Rapid ionic current phenotyping (RICP) identifies mechanistic underpinnings of iPSC-CM AP heterogeneity

Figure 1:. Current clamp recordings illustrate baseline heterogeneity. Spontaneously beating (n=27) and quiescent depolarized cells (n=12) from current clamp recordings of iPSC-CMs. If reported as mean± SEM, the variation in MP (−52± 1.6 mV), APD90 (132± 14 ms), and dV/dtmax (10.4± 1.2 V/s) appears quite small.

Figure 2:. Variations in AP features from 24 independent datasets. The numbered studies on the right are independent datasets, named according to the first author (see references for full bibliographic information), and the numbers correspond to their position on the x-axis. Studies labeled with (N), (A), or (V) used AP features to sort cells into nodal, atrial, or ventricular groupings. The datasets from Lee et al. (2017) used differentiation protocols to induce either atrial-like (AI) or ventricular-like (VI) phenotypes. The datasets in this figure, including our own (position 5), are sorted in order from most to least depolarized MP.

Figure 3:. VC variations and membrane potential in 39 cells. A, The optimized voltage clamp protocol (top), with lines overlaid that indicate time points designed to isolate each current. Responses to the voltage clamp protocol (bottom) for spontaneous (black), quiescent (blue), and the outlier (pink). B, APs from spontaneously beating cells (n=27). The pink AP is an outlier with an APD90 >2x longer than the rest of the population. C, Voltage recordings from quiescent and depolarized cells (n=12). The voltage recordings in B and C are the same as those shown in Figure 1 but are here separated for clarity.

Figure 4:. Iout distribution at the eight current-isolating time points. Histograms display the distribution of recorded current values at each of the eight time points. The pink dashed line shows Iout values for the outlier cell.

Figure 5:. IcaL helps drive upstroke in depolarized iPSC-CMs. A, There is no correlation between Iout from the eight current-isolating segments and dV/dtmax. B, dV/dtmax decreases as MP depolarizes. The red boxes denote the two cells with the most hyperpolarized MP and largest dV/dtmax. C, The two highlighted cells from panel B share few AP commonalities with one another other than their relatively hyperpolarized MP and large dV/dtmax. D, A trend emerges between Iout during the IcaL-isolating segment and dV/dtmax when the two hyperpolarized cells are removed from the analysis.

Figure 6:. Correlations between ionic current segments and MP. A, Iout from the IKr, Ito, IK1, If, and IKs segments correlate significantly (p<.05) with MP. B, Current clamp recordings from all cells. The iPSC-CMs with the most hyperpolarized (blue, Cell 1) and depolarized (green, Cell 2) MP values are compared. C, Traces from the IKr-isolating segment of the VC protocol for all cells. The VC protocol (top) includes a 750 ms prestep at 6 mV, followed by a 7 ms step at −41 mV, and then a step to 9 mV. The VC protocol was designed to isolate IKr at 1262 ms (red dashed line). Cell 1 (blue) and Cell 2 (green) are highlighted.

Figure 7:. AP simulations of a Kernik-Clancy model with a 2 GΩ seal resistance and varying levels of gKr A, APs generated by running the model with varying levels of gKr from 0.1 to 1.6 times the baseline Kernik-Clancy value (0.218025 nS/pF). The arrow indicates that depolarized cells have less IKr (i.e., smaller gKr). B, Relationship between gKr and MP for cells plotted in A. The yellow highlighted region represents the 10% to 90% range of MP values from cells in this study.

Figure 8:. A population of Paci models compared to experimental data. 500 Paci individuals were generated by varying ionic current conductances and experimental artifact parameters (see Methods). The dashed orange line shows the average Iout for all models, and the shaded region is the range of values for all models. The experimental data are displayed in gray.

Figure 9:. Ito, IK1, If, and IKs-isolating segments are sensitive to seal-leak current. Correlations calculated between model parameter values (primarily conductances; e.g., gKr) and Iout during each of these four segments. Each current is sensitive to its own conductance, with outward currents (Ito and IKs) having a positive correlation and inward currents (IK1 and If) showing as a negative correlation to their own conductance. Each current is also sensitive to gleak. Both Ito and IKs have a surprisingly strong sensitivity to gNaCa.

Figure 10:. Correlations between ionic currents and APD90 A, Iout from the I6mV, IK1, If, and IKs segments correlate significantly (p<.05) with APD90. B, Cells that generated the shortest (Short) and second longest (Long) APs were investigated. C-D,Current responses from cells during I6mV (C) and IKs (D) step.

Figure 11:. Comparison of Paci and experimental I6mV. Experimental (gray) and range of Paci model traces (shaded orange) during I6mV voltage step.