Human stem cell-derived cardiomyocytes (hSC-CMs) hold great promise as in vitro models to study the electrophysiological effects of novel drug candidates on human ventricular repolarization. Two recent large validation studies have demonstrated the ability of hSC-CMs to detect drug-induced delayed repolarization and "cellrhythmias" (interrupted repolarization or irregular spontaneous beating of myocytes) linked to Torsade-de-Pointes proarrhythmic risk. These (and other) studies have also revealed variability of electrophysiological responses attributable to differences in experimental approaches and experimenter, protocols, technology platforms used, and pharmacologic sensitivity of different human-derived models. Thus, when evaluating drug-induced repolarization effects, there is a need to consider 1) the advantages and disadvantages of different approaches, 2) the need for robust functional characterization of hSC-CM preparations to define "fit for purpose" applications, and 3) adopting standardized best practices to guide future studies with evolving hSC-CM preparations. Examples provided and suggested best practices are instructional in defining consistent, reproducible, and interpretable "fit for purpose" hSC-CM-based applications. Implementation of best practices should enhance the clinical translation of hSC-CM-based cell and tissue preparations in drug safety evaluations and support their growing role in regulatory filings.

Treatment of arrhythmias evoked by hypothermia/rewarming remains challenging, and the underlying mechanisms are unclear. This in vitro experimental study assessed cardiac electrophysiology in isolated rabbit hearts at temperatures occurring in therapeutic and accidental hypothermia. Detailed ECG, surface electrogram, and panoramic optical mapping were performed in isolated rabbit hearts cooled to moderate (31°C) and severe (17°C) hypothermia. Ventricular activation was unchanged at 31°C while action potential duration (APD) was significantly prolonged (176.9 ± 4.2 ms vs. 241.0 ± 2.9 ms, P < 0.05), as was ventricular repolarization. At 17°C, there were proportionally similar delays in both activation and repolarization. These changes were reflected in the QRS and QT intervals of ECG recordings. Ventricular fibrillation threshold was significantly reduced at 31°C (16.3 ± 3.1 vs. 35 ± 3.5 mA, P < 0.05) but increased at 17°C (64.2 ± 9.9, P < 0.05). At 31°C, transverse conduction was relatively unchanged by cooling compared to longitudinal conduction, but at 17°C both transverse and longitudinal conduction were proportionately reduced to a similar extent. The gap junction uncoupler heptanol had a larger relative effect on transverse than longitudinal conduction and was able to restore the transverse/longitudinal conduction ratio, returning ventricular fibrillation threshold to baseline values (16.3 ± 3.1 vs. 36.3 ± 4.3 mA, P < 0.05) at 31°C. Rewarming to 37°C restored the majority of the electrophysiological parameters. Moderate hypothermia does not significantly change ventricular conduction time but prolongs repolarization and is pro-arrhythmic. Further cooling to severe hypothermia causes parallel changes in ventricular activation and repolarization, changes which are anti-arrhythmic. Therefore, relative changes in QRS and QT intervals (QR/QTc) emerge as an ECG-biomarker of pro-arrhythmic activity. Risk for ventricular fibrillation appears to be linked to the relatively low temperature sensitivity of ventricular transmural conduction, a conclusion supported by the anti-arrhythmic effect of heptanol at 31°C.

Cardiac electrophysiological heterogeneity includes: (i) regional differences in action potential (AP) waveform, (ii) AP waveform differences in cells isolated from a single region, (iii) variability of the contribution of individual ion currents in cells with similar AP durations (APDs). The aim of this study is to assess intra-regional AP waveform differences, to quantify the contribution of specific ion channels to the APD via drug responses and to generate a population of mathematical models to investigate the mechanisms underlying heterogeneity in rabbit ventricular cells. APD in ∼50 isolated cells from subregions of the LV free wall of rabbit hearts were measured using a voltage-sensitive dye. When stimulated at 2 Hz, average APD₉₀ value in cells from the basal epicardial region was 254 ± 25 ms (mean ± standard deviation) in 17 hearts with a mean interquartile range (IQR) of 53 ± 17 ms. Endo-epicardial and apical-basal APD₉₀ differences accounted for ∼10% of the IQR value. Highly variable changes in APD occurred after IK(r) or ICa(L) block that included a sub-population of cells (HR) with an exaggerated (hyper) response to IK(r) inhibition. A set of 4471 AP models matching the experimental APD₉₀ distribution was generated from a larger population of models created by random variation of the maximum conductances (G_max) of 8 key ion channels/exchangers/pumps. This set reproduced the pattern of cell-specific responses to ICa(L) and IK(r) block, including the HR sub-population. The models exhibited a wide range of G_max values with constrained relationships linking ICa(L) with IK(r), ICl, INCX, and INaK. Modelling the measured range of inter-cell APDs required a larger range of key G_max values indicating that ventricular tissue has considerable inter-cell variation in channel/pump/exchanger activity. AP morphology is retained by relationships linking specific ionic conductances. These interrelationships are necessary for stable repolarization despite large inter-cell variation of individual conductances and this explains the variable sensitivity to ion channel block.

