Automated patch-clamp for high throughput characterization of subtype-specific induced pluripotent stem cell derived cardiomyocytes

Abstract

Atrial fibrillation (AF) represents the most prevalent cardiac arrhythmia observed in clinical practice. The efficacy of therapeutic interventions for AF remains suboptimal, and the exploration of ion channel remodeling mechanisms in AF is restricted by both the lack of atrial specificity in standard cell lines and the low throughput of ‘gold standard’ methodologies. Significant progress in the development of high throughput automated patch-clamp (APC) systems could be extremely beneficial for accelerating electrical investigations into the mechanisms of atrial-specific disorders such as AF. Human induced pluripotent stem cells (iPSC) were derived from dermal fibroblasts of a healthy male donor. Factor-free cardiac differentiation into iPSC derived cardiomyocytes (iPSC-CM) was achieved via small molecule temporal modulation of wnt signaling. Atrial specificity was induced through Retinoic acid (1 μM) application between day 3 and 6 of cardiac cell differentiation as has been previously shown (Seibertz et al., 2023). We describe the application of a high throughput APC device (SyncroPatch 384) to characterize key ionic currents and action potentials (AP) in human atrial- and ventricular-specific iPSC-CM. Thin borosilicate glass 384-well planar chips (1xS-type NPC-384 T) were used for all experiments. Recordings were excluded if they showed a seal resistance of <250 MΩ or a peak current of <50 pA. All measurements were carried out at room temperature. Robust current-clamp measurements of cardiac action potential revealed subtype-specific characteristics with shorter action potentials recorded in atrial iPSC-CM. High throughput voltage-clamp measurements of sodium current (INa), L-type calcium current (ICa,L), the rapid component of the delayed rectifier (IKr), and basal inward rectifier potassium current (IK1) were able to be measured in both cell types. In addition, activation of the atrial-specific acetylcholine-activated inward rectifier potassium current (IK,ACh) was exclusively observed in atrial but not in ventricular iPSC-CM following application of the M-receptor agonist carbachol. The successful application of a high throughput APC-system for the recording and characterization of atrial and ventricular APs and ionic currents in highly scalable iPSC-CM models implies that APC represents a powerful tool for future studies of atrial-specific pathologies such as AF.