The rhythmic contraction of our heart is controlled by electrical impulses that travels throughout the cardiac muscle. The signal is generated by the sinus node, which can be thought of as the natural pacemaker of our heart. The question is, what it is that makes the sinus node unique, enabling it to generate a repetitive firing of electrical signals thereby ensuring the foundation for the rhythmic cardiac contraction? How is dysregulation of the molecular build-up of the sinus node linked to cardiac disorders? By Associate Professor Alicia Lundby, Department of Biomedical Sciences, University of Copenhagen Every heartbeat is initiated and coordinated by the cardiac conduction system, which consists of the sinus node (generating pacemaker potentials), the atrioventricular node and His-Purkinje system. The sinus node The rhythmic contraction of our heart is controlled by electrical impulses that travels throughout the cardiac muscle, thereby stimulating the heart to contract and pump blood. The electrical signal is generated by the sinus node, which can be thought of as the natural pacemaker of our heart. The sinus node consists of a small cluster of highly specialized cells located in the upper part of the right atrium. The function of the sinus node is primarily electrical, whereas the function of the atrial and ventricular muscle is primarily contractile. Although the sinus node is primarily comprised of myocytes and fibroblasts like the rest of the cardiac tissue, the protein expression landscape of the sinus node differs from the surrounding tissue endowing it with its unique ability to regulate heart rate. With the research funding from the Carlsberg Foundation, the first question my group set out to address was: What makes the particular heart region encompassing the sinus node unique? What is it that enables this particular region to generate a repetitive firing of electrical signals thereby ensuring the foundation for the rhythmic cardiac contraction? To study this, we performed in-depth investigations of the molecular composition of the cardiac sinus node using mass spectrometry-based proteomics strategies. High-resolution proteomics Proteomics technologies based on liquid chromatography tandem mass spectrometry (LC-MS/MS) has advanced many areas of biological research concerning cellular signalling in the last decade. The power of the LC-MS/MS technology lies in its ability to present an unbiased approach to investigate the protein architecture of a given cellular system. In a typical experiment we can measure and quantify on the order of 10,000 proteins. Using this technology, we can investigate the protein landscape in tiny tissue biopsies and, importantly, quantify changes under different physiological conditions. High-resolution mass spectrometry offers the possibility to measure and quantify a comprehensive proteome, thereby enabling an unbiased investigation of the protein landscape of a given tissue. Earlier this year, we published a study in Nature Communications, where we used quantitative proteomics to measure 7,248 proteins in sinus nodes from mouse hearts. Mouse sinus node is quite small, approximately 1x1 mm. Figure 1: Localisation of sinus node in heart tissue. The location of the sinus node is indicated by an orange circle in the drawing of a heart (left). The image shows a tissue preparation from a mouse heart, where the sinus node is localised to the area indicated by the stipulated orange circle (middle). Localisation is confirmed by immunohistochemistry using sinus node marker, HCN4 for positive confirmation of sinus node and atrial marker protein CX43 as negative marker (right). Proteins extracted and analysed by high-resolution mass spectrometry from such tiny tissue samples allow us to quantify thousands of proteins. Despite limited tissue material, we acquired high-quality quantitative data. For all the proteins we compared the protein abundances in the sinus node with the neighbouring atrial tissue. We included cell-type information by also acquiring single nucleus transcriptomics data. The combined dataset of quantitative proteomics and single nucleus transcriptomics allowed us to generate a detailed molecular map of the sinus node. This map of the cardiac pacemaker offers a unique insight into the biology of the sinus node. For example, we outlined more than fifty ion channels in the mouse sinus node going much beyond what was known. By computational modelling we confirmed that our measured channel abundance profiles indeed recapitulated the pacemaking ability of the sinus node. Figure 2: Based on our proteomics dataset we quantified the abundance of all proteins involved in the Ca2+ clock and the membrane clock, respectively, in the sinus node of the heart. a Left: Illustration of the two signalling mechanisms suggested to