Cardiac glycosides (CG) like 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, if treatment with the selective ouabain antagonist rostafuroxin prevents CG-mediated electrophysiological derangements and arrhythmia by restoring electrolyte homeostasis. We employed incubated chicken eggs (iCEs), a 3R-compliant model, for which we established Electrocardiograms (ECGs). ECGs were recorded under i) baseline conditions, ii) after treatment with ouabain and iii) after co-treatment 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. Co-treatment of ouabain and rostafuroxin led to reduced HRV and ameliorated the frequency and duration of transient sinus arrest, while 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 3R compliant manner. We found, that rostafuroxin treatment ameliorated ouabain-induced electrophysiological disturbances, suggesting rostafuroxin as a potential therapeutic intervention for ouabain mis- or overdosing.
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