Rostafuroxin ameliorates cardiac glycoside-induced cardiomyocyte electrolyte imbalances and arrhythmia in ovo

Cardiac glycosides (CGs) such as ouabain exert positive inotropic effects by inhibiting the Na⁺-K⁺-ATPase. CGs' wide spread use is limited by CGs' narrow therapeutic window. Mis- or overdosing with CGs may cause cardiac arrhythmias, resulting from electrolyte disturbances. To study the ethically challenging topic of CG overdosing, we here optimized the in ovo platform to test whether treatment with the selective ouabain antagonist rostafuroxin prevents CG-mediated electrophysiological derangements and arrhythmia by restoring electrolyte homeostasis. We used incubated chicken eggs (iCEs), a 3 R-compliant model, for which we established electrocardiograms (ECGs). ECGs were recorded under <i>1</i>) baseline conditions, <i>2</i>) after treatment with ouabain, and <i>3</i>) after cotreatment with rostafuroxin. Underlying mechanisms of ouabain and rostafuroxin effects were studied using blood gas analysis and fluorescence microscopy. Isolated murine and human cardiomyocytes served as an independent model to confirm in ovo results. Ouabain treatment resulted in increased heart rate variability (HRV), transient sinus arrest, and atrio-ventricular dyssynchrony, accompanied by plasma hyperkalemia and cardiomyocyte Na⁺ overload. Cotreatment of ouabain and rostafuroxin led to reduced HRV and ameliorated the frequency and duration of transient sinus arrest, whereas plasma K⁺ levels remained unchanged. In isolated cardiomyocytes, ouabain treatment induced intracellular Na⁺ overload, which was abolished by additional rostafuroxin treatment. Our work demonstrates the in ovo platform and corresponding readouts as a suitable tool to study cardiac electrophysiology in a 3 R-compliant manner. We found that rostafuroxin treatment ameliorated ouabain-induced electrophysiological disturbances, suggesting rostafuroxin as a potential therapeutic intervention for ouabain mis- or overdosing.<b>NEW & NOTEWORTHY</b> This study evaluates rostafuroxin, a selective ouabain inhibitor, for its potential to antagonize electrophysiological derangements in ouabain overdosing. Methodologically, the study uses the iCE model, previously introduced as a suitable 3 R-compliant cardiovascular research platform. We developed and validated a comprehensive electrophysiological workflow in iCEs to perform our investigations. Ouabain increased heart rate variability, induced arrhythmia and electrolyte imbalances in iCEs, whereas rostafuroxin largely protected them from these effects.