Long QT syndrome (LQTS) carries a risk of life-threatening polymorphic ventricular tachycardia (Torsades de Pointes, TdP) and is a major cause of premature sudden cardiac death. TdP is induced by R-on-T premature ventricular complexes (PVCs), thought to be generated by cellular early-afterdepolarisations (EADs). However, EADs in tissue require cellular synchronisation, and their role in TdP induction remains unclear. We aimed to determine the mechanism of TdP induction in rabbit hearts with acquired LQTS (aLQTS). Optical mapping of action potentials (APs) and intracellular Ca²⁺ was performed in Langendorff-perfused rabbit hearts (n = 17). TdP induced by R-on-T PVCs was observed during aLQTS (50% K⁺/Mg⁺⁺ & E4031) conditions in all hearts (P < 0.0001 vs. control). Islands of AP prolongation bounded by steep voltage gradients (VGs) were consistently observed before arrhythmia and peak VGs were more closely related to the PVC upstroke than EADs, both temporally (7 ± 5 ms vs. 44 ± 27 ms, P < 0.0001) and spatially (1.0 ± 0.7 vs. 3.6 ± 0.9 mm, P < 0.0001). PVCs were initiated at estimated voltages of ∼ −40 mV and had upstroke dF/dt_max and V_m-Ca²⁺ dynamics compatible with I_CaL activation. Computational simulations demonstrated that PVCs could arise directly from VGs, through electrotonic triggering of I_CaL. In experiments and the model, sub-maximal L-type Ca²⁺ channel (LTCC) block (200 nM nifedipine and 90% gCaL, respectively) abolished both PVCs and TdP in the continued presence of aLQTS. These data demonstrate that I_CaL activation at sites displaying steep VGs generates the PVCs which induce TdP, providing a mechanism and rationale for LTCC blockers as a novel therapeutic approach in LQTS. (i) Pseudo-ECG recorded from a Langendorff-perfused rabbit heart under aLQTS conditions during pacing at 2000ms showing induction of TdP by spontaneous PVCs. (ii) Contour maps showing APD90 for a paced beat and AT of the subsequent PVC with contiguous single-pixel AP traces taken from dashed line plotted with estVm and expanded to show the temporal relationship between the peak of the EAD (blue dashed line), the peak VG (red dashed line) and the earliest upstroke of the PVC (dark grey dashed line). (iii) Simulations of the border of a prolonged AP island, showing the initiation of a PVC at the border of the long APD region with propagation into the normal APD region with ICaL and INa traces sampled at 1mm spacing from the border. (iv) Superimposed AP traces showing abolition of PVCs with nifedipine in the presence of EADs and APD prolongation, with dose-response for anti-arrhythmic effect of nifedipine and recapitulation with reduction of gCaL in simulations.

Recent high-throughput experiments unveil substantial electrophysiological diversity among uncoupled healthy myocytes under identical conditions. To quantify inter-cell variability, the values of a subset of the parameters in a well-regarded mathematical model of the action potential of rabbit ventricular myocytes are estimated from fluorescence voltage measurements of a large number of cells. Statistical inference yields a population of nearly 1200 cell-specific model variants that, on a population-level replicate experimentally measured biomarker ranges and distributions, and in contrast to earlier studies, also match experimental biomarker values on a cell-by-cell basis. This model population may be regarded as a random sample from the phenotype of healthy rabbit ventricular myocytes. Univariate and bivariate joint marginal distributions of the estimated parameters are presented, and the parameter dependencies of several commonly used electrophysiological biomarkers are determined. Parameter values are weakly correlated, while summary metrics such as the action potential duration are not strongly dependent on any single electrophysiological characteristic of the myocyte. Our results demonstrate the feasibility of accurately and efficiently fitting entire action potential waveforms at scale.

Aims: Electrical integration of human-induced pluripotent stem-cell-derived cardiomyocyte (hiPSC-CM)-based tissue with the host myocardium is a requirement of successful regeneration therapy. This study was designed to identify electrical coupling in the acute phase (1–2 h) post-grafting using an ex vivo model. Methods and results: Small, engineered heart tissues (mini-EHTs), consisting of ∼50 000 hiPSC-CMs on a hydrogel (spontaneous rate 0.34 ± 0.05 Hz), were loaded with Cal520-AM. EHTs were implanted sub-epicardially into a Langendorff-perfused rabbit heart after blebbistatin treatment. For up to 100 min, a continuous pseudo-electrocardiogram was recorded during sinus rhythm (rate 2.0–3.5 Hz). At 25 min intervals, EHT calcium transients (CaTs) were recorded for 10–20 s (no contraction group). To study the influence of mechanical activity, blebbistatin was washed off after implantation (contraction recovery group). Periodic entrainment of EHTs with the myocardium was detected less often (P = 0.011) in the no contraction group (1/9 hearts) than in the contraction recovery group (5/6 hearts). The average coupling delay (QRS-CaT) and the difference in consecutive delays (Δdelay) were 89 ± 50 and 10 ± 3 ms, respectively (n = 12 traces; N = 6 hearts). Coupling ratios (QRS:CaT) varied from 2:1 to 4:1. These coupling parameters were not significantly different in the two experimental groups. Modelling of hiPSC-CM tissue separated by a 25 μm saline gap from the myocardium demonstrated field-effect coupling with similarly variable activation delays. Importantly, coupling failed with a gap of 100 μm. Conclusion: EHT entrainment is possible immediately after grafting and has features compatible with field-effect coupling. Sensitivity to the gap dimensions may explain why entrainment is more common in actively contracting myocardium.