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Characterizing Nav1.5 Expression, Organization, and Electrical Behavior in Cardiomyocyte Domains

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Characterizing Nav1.5 Expression, Organization, and Electrical Behavior in Cardiomyocyte Domains Graduate School for Cellular and Biomedical Sciences University of Bern Characterizing Nav1.5 expression, organization, and electrical behavior in cardiomyocyte domains PhD Thesis submitted by Sarah Helena Vermij from the Netherlands for the degree of PhD in Biomedical Sciences Supervisor Prof. Dr. Hugues Abriel Institute of Biochemistry and Molecular Medicine Faculty of Medicine of the University of Bern Co-advisor Prof. Dr. Stephan Rohr Department of Physiology Faculty of Medicine of the University of Bern Urheberrechtlicher Hinweis Dieses Dokument steht unter einer Lizenz der Creative Commons Namensnennung-Keine kommerzielle Nutzung-Keine Bearbeitung 2.5 Schweiz. http://creativecommons.org/licenses/by-nc-nd/2.5/ch/ Sie dürfen: dieses Werk vervielfältigen, verbreiten und öffentlich zugänglich machen Zu den folgenden Bedingungen: Namensnennung. Sie müssen den Namen des Autors/Rechteinhabers in der von ihm festgelegten Weise nennen (wodurch aber nicht der Eindruck entstehen darf, Sie oder die Nutzung des Werkes durch Sie würden entlohnt). Keine kommerzielle Nutzung. Dieses Werk darf nicht für kommerzielle Zwecke verwendet werden. Keine Bearbeitung. Dieses Werk darf nicht bearbeitet oder in anderer Weise verändert werden. Im Falle einer Verbreitung müssen Sie anderen die Lizenzbedingungen, unter welche dieses Werk fällt, mitteilen. Jede der vorgenannten Bedingungen kann aufgehoben werden, sofern Sie die Einwilligung des Rechteinhabers dazu erhalten. Diese Lizenz lässt die Urheberpersönlichkeitsrechte nach Schweizer Recht unberührt. Eine ausführliche Fassung des Lizenzvertrags befindet sich unter http://creativecommons.org/licenses/by-nc-nd/2.5/ch/legalcode.de Accepted by the Faculty of Medicine, the Faculty of Science and the Vetsuisse Faculty of the University of Bern at the request of the Graduate School for Cellular and Biomedical Sciences Bern, Dean of the Faculty of Medicine Bern, Dean of the Faculty of Science Bern, Dean of the Vetsuisse Faculty Bern ABSTRACT Proper function of the heart depends on the function of voltage-gated ion channels. These channels open and close in a tightly regulated way. The resulting ion currents change the membrane potential and shape the action potential, which initiates cardiac muscle contraction. The sodium channel Nav1.5 is especially important as it generates the initial upstroke of the action potential. In cardiomyocytes, it is expressed in different membrane domains, including the intercalated disc, where two cardiomyocytes are mechanically and electrically coupled; the lateral membrane; and possibly at the T-tubules, which are invaginations of the lateral membrane. Many different proteins and molecules bind Nav1.5, together forming a macromolecular complex, and modulate Nav1.5 expression and/or function. Mutations in the Nav1.5-encoding gene SCN5A can confer a loss or gain of channel function, and are associated with several heart rhythm disorders, including Brugada syndrome, long-QT syndrome type 3 (LQTS3), and sick-sinus syndrome. The mechanisms that lead to this phenotypic variability remain unknown. Since Nav1.5 occurs at different cardiomyocyte membrane domains and several interacting proteins are specific to a domain, we hypothesize that the effects of a mutation depend on the subcellular location of Nav1.5 and the composition of the macromolecular complex. This thesis aims to (1) determine Nav1.5 cluster organization in cardiomyocyte membrane domains from mice with different genetic backgrounds using single-molecule localization techniques; (2) contribute to the fundamental understanding of cardiomyocyte excitability by assessing voltage-gated ion channel expression with next-generation RNA sequencing; and (3) assess passive electrical properties of T-tubules and the effects of a large T-tubular sodium current with an in silico model. In addition, this thesis contains two thorough literature reviews on the cardiac intercalated disc and T- tubules. Firstly, we show that Nav1.5 is expressed in T-tubules of wild-type cells using single-molecule localization microscopy and computational modeling techniques. We observed that Nav1.5 cluster organization and density partly depend on the presence of the large scaffolding protein dystrophin and on the three C-terminal amino acids of Nav1.5, Ser-Ile-Val. Cardiomyocytes expressing C-terminally truncated Nav1.5 (ΔSIV) display a loss of Nav1.5 expression at the lateral membrane and particularly at the lateral membrane groove compared to wild-type cells. Dystrophin-deficient cardiomyocytes also display a reduction of Nav1.5 expression at the lateral membrane, but no groove-specific reduction, and most notably an increase of