Introduction:
Due to their narrow therapeutic window, cardiac glycosides (CGs) have been used less frequently in heart failure (HF). Recent evidence from the DIGIT-HF (DIGitoxin to Improve ouTcomes in patients with advanced chronic Heart Failure) clinical trial, however, has brought CGs back into the clinical management of HF by showing reduced hospitalization and mortality in patients with HF and atrial fibrillation. We investigated the effects of overdosing CGs, such as ouabain, on the heart of the incubated Chicken Egg (iCE), hypothesizing that CG overdose induces electrophysiological disturbances in iCEs that can be measured via ECG, supporting its use as a tool in electrophysiological cardiovascular basic research. iCEs provide an ethical alternative to small animals, as they cannot experience pain for the first 15 days of embryonic development (ED) and have a four-chambered heart. Additionally, we hypothesize that the ouabain antagonist rostafuroxin, previously analyzed in the context of hypertension, has the potential to prevent CG-induced electrophysiological abnormalities.
Methods:
Electrocardiograms (ECGs) were recorded in fertilized Lohmann Selected Leghorn-Classic/Brown-Classic eggs on ED 13, and reference parameters of standard ECG metrics were created. We injected ouabain (35 µg, i.v.; n = 13) to assess spontaneous arrhythmia occurrence. Simultaneous ECG and echocardiographic recordings were used to determine the effects of arrhythmia on cardiac function. As the observed arrhythmia seemed to originate in the sinus node itself, excised iCE hearts were imaged using a widefield fluorescence microscope to analyze cardiac membrane potential in the right atrium, as the location of the sinus node (n = 4-7). Intracellular Na+ levels after ouabain overdose were measured in isolated murine (n = 4 animals, 48 cells per group) and human cardiomyocytes from heart transplantation (n = 2 hearts, 12-16 cells per group) using patch-clamping to verify our findings in established models.
Results:
ECG reference values of iCEs, like P-wave, T-wave and ventricular complex, proved to be closer to human reference values than murine values, as the average heart rate was slower than the heart rate of mice and rats. Ouabain, digoxin, and digitoxin treatments induced arrhythmia in iCE, while no arrhythmia occurred in the vehicle-treated control group. The observed arrhythmia phenotypes were transient sinus arrest and atrio-ventricular dyssynchrony. Ouabain treatment increased intracellular Na+ in human and murine cardiomyocytes and induced hyponatremia in the blood gas analysis of iCEs, verifying the inhibition of the Na+-K+-ATPase. In fluorescence microscopy, ouabain treatment resulted in reduced relative fluorescence in the right atrium, demonstrating reduced electrical activity of the right atrium. A combined injection of ouabain and rostafuroxin led to a reduction in transient sinus arrest in iCEs and prevented intracellular sodium accumulation in cardiomyocytes.
Conclusion:
iCEs proved to be a novel 3R-compliant model in cardiovascular basic research by robustly responding to the induction of arrhythmia using CGs. Ouabain-associated arrhythmia burden was partially prevented by treatment with the ouabain-inhibitor rostafuroxin, indicating that rostafuroxin works against ouabain overdose not just in the setting of hypertension, and, if modified to antagonize digitoxin or digoxin, might be an interesting target in treating CG overdose.
