✍🏼 Madison Hess, Sanya, Sei Kim, Nathan Grainger

🏠 Department of Physiology & Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA

📑 ScholarWolf, the institutional repository for the University of Nevada, Reno (2025)

Abstract
The sinoatrial node (SAN) is responsible for generating action potentials that travel through the atria and ventricles of the heart, producing a heartbeat. SAN cells undergo significant changes to their metabolism during periods of heart failure. However, to understand these changes more clearly, we must ascertain the metabolic needs of SAN cells under healthy conditions. Our investigation focused on using gentle microfluidic cell sorting to isolate SAN cells from other cell types that occupy the region (i.e. cardiomyocytes, fibroblasts, endothelial cells, immune cells, etc.). We also aimed to distinguish between the live and dead cell populations using a cell viability dye, namely Sytox Green, which stains the nuclei of dead cells with GFP. Finally, we focused on optimizing two techniques on wild type cells that will be applied to SAN cells in the future: cell culturing and measuring oxygen consumption rates on the Seahorse Bioanalyzer, stimulated with isoproterenol.

How the WOLF is used in this study
In this poster, the WOLF G2 microfluidic cell sorter is used to gently isolate and purify fluorescently tagged sinoatrial node (SAN) pacemaker cells from digested cardiac tissue for downstream metabolic analysis. Following enzymatic and mechanical dissociation of SAN tissue, isolated cells are introduced into the WOLF G2, where they are analyzed by a 488 nm laser and sorted based on red fluorescent protein (RFP) expression from the HCN4-tdTomato transgenic model. The system operates at <2 psi using a piezoelectric push-pull sorting mechanism, which preserves membrane integrity and minimizes cell death, enabling recovery of highly viable SAN cells. Additional forward- and side-scatter gating enriches for large, complex pacemaker cells, while Sytox Green staining confirms viability post-sort. The purified SAN cells are then cultured and analyzed using the Seahorse Bioanalyzer to measure mitochondrial function and oxygen consumption rate (OCR), establishing baseline metabolic characteristics of healthy pacemaker cells for future heart failure studies.