T-tubular Nav1.5 expression. Nav1.5 cluster shapes are less complex in dystrophin-deficient cells at the lateral membrane and inside the cell compared to wild type. ΔSIV cells show this effect only inside the cells, not at the lateral membrane. Secondly, we show in murine cardiomyocytes of Black/6J mice that of the voltage-gated sodium channels, mRNAs are expressed encoding mainly Nav1.5 and Nav1.4, and a small amount of Nav2.1- 5 encoding mRNA. No other isoforms were detected. Of the β-subunits, only β1- and β4-encoding mRNA are found. Thirdly, we assessed electrical properties of T-tubules. We compared the depolarization delay of a deep T-tubular segment to the mouth of a T-tubule upon a large depolarizing voltage step reminiscent of the upstroke of the cardiac action potential. We chose to compare the time to reach the activation threshold of voltage-gated calcium channels as these channels are highly expressed in T-tubules and crucial for initiating cardiomyocyte contraction. Deep inside the T-tubule, the activation threshold of voltage-gated calcium channels was reached only 10 microseconds later than at the mouth of the T- tubule. This delay increased 10-20 times when we introduced constrictions. Then, we introduced a large sodium current to the model. We show that the sodium current is smaller deep inside the T- tubule than at the mouth due to the positive extracellular potential, which decreases the driving force of the channels. In the constricted tubules, we observed a stronger sodium current self-attenuation, but an increase of peak sodium current in the first constriction due to an increase in open probability and driving force. In conclusion, these studies contribute to the fundamental understanding of voltage-gated sodium channel composition, organization, and function in cardiomyocytes, with a focus on Nav1.5. Exciting subjects of further study include the functional implications of the changes in Nav1.5 cluster organization in ΔSIV and dystrophin-deficient mice, the functional contributions of Nav1.4, and β1- and β4-subunits to murine cardiomyocyte function, and the composition of voltage-gated ion channels in human cardiomyocytes. 6 TABLE OF CONTENTS ABSTRACT .................................................................................................................................................................. 5 1. INTRODUCTION ............................................................................................................................................... 11 1.1 Basic anatomy and function of the heart ................................................................................................. 11 1.2 From poetry to science: A short history of the heart ......................................................................... 11 1.2.1 Ancient Egypt up to the Renaissance ................................................................................................ 13 1.2.2 Renaissance, Scientific Revolution and beyond .............................................................................. 15 1.2.2.1 Sixteenth century .......................................................................................................................... 16 1.2.2.2 Seventeenth century ..................................................................................................................... 17 1.2.2.3 Eighteenth century ........................................................................................................................ 19 1.2.2.4 Nineteenth century ....................................................................................................................... 20 1.3 Cell types in the heart .................................................................................................................................. 22 1.3.1 The ventricular cardiomyocyte .......................................................................................................... 23 1.4 Cardiac conduction ....................................................................................................................................... 25 1.4.1 The cardiac action potential ............................................................................................................... 26 1.4.2 Electrotonic versus ephaptic conduction ........................................................................................ 27 1.5 Cardiac arrhythmias...................................................................................................................................... 28 1.6 Nav1.5 ............................................................................................................................................................... 29 1.6.1 Nav1.5 states ..........................................................................................................................................
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