contribute to pacemaking potentials. Right: Proteins measured in sinus node (x-axis) ranked from lowest to highest mean protein abundance (y-axis). Proteins involved in membrane clock (green) are less abundant than those involved in the Ca2+ clock (yellow). b Illustration of proteins involved in pacemaker generation in the sinus node. Proteins are coloured by abundance in favour of sinus node (red) or atrial muscle (blue). Ca2+ clock proteins are evenly expressed in atria and sinus node, whereas the ion channels of the membrane clock show differential expression. Photo: Linscheid et al. With the computational modelling, we used our data on ion channels to estimate copy numbers per sinus node myocyte of the respective channels, which is a fundamentally important insight to obtain. In addition to answering some questions, the work also posed many new questions. E.g. we observed that the extracellular matrix of the sinus node is more elastic than anticipated, and we observed that the metabolic profile differs from that of cardiac muscle. We confirmed the first by orthogonal experimental approaches, and for the latter we observed by electron microscopy that the sinus node myocytes have lipid vesicles. With that study, we characterised the unique protein expression landscape of the natural cardiac pacemaker. The sinus node molecular architecture in human hearts Our efforts studying mouse heart sinus nodes have been important to address fundamental questions in cardiac physiology, but it has been equally important in the establishments of methodologies. We are now confident that we have the technical expertise to perform in-depth quantitative analyses of sinus nodes, and with this expertise, we aim to outline the molecular build-up of human heart sinus node. This is important, among others to know to which extend the cardiac conduction system can be recapitulated by studies in model organisms. With this, we aim to identify cardiac conduction system proteins involved in heart failure The conduction system and heart failure We are expanding our efforts to address molecular changes underlying pathological states of the conduction system. The diseased conduction system is an important cause of life-threatening brady- and tachyarrhythmias, which are currently treated with implantation of electronic pacemakers. We envision that understanding the molecular underpinnings of the remodelling of the conduction system in disease is key for identification of new therapeutic targets. The first disease-centric study we undertake is focused on an animal model of heart failure, where we investigate regulation of the sinus node in heart failure. This will be a first in-depth molecular characterisation of the remodelling of the cardiac conduction system in heart disease. With this, we aim to identify cardiac conduction system proteins involved in heart failure, which would establish the scientific foundation to understand which particular parts of the cardiac conduction system protein network is re-wired in heart failure. This work is based on the hypothesis that new heart failure treatments could be developed for the underlying dysfunction of the cardiac conduction system. Outlook The research outcome of our efforts will contribute to clarification of the molecular players underlying the rhythmic contraction of our heart, and hence form a foundation for addressing protein dysfunctionalities in sinus node pathologies and rhythm disorders. It will explain fundamental biology of pacemaker activity of our hearts and aims to use this information to explain molecular underpinnings of heart failure. If successful, a molecular link between sinus node function and heart failure is found, which would present a potential means for disease intervention. References Lundby, A. et al. In vivo phosphoproteomics analysis reveals the cardiac targets of β-adrenergic receptor signaling. Sci. Signal. 6, rs11 (2013). Lundby A et al. Cell. 2019 Oct 3;179(2):543-560.e26. doi: 10.1016/j.cell.2019.09.008. Oncogenic Mutations Rewire Signaling Pathways by Switching Protein Recruitment to Phosphotyrosine Sites. Linscheid et al, Quantitative proteomics and single-nucleus transcriptomics of the sinus node elucidates the foundation of cardiac pacemaking, Nature Communications volume 10, Article number: 2889 (2019) Other references Dobrzynski, H. et al. Structure, function and clinical relevance of the cardiac conduction system, including the atrioventricular ring and outflow tract tissues. Pharmacol. Ther. 139, 260–288 (2013). Lakatta, E. G. & DiFrancesco, D. What keeps us ticking: a funny current, a calcium clock, or both? J. Mol. Cell Cardiol. 47, 157–170 (2009). Bekker-Jensen, D. B. et al. An Optimized Shotgun Strategy for the Rapid Generation of Comprehensive Human Proteomes. Cell Syst. 4, 587–599 (2017). e4.