Is increased Na+ influx a cause of mitochondrial redox stress, cardiomyopathy, and a therapeutic target?
by Mufassra Mushtaq
Date of Examination:2023-10-13
Date of issue:2023-11-09
Advisor:Prof. Dr. Stephan E. Lehnart
Referee:Prof. Dr. Stephan E. Lehnart
Referee:Prof. Dr. Dörthe Katschinski
Referee:Prof. Dr. Niels Voigt
Referee:Prof. Dr. Silvio Rizzoli
Referee:Prof. Dr. Ralf Dressel
Referee:Prof. Dr. Ralph Kehlenbach
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EnglishMaintaining balanced Na+ levels is critical for normal functioning of cardiomyocytes, including contractions, excitation-contraction coupling (ECC), and metabolism. The NaV1.5-F1759A (human) mutation, which increases intracellular Na+, has been associated with the development of atrial fibrillation (AF). We propose that excessive intracellular Na+ entry could interfere with the activity of plasma and mitochondrial matrix Na+/Ca2+ exchangers (NCX). This disruption could lead to increased cytosolic Ca2+, which may cause the activation of calpain proteases known to cleave junctophilin-2 (JP2), as well as mitochondrial dysfunction, ultimately increasing the generation of reactive oxygen species (ROS). To test this hypothesis, we conducted a study using a newly developed double-transgenic (DTG) mouse model, FLAG-NaV1.5-F1759A. These mice express transgenes controlled by the cardiac-specific αMHC promoter, and the background was changed to C57Bl/6N, which is known for its mitochondrial redox competence. Kaplan-Meier survival analysis of 50 WT and 158 DTG mice revealed an overall mortality rate of 35% after 12 weeks, with a more pronounced effect in male mice. Intermittent AF episodes were confirmed in 89% of the DTG mice during 10-minute of 6-lead ECG recordings. Immunoblotting confirmed NaV1.5-F1759A expression in the atria and ventricles of the DTG mice. Transthoracic echocardiography measurements of 8-week-old mice demonstrated significant structural and functional changes in the atria, including increased left atrial inner diameters (WT:2.04 ± 0.03 vs. DTG:2.75 ± 0.08, P <0.0001) and depressed left atrial fractional shortening (WT:17.78 ± 0.90 in DTG: 5.58 ± 0.63, P <0.0001). Confocal imaging of the isolated atrial myocytes revealed increased cellular size and length, suggesting cellular hypertrophy. Changes in the transverse-axial tubule network, characterized by decreased transverse and increased axial tubule components, were also observed, implying subcellular excitation-contraction coupling defects. Intracellular Na+ measurements using 23Na NMR confirmed higher levels of intracellular Na+ in DTG mice, which contributed to the AF phenotype. TMT labelling revealed altered cardiac metabolism in DTG mice, that is, relying on glycolysis and ketone bodies for ATP synthesis instead of fatty acid oxidation. Metabolic profiling of ventricular tissue using 1H NMR showed elevated lactate levels and reduced high-energy metabolites (ATP and NADP) in DTG mice, indicating metabolic impairments, which is in line with proteomics data. Additionally, live-cell redox biosensor imaging showed a significantly decreased EGSH (TTG-mito-roGFP: -296.30 ± 2.0mV vs. mito-roGFP: -284.90 ± 1.5 mV, P <0.0001) in atrial myocytes of triple transgenic mitochondrial redox biosensor mice (TTG-mito-roGFP), indicating increased mitochondrial matrix oxidation and compensation for ROS production. DTG mice showed a significant reduction of JP2 in the ventricular tissue, but the triple transgenic junctophilin overexpression mice (TTG-JP2OE) showed increased JP2 expression, thereby rescuing cellular hypertrophy, atrial contractile function, and TAT network reorganization. These findings support our hypothesis that increased intracellular Na+ [Na+]i via NaV1.5 leads to oxidative stress, mitochondrial dysfunction, and altered energy metabolism, ultimately contributing to maladaptive atrial remodelling and the perpetuation of atrial fibrillation. JP2OE significantly rescued the complex AF phenotype, providing a novel therapeutic strategy for persistent Na+-current [Na+]i-mediated cardiomyopathy.
Keywords: Atrial fibrillation, sodium influx, cardiomyopathy