Anatomical and Molecular Mapping of The Human Cardiac Conduction System - With Aims to Further Understand Its Function

Abstract

Background: Worldwide, cardiovascular diseases (CVDs) remain a leading cause of death, with ageing and obesity being common risk factors. CVDs cause structural and functional remodelling of the heart – hence further cardiac dysfunctions. The cardiac conduction system (CCS) is a collection of specialised cardiomyocytes that work together to regulate and propagate electrical impulses around the heart. The CCS consists of the sinus node (SN, the heart’s primary pacemaker), the atrioventricular conduction axis, and the His-Purkinje network. Dysfunction of this system leads to CVDs such as arrhythmia and heart failure. Therefore, it is vital to further our anatomical understanding of key cardiac structures and the CCS in healthy, aged, and obese hearts. Expanding our current understanding of the human SN's molecular mechanisms is equally vital. Dysfunction of key molecular pathways in the SN results in SN dysfunctions such as bradycardia. Aims: The studies described in this thesis aimed to contribute to the current understanding of anatomical and molecular interactions of the working myocardium and CCS of the human heart from healthy, aged, and obese individuals. The study aimed to create 3D reconstructions of the whole heart, major blood vessels, valves, and components of the CCS of the human heart from normal (healthy), aged, and obese individuals. Another aim of using healthy human SN samples was to apply bioinformatics to predict interactions between microRNAs, transcription factors (TFs), and cell markers that regulate SN function. Methods: 1) Normal, aged, and obese whole hearts and tissue blocks were micro-CT scanned following iodine potassium-iodide (I2KI) or graphene oxide (GO, a novel contrast agent) staining. Some samples were frozen before sectioning and then stained using Masson’s trichrome. 2) RNA-sequencing and bioinformatics were used to analyse the expression profile and predict interactions of key TFs and cell markers mRNAs with key microRNAs in the healthy adult human SN. 3) Luciferase assay experiments were performed to validate the predicted significant downregulation of mRNAs by microRNAs. Results: 1) Successfully used I2KI and a novel contrast agent (GO) for high-resolution micro-CT to visualise and create 3D reconstructions of the whole myocardium, major blood vessels, coronary artery network, and valves from whole normal, aged, and obese hearts. Created 3D reconstructions of Purkinje fibres and the region containing the SN from these hearts. 2) Embryonic TFs (e.g., ISL1) and ‘novel’ TFs (e.g., RUNX1-2) are significantly more abundant in the SN vs. atrial tissue. These TFs were predicted to regulate HCN4 expression and different cell markers (e.g., COL1A1, a fibroblast marker; and TPSAB1, a mast cell marker) in the SN. Key microRNAs significantly less abundant in the SN (e.g., miR-486-3p and miR-938) were predicted to downregulate mRNAs of pacemaking ion channels. 3) MiR-486-3p significantly downregulates the luciferase activity of Cav1.3, Cav3.1, and TPSAB1. Conclusions: This thesis contributes to and expands the current understanding of myocardium and CCS variations between healthy, aged, and obese individuals. The novel insights into the complex molecular interactions presented in this thesis can be targeted in treating SN dysfunctions. The data presented can aid in developing cardiac models.

Date of Award	31 Dec 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Delvac Oceandy (Supervisor), Halina Dobrzynski (Supervisor) & Alicia D'Souza (Supervisor)