Asymmetric biphasic electric stimulation supports cardiac maturation and functionality

Two-dimensional (2D) cardiac models are widely used for cardiotoxicity screening but often lack structural and functional maturity of adult native tissue. Electrical stimulation (ES) enhances in vitro maturation, yet conventional waveforms (monophasic and symmetric biphasic) have shown limitations, including charge accumulation and possible cell hyperpolarization. Here, we introduce for the first time an asymmetric biphasic ES waveform that combines the advantages of monophasic and symmetric biphasic stimulation by reversing the current and reducing residual voltage. Asymmetric biphasic stimulation improved electrical functionality, calcium handling and contractility of neonatal rat cardiac cells, without triggering cellular stress. Additionally, cells subjected to asymmetric biphasic ES displayed a metabolic shift toward fatty acid oxidation, a hallmark of mature cardiomyocytes. Taken together, these findings highlight the novelty and efficacy of asymmetric biphasic stimulation in generating more physiologically relevant in vitro cardiac models, providing a promising alternative to standard ES protocols.