Nanodomains of ROS production control cardiomyocyte metabolism and contractility

• Applicants: Privatdozent Dr. Sören Brandenburg; Professorin Dr. Dörthe M. Katschinski
• Subject Area: Anatomy and Physiology ; Cardiology, Angiology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 545018649

Heart failure triggers structural changes, alters energetics, and reconfigures metabolism within cardiomyocytes. Reactive oxygen species (ROS) have long been associated with heart failure due to their impact on these changes. However, a clear functional mechanism remains elusive, suggesting a complex and not entirely understood role of ROS in cardiomyocytes. Once generated, ROS diffuse within the cell. Understanding the spatiotemporal organization of this diffusion is crucial, considering compartmentalization and specific ROS-induced oxidation protein targets. These targets may vary depending on the site of ROS production, controlled by diffusion rates. To analyze ROS nanodomains regarding their production sites and oxidation targets, we propose employing D-amino acid oxidase (DAO). DAO enables the use of D-amino acids for carbon and energy in yeast. When expressed in mammalian cells, DAO acts as a chemogenetic tool, enhancing precise and dynamic H2O2 production upon exposure to D-amino acids. Recently, we've demonstrated the feasibility of applying DAO alongside the H2O2 sensor HyPer in transgenic mice to identify specific H2O2 protein targets in cardiomyocytes. Apart from developing DAO-expressing mice, we've established a redox proteome workflow. To explore compartment-specific ROS effects, we've generated induced pluripotent stem cells (iPSCs) expressing HyPer-DAO in the cytoplasm (NES), nucleus (NLS), or mitochondrial matrix (MLS). Our goal is to use iPSC-derived cardiomyocytes expressing HyPer-DAO in different subcellular domains to understand the spatiotemporal distribution of compartmentalized ROS production. This approach will be supplemented by in-depth ROS gradient analysis in healthy and diseased murine adult cardiomyocytes using high resolution nanoscopy. Applying our established redox proteome workflow, we aim to characterize cardiac ROS target proteins in subcellular compartments and their impact on heart failure development, comparing iPSC-derived cardiomyocytes expressing compartmentalized HyPer-DAO to diseased heart samples. Ultimately, our objective is to unveil how ROS dynamics affect cardiomyocyte energetics and contractility. Overall, this project aims to significantly enhance our understanding of how cardiomyocytes employ ROS nanodomains spatially to influence signal propagation, regulate cardiac function, and respond when ROS levels exceed thresholds. This knowledge will shed light on the physiological function of ROS and their involvement in misdirected pathways within cardiomyocytes.

• DFG Programme: Research Grants