Multi-scale electrophysiological characterization of human cardioids reveals progressive maturation
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Background
Human induced pluripotent stem cell-derived cardiomyocytes are valuable for cardiac disease modelling and drug testing but remain limited by immature structural and electrophysiological properties in conventional two-dimensional cultures. Three-dimensional cardiac models, including cardioids, provide a more physiological environment through multicellular organization and enable tissue-level functional analysis. Their electrophysiological properties and maturation, however, remain incompletely characterized.
Methods
We performed an integrated electrophysiological characterization of human cardioids over the first three weeks of maturation using sharp microelectrode recordings, calcium transient measurements, whole-cell patch clamp recordings, immunofluorescence, transcriptomic analysis, and pharmacological interventions.
Results
During maturation, cardioids exhibited progressive electrophysiological maturation, characterized by more physiological resting membrane potentials (−54.0±3.1mV to −68.1±1.2mV), increased action potential amplitude (73.5±4.8mV to 101.7±2.1mV) and upstroke velocity (44.3±5mV/ms to 86.6±8.4mV/ms), and shorter action potential duration (178.9±8.7ms to 144.0±4.8ms at 1Hz stimulation). These changes were associated with progressively increased I Na density and enhanced SCN5A mRNA and Na v 1.5 protein expression, consistent with progressive maturation of cardiac excitability. I Kr current and KCNH2 /hERG expression were already present at early maturation stages and remained stable, as evidenced by preserved sensitivity to dofetilide-blockade. Pharmacological inhibition of barium- and HMR-sensitive currents altered resting membrane and/or action potential characteristics in intact cardioids, whereas in isolated cardiomyocytes classical I K1 remained undetectable and I Ks was small, highlighting a certain degree of persisting immaturity of the model.
Conclusions
These findings demonstrate that human cardioids undergo progressive electrophysiological maturation and provide complementary tissue- and cellular-level insights into cardiac function, establishing their suitability for investigating I Na and I Kr -related cardiac pathologies.
