoviral gene transfer indicating a reduction in Na+ current density. Voltage-dependent Na+ channel activation and inactivation curve was shifted to more positive potentials with overexpression of DPP10, resulting in enhanced availability of Na+ channels for activation, along with increasing window Na+ current. Thus, we assumed a role of DPP10 on promotion of arrhythmias via interaction with Nav1.5. The results of this study can help to understand the complex interaction pattern between Nav and Kv channels and the role of their β-subunits, especially DPP10. In conclusion, DPP10 was identified as a new modulator of Kv and Nav currents in the human heart, suggesting that this β-subunit may contributes to cardiac arrhythmias and might be a new therapeutic target.:1 Introduction 1.1 The cardiac action potential 1.2 Cardiac potassium channels 1.2.1 The Kv4.3 channel complex 1.2.2 Accessory β subunits of K+ channel 1.2.3 The Kv1.5 channel 1.2.4 Separation of Ito and IKur in native cardiomyocytes 1.3 Cardiac sodium channels 1.3.1 Molecular construction of Nav1.5 channel 1.3.2 Accessory β subunits of Na+ channel 1.3.3 The role of Nav1.5 in cardiac electrical disorders 1.4 Aim of the thesis and systematic approach 2 The research articles 3 Summary 4 Zusammenfassung 6 References 7 Appendices 7.1 Abbreviations

La plupart des maladies cardio-vasculaires sont étroitement liées à l’architecture 3D des faisceaux de cardiomyocytes du myocarde humain. Connaitre en détail cette architecture permet de lever un verrou scientifique sur l’organisation spatiale complexe des faisceaux de cardiomyocytes, et offre des pistes pour trouver des solutions pertinentes permettant de guérir ces maladies. A cause de la nature biréfringente des filaments de myosine qui se trouvent dans les cellules cardiomyocyte, l’Imagerie en Lumière Polarisée (ILP) se révèle comme la seule méthode existante permettant d’étudier en détail, l’architecture et l’orientation des faisceaux de cardiomyocytes au sein de la masse ventriculaire. Les filaments de myosine se comportent comme des cristaux uni-axiaux biréfringents, ce qui permet de les modéliser comme les cristaux uni-axiaux biréfringents. L’ILP exploite les propriétés vibratoires de la lumière car l’interaction photonique et atomique entre la lumière et la matière permet de révéler l’organisation structurelle et l’orientation 3D des cardiomyocytes. Le présent travail se base sur la modélisation des différents comportements de la lumière après avoir traversé des faisceaux de cardiomyocytes. Ainsi, un volume 100×100×500 µm3 a été décomposé en plusieurs éléments cubiques qui représentent l'équivalent de l'intersection des cellules de diamètre de 20 µm chacune. Le volume a été étudié dans différentes conditions imitant l’organisation 3D des cardiomyocytes dans différentes régions du myocarde. Les résultats montrent que le comportement du volume change suivant l’arrangement spatial des cardiomyocytes à l’intérieur du volume. Grâce à un modèle analytique développé à l’aide des simulations, il a été possible de connaitre en tout point, l’orientation 3D des cardiomyocytes dans tout le volume. Ce modèle a été implémenté dans un greffon logiciel. Puis, il a été validé avec les piliers des valves auriculo-ventriculaire en comparant les courbes obtenues en simulation numérique à celles obtenues dans la phase expérimentale. De plus, il a été possible de mesurer l’orientation 3D des faisceaux de cardiomyocytes à l’intérieur du pilier. Après cette validation, le modèle a été utilisé sur un cœur humain (sain) en entier. Puis, nous avons extrait les cartographies des orientations 3D (angle azimut, angle d’élévation) des cardiomyocytes, ainsi que la cartographie des niveaux d’homogénéité du myocarde en entier. Pour une confrontation qualitative des mesures de l’orientation 3D obtenues en ILP avec celles en IRM, un cœur humain sain d’un enfant de 14 mois a été prélevé lors de l’autopsie, fixé dans du formol, puis imagé en entier par IRM puis en ILP. Malgré la faible résolution des images en IRM, les résultats obtenus montrent que les mesures de l’orientation 3D des cardiomyocytes issues de ces deux méthodes d’imageries se révèlent quasiment identiques. / Most cardiovascular diseases are closely linked to the 3D cardiomyocytes bundles of the human myocardium. Knowing in detail this architecture allows us to overcome a scientific bottleneck on the complex spatial organization of cardiomyocytes, and offers ways to find appropriate solutions to treat these diseases. The goal of present thesis is then to develop methods and techniques that allow gaining insights into the geometric arrangement of cardiomyocytes or cardiomyocytes bundles in the myocardium. Due to the birefringent nature of myosin filaments that are found in myocardial cells, the Polarized Light Imaging (PLI) appears as the only existing method for studying in detail the architecture and cardiomyocytes bundle orientation in ventricular mass. Myosin filaments react as uniaxial birefringent crystal; thereby it has been modeled as the uniaxial birefringent crystal. The PLI uses the vibration properties of light; the photonic and atomic interaction between light and matter can reveal the structural organization and the 3D cardiomyocytes orientation of the myocardium. The present work is based on modeling the behavior of the light after passing through a cardiomyocytes bundle. Thus, a volume 100 × 100 × 500 μm3 has been decomposed in a number of cubic elements which are equivalent to cardiac cells of diameter of 20 microns. The volume was studied under different conditions to emulate the organization of cardiomyocytes in different regions in human myocardium: isotropic region, heterogeneous region, region with cardiomyocytes bundle crossing. The results showed that the behavior of the volume changes according to the spatial arrangement of cardiomyocytes within the volume. Through an analytical model developed using simulation, it has been possible to know the 3D orientation of cardiomyocytes at any region throughout the volume. This model has been implemented in software as a plugin. Then, it has been validated with the pillars of atrio-ventricular valves by comparing the curves obtained by numerical simulation with those obtained in the experimental phases. Moreover, it has been possible to measure the 3D orientation of cardiomyocytes bundles within the pillars. After validation, the model was applied to an entire human healthy heart. Then, we extracted the mapping of the 3D orientations (azimuth angle, elevation angle) of cardiomyocytes bundles, as well as the mapping of the homogeneity levels of the entire myocardium. For a qualitative comparison of the 3D orientation measurements obtained with the PLI and Magnetic Resonance Imaging (MRI), the healthy human heart of a 14 month old child was extracted at autopsy, then fixed in formalin, and finally imaged by MRI and PLI. Despite the low spatial resolution of MRI images, the results showed that the 3D orientations of cardiomyocytes bundles measured from these two imaging methods appeared almost identical.

