A light-inducible Na+ pump to study the role of intracellular Na+ accumulation in heart failure
by Svenja Mareike Kiehn
Date of Examination:2024-06-07
Date of issue:2024-10-10
Advisor:Prof. Dr. Tobias Brügmann
Referee:Prof. Dr. Tobias Brügmann
Referee:Prof. Dr. Susanne Lutz
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Abstract
English
Elevated Nai levels have been identified as a significant factor in the development of heart failure, observed across multiple species. This is caused by downregulated NKA, increased NHE activity and elevated late Na+ current, which disrupts ionic balance and metabolism. Consequently, NCX reverse activity is increased, leading to impaired Ca2+ efflux and increased SR Ca2+ load, ultimately leading to arrhythmias. Elevated levels of Nai also lead to increased activity of NCLX, which results in a depletion of Ca2+ in the mitochondria. As ATP supply and ROS regulation depend on Ca2+ in the mitochondria, it is essential to maintain proper levels of Camito. Depletion may result in increased energy demand and overflow of ROS, which can contribute to the devel- opment of heart failure. However, there is no evidence that reversing Nai elevation improves heart failure outcomes yet. Therefore, it is essential to find a tool that can effectively lower Nai without disturbing ion homeostasis to investigate its impact on heart failure. As pharmacological approaches have not yet been successful in lowering Nai, this thesis will focus on a recently discovered enhanced variant of a light-driven Na+ pump eKR2 to investigate its effectiveness in lowering Nai in HEK293 cells and its impact on hiPSC derived cardiomyocytes. Upon illumination with green light, eKR2 is activated, resulting in the efflux of Na+. These Na+ efflux currents were investigated in HEK293 cells using patch clamp. Fur- thermore, the effect of long-term activation of eKR2 in HEK293 cells on Nai was ex- amined using the ratiometric Na+ dye SBFI and 23Na-NMR spectroscopy. Experiments using SBFI revealed a decrease in Nai upon illumination of HEK293 cells expressing eKR2 in comparison to non-illuminated cells or WT controls. Additionally, investiga- tion of Camito upon increased Nai could be important to elucidate the effects on ATP demand in heart failure. To achieve this, a new technique using a chemical dye coupled to an expressed HaloTag developed by a collaboration partner was used to measure Camito upon ATP application in HEK293 cells. Furthermore, the optical pacing ability iv Abstract of hiPSC-derived cardiomyocytes expressing eKR2 by an anode break excitation was analysed. For further experiments, a heart failure assay was established using hiPSC- derived cardiomyocytes treated with ET-1 to induce hypertrophy, which was analysed by fluorescent antibody staining against proBNP and BNP measurements by ELISA. To sum up, as light activation of eKR2 is sufficient to lower Nai in HEK293 cells, it could be a powerful tool for future studies involving Nai analysis in cardiomyocytes. Its ability to lower Nai can be used in various experiments to investigate the impact of elevated Nai on ATP demand, ROS overflow and SR Ca2+ in heart failure. Ultimately, it could prove that lowering Nai is able to reverse markers of heart failure. Abbreviations: ABE: anode break excitation, ATP: adenosine triphosphate, BNP: brain natriuretic peptide, Ca2+: calcium ions, eKR2: enhanced Krokinobacter eikastus Rhodopsin 2, ELISA: enzyme-linked immunosorbent assay, ET-1: endothelin-1, HEK: human embryonic kidney-derived cells, hiPSC: human induced pluripotent stem cell, MOCA: myocyte contraction analysis, Na+: sodium ions, Nai: intracellular sodium, NCLX: mitochondrial sodium-calcium exchanger, NCX: sarcolemmal sodium-calcium exchanger, NHE: sodium proton exchanger, NKA: sodium-potassium ATPase, NMR: nuclear magnetic resonance, ROS: reactive oxygen species, SBFI: sodium benzofuran isophthalate SR: sarcoplasmic reticulum
Keywords: optogenetic; heart failure; sodium elevation; iPSC derived cardiomyocytes; eKR2