Voltage Imaging of Cardiac Cells and Tissue Using the Genetically Encoded Voltage Sensor Archon1

Figures

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  Figure 1. Demonstration of Archon1 for Monitoring Action Potentials of iPSC-Derived Cardiomyocytes (A) Diagrams of Cre-recombinase AAV system used to infect CMs with Archon1. KGC, linker; ER2, ER export trafficking sequence. (B) Single-frame images of CM expressing Archon1 at different phases of an AP as shown in (C). Scale bar, 50 μm. See Video S1. (C) Left, optically recorded raw voltage trace for cell shown in (B). Right, quantification of APD90, as defined in (D), per voltage trace for unpaced CMs (n = 31 cells). (D) An example AP waveform with APD90, APD50, and rise time (trise) defined. (E) Representative normalized fluorescence voltage traces from CM electrically paced at 0.5 Hz (blue), 1 Hz (orange), and 2 Hz (yellow). (F) Corresponding APD90 box plot for each pacing shown in E (from n = 15, 12, 9, cells, respectively). Box plots represent 25th to 75th percentiles, with whiskers extending 1.5x the interquartile range; horizontal line represents median; each dot represents data from a single cell. All optical traces obtained at image acquisition rate of 100 Hz.

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  Figure 2. Characterization of Archon1 in iPSC-derived Cardiomyocyte Monolayers (A) Representative overlay of single AP waveform simultaneously measured using patch-clamp electrophysiology (blue) and Archon1 fluorescence (orange). (B) Normalized-intensity Archon1 fluorescence trace versus membrane potential recorded via electrophysiology for cell simultaneously recorded in (A). Trendline equation and R2 value shown on chart. (C) Normalized intensity Archon1 fluorescence traces versus normalized membrane potential from all simultaneously recorded cells (n = 4). Trendline equation and R2 value shown on chart. (D) Quantification of rise time, as defined in Figure 1D, per cell for patch-clamp electrophysiology (Ephys; acquired at 10–20kHz), down-sampled electrophysiology (Ephys-DS; same electrophysiology data down-sampled to 100 Hz to match the acquisition rate of Archon1), and Archon1 (Archon1; acquired at 100 Hz) from n = 17, 17, and 67, cells, respectively. ∗: p < 0.05 in two-tailed Student’s t test, ns: p > 0.05 in two-tailed Student’s t test. (E) Representative overlay of single AP waveform measured with Di-8-ANEPPS (blue) versus Archon1 (orange). (F) APD50 values per cell for Archon1…

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  Figure 3. Archon1 Can Reliably Detect Changes in AP Waveforms Induced by K+ and Ca2+ Ion Channel Inhibitors E-4031 (K+ Channel Blocker) and Nifedipine (Nif; Ca2+ Channel Blocker) compared with DMSO control; electrically paced at 1 Hz. (A–C) (A) Average normalized Archon1 fluorescence intensity traces for DMSO control (n = 10 cells), (B) E-4031 (3, 10, 30 nM; n = 10 cells), and (C) Nifedipine (10, 30, 100 nM; n = 10 cells) before (blue) and after (red) drugs, SD (dotted lines). (D and E) (D) %ΔAPD90 for E-4031 APs from pre-drug condition. (E) %ΔAPD50 for Nifedipine APs from pre-drug condition where a, b, c, and d represent different statistical groups (all pairs Tukey HSD test). For full statistics see Table S2. Box plots displayed as described in Figure 1. All optical traces obtained at image acquisition rate of 100 Hz.

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  Figure 4. Archon1 Enables Monitoring of APs in μTUG hMSC-Cardiomyocyte Tissues (A) Brightfield image of μTUG fibroblast-CM tissues (left). Scale bar, 250 μm. See Video S5. Maximum-minus-minimum projection image of CM within μTUG tissue expressing Archon1 (top right), with corresponding ROI overlaid (bottom right). Scale bar, 20 μm. (B) Optically recorded voltage trace for the cell shown in (A). Image acquisition rate, 50 Hz. (C) Quantification of APD90 for CMs within tissues (n = 5, 7, 4, cells for Tissue 1 (red), 2 (blue), 3 (yellow), respectively). (D) Quantification of SNR for CMs within tissues (n = 5, 7, 4, cells for Tissue 1 (red), 2 (blue), 3 (yellow), respectively). (E) Quantification of rise time for CMs within tissues (n = 5, 7, 4, cells for Tissue 1 (red), 2 (blue), 3 (yellow), respectively). Box plots displayed as described in Figure 1; image acquisition rate, 50 Hz. (F) Max projection image of μTUG tissue (top), with cells expressing Archon1 identified in yellow (bottom). Scale bar, 50 μm. (G) Corresponding optically recorded voltage traces for cells identified in (F), with gray dashed vertical lines demonstrating AP synchrony. Image acquisition rate, 100 Hz. (H) Overlay of…