Kürzlich wurde mithilfe von Ribo-seq Experimenten die Translation hunderter Mikroproteine in menschlichen Herzen entdeckt. Diese blieben zuvor aufgrund ihrer geringen Größe (< 100 Aminosäuren) unentdeckt, und ihre physiologische Rolle ist noch weitgehend unbekannt. Ziel dieser Promotionsarbeit ist es, potentielle Funktionen dieser neuartigen Mikroproteine zu entschlüsseln. Dabei sollen insbesondere die Aufklärung ihrer evolutionären Konservierungssignatur, subzellulären Lokalisierung und ihres Proteininteraktoms helfen. Die Konservierungsanalyse ergab, dass fast 90% der Mikroproteine nur in Primaten konserviert ist. Weiterhin konnte ich die Produktion von Mikroproteine in vitro und in vivo nachweisen, die subzelluläre Lokalisierung von 92 Mikroproteinen definieren, und Interaktionspartner für 60 Mikroproteine identifizieren. Dutzende dieser Mikroproteine lokalisieren in Mitochondrien. Dazu gehörte ein im Herzen angereichertes Mikroprotein, das aufgrund der Interaktions- und Lokalisationsdaten einen neuartigen Modulator der mitochondrialen Proteintranslation darstellen könnte. Der Interaktom-Screen zeigte außerdem, dass evolutionär junge Mikroproteine ähnliche Interaktionsfähigkeiten wie konservierte Kandidaten haben. Schließlich wurden kurze Sequenzmotive identifiziert, die Mikroprotein-Protein-Wechselwirkungen vermitteln, wodurch junge Mikroproteine mit zellulären Prozessen – wie z.B. Endozytose und Spleißen – in Verbindung gebracht werden konnten. Zusammenfassend wurde die Produktion vieler kleiner Proteine im menschlichen Herzen bestätigt, von denen die meisten lediglich in Primaten konserviert sind. Zusätzlich verknüpften umfangreiche Lokalisierungs- und Interaktionsdaten mehrere Mikroproteine mit Prozessen wie Spleißen, Endozytose und mitochondrialer Translation. Weitere Untersuchungen dieses zuvor verborgenen Teils des Herzproteoms werden zu einem besseren Verständnis von evolutionär jungen Proteinen und kardiologischen Prozessen beitragen. / Recently, the active translation of hundreds of previously unknown microproteins was detected using ribosome profiling on tissues of human hearts. They had remained undetected due to their small size (< 100 amino acids), and their physiological roles are still largely unknown. This dissertation aims to investigate these novel microproteins and validate their translation by independent methods. Particularly, elucidating their conservation signature, subcellular localization, and protein interactome shall aid in deciphering their potential biological role. Conservation analysis revealed that sequence conservation of almost 90% of microproteins was restricted to primates. I next confirmed microprotein production in vitro and in vivo by in vitro translation assays and mass spectrometry-based approaches, defined the subcellular localization of 92 microproteins, and identified significant interaction partners for 60 candidates. Dozens of these microproteins localized to the mitochondrion. These included a novel cardiac-enriched microprotein that may present a novel modulator of mitochondrial protein translation based on its interaction profile and subcellular localization. The interactome screen further revealed that evolutionarily young microproteins have similar interaction capacities to conserved candidates. Finally, it allowed identifying short linear motifs that may mediate microprotein-protein interactions and implicated several young microproteins in distinct cellular processes such as endocytosis and splicing. I conclude that many novel small proteins are produced in the human heart, most of which exhibit poor sequence conservation. I provide a substantial resource of microprotein localization and interaction data that links several to cellular processes such as splicing, endocytosis, and mitochondrial translation. Further investigation into this hidden part of the cardiac proteome will contribute to our understanding of recently evolved proteins and heart biology.