DOI: 10.5061/dryad.b8gtht7mz
title: Data from: Robotic manipulation of cardiomyocytes to identify gap junction modifiers for arrhythmogenic cardiomyopathy

description: Arrhythmogenic cardiomyopathy (ACM) is a leading cause of sudden cardiac
 death among young adults. Aberrant gap junction remodeling has been linked
 to disease-causative mutations in plakophilin-2 (PKP2). Although gap
 junctions are a key therapeutic target, measurement of gap junction
 function in preclinical disease models is technically challenging. To
 quantify gap junction function with high precision and high consistency,
 we developed a robotic cell manipulation system with visual feedback from
 digital holographic microscopy (DHM) for three dimensional and label-free
 imaging of human induced pluripotent stem cell-derived cardiomyocytes
 (iPSC-CMs). The robotic system can accurately determine the dynamic height
 changes in the cells’ contraction and resting phases, microinject
 drug-treated healthy and diseased iPSC-CMs in their resting phase with a
 constant injection depth across all cells, and deposits a
 membrane-impermeable dye that solely diffuses between cells through gap
 junctions for measuring the gap junction diffusion function. The robotic
 system was applied towards a targeted drug screen to identify
  gap junction modulators and potential therapeutics for ACM. Five
 compounds were found to dose-dependently enhance gap junction permeability
 in cardiomyocytes with PKP2 knockdown. Additionally, PCO 400 (Pinacidil)
 reduced beating irregularity in a mouse model of ACM expressing mutant
 PKP2 (R735X). These results highlight the utility of the robotic cell
 manipulation system to efficiently assess gap junction function in a
 relevant preclinical disease model, thus providing a technique to advance
 drug discovery for ACM and other gap junction-mediated diseases.
description: Please refer to the Materials and Methods section in the
 paper.
description: # Robotic cell manipulation to identify gap junction modifiers in
 cardiomyocytes and potential therapeutics for arrhythmogenic
 cardiomyopathy AUTHOR INFORMATION: Corresponding Investigators Name: Xinyu
 Liu, Yu Sun, Jason T. Maynes Institution: University of Toronto; Hospital
 for Sick Children Email:
 [xyliu@mie.utoronto.ca](mailto:xyliu@mie.utoronto.ca),
 [yu.sun@utoronto.ca](mailto:yu.sun@utoronto.ca),
 [jason.maynes@sickkids.ca](mailto:jason.maynes@sickkids.ca) DATA &
 FILE OVERVIEW: 1. Fig.1D(ii)_cell height from Live staining image .fig ---
 Live staining of iPSC-CM monolayer by calcian (green) and Hoechst (blue)
 to quantify cell height as the benchmark for calculating the mean
 intracellular refractive indexes of iPSC-CMs. 2. Fig.1E cell height phase
 image.xlsx ---Cross-sectional cell height of the same cardiomyocytes
 obtained by DHM phase imaging. 3. Fig. 1F heights of nucleus and cytoplasm
 region.xlsx --- Heights of the nucleus region and the cytoplasm region in
 the resting phase and in the contraction phase when the cell beating
 amplitude reached maximum. 4. Fig. 2B Depth vs. Dye Transfer.xlsx --- Dye
 diffusion through gap junctions is negatively correlated with injection
 depth 2 minutes post-injection; however, cells recover full gap junction
 function by 6 minutes post-injection. 5. Fig. 2C Temp vs. Dye transfer.csv
 --- Quantification of dye transfer in microinjected iPSC-CMs under
 different temperatures. Dye diffusion through gap junctions increased as
 the temperature increased from 22.0°C to 37.5°C, and then decreased as the
 temperature further increased to 39.5°C. 6. Fig. 2D Temp vs. viability.csv
 --- Cell viability also increased from 22.0°C to 37.5°C and then decreased
 at higher temperatures. 7. Fig, 2E Gap junction modifiers.csv --- Dye
 diffusion was measured in iPSC-CMs treated with known enhancers of gap
 junction activity. 8. Fig. 3A (i)_PKP2 KD.csv --- Reduction in
 GAPDH-normalized PKP2 protein levels were observed by Western blot 14 days
 post-transduction, relative to the non-silencing (N/S) control. Protein
 levels were quantified by densitometry. 9. Fig. 3B Relative beat
 amplitude.csv --- Analysis of iPSC-CM beat amplitude (contractility). PKP2
 knockdown was found to decrease beat amplitude compared to iPSC-CMs
 transduced with a N/S control. 10. Fig. 3C Dye transfer (PKP2 KD).csv ---
 Measurement of gap junction permeability in iPSC-CMs 14 days
 post-transduction. Compared to N/S control cells, PKP2 knockdown causes a
 reduction in dye transfer to neighboring cardiomyocytes. 11. Fig. 4
 Relative dye transfer drug screening.csv --- Measurement of gap junction
 permeability in iPSC-CMs 14 days post-transduction. Compared to N/S
 control cells, PKP2 knockdown causes a reduction in dye transfer to
 neighboring cardiomyocytes. 12. Fig. 5A Mean heart rate (Flecainide).csv
 --- Mean heart rate (bpm) for control AAV-PKP2 and mutant AAV-R735X
 transduced anesthetized mice at specific time points after a single dose
 of flecainide (20 mg/kg). 13. Fig. 5B Mean heart rate (PCO 400).csv ---
 Mean heart rate (bpm) for control AAV-PKP2 and mutant AAV-R735X transduced
 anesthetized mice at specific time points after a single dose of PCO 400
 (1 mg/kg). 14. Fig. 5C Beating irregularity (Flecainide).csv --- heart
 rate variation (RMSSD) for control AAV-PKP2 and mutant AAV-R735X
 transduced anesthetized mice at specific time points after a single dose
 of flecainide (20 mg/kg). 15. Fig. 5D Beating irregularity (PCO 400).csv
 --- heart rate variation (RMSSD) for control AAV-PKP2 and mutant AAV-R735X
 transduced anesthetized mice at specific time points after a single dose
 of PCO 400 (1 mg/kg). 16. Fig. 5E QRS duration (Flecainide).csv --- QRS
 duration (ms) or control AAV-PKP2 and mutant AAV-R735X transduced
 anesthetized mice at specific time points after a single dose of
 flecainide (20 mg/kg). 17. Fig. 5F QRS duration (PC0400).csv --- QRS
 duration (ms) or control AAV-PKP2 and mutant AAV-R735X transduced
 anesthetized mice at specific time points after a single dose of PCO 400
 (1 mg/kg). 18. Fig. S5. Error of injection depth. 19. Fig. S6 time
 dependent dye spreading.xlsx --- Quantification of dye transfer in
 microinjected iPSC-CMs with increase of time. The fluorescence images were
 captured every 30 s for 8 min after the microinjection of each cell. The
 number of neighboring cells with nuclei exhibiting a positive fluorescent
 signal continuously increased throughout the 8 min period. 20. Fig. S9
 Relative beat rate.csv --- PKP2 knockdown was found to cause a
 time-dependent increase in the spontaneous beat rate of iPSC-CMs, relative
 to cardiomyocytes transduced with a non-silencing control. 21. Fig. S10
 Flecainide.csv --- Dose-dependent enhancement of gap junction function by
 Flecainide, (Effective concentration) EC50=0.01 µM. 22. Fig. S10 PCO
 400.csv --- Dose-dependent enhancement of gap junction function by PCO
 400, EC50=0.04 µM. 23. Fig. S10 PQ15.csV --- Dose-dependent enhancement of
 gap junction function by PQ15, EC50=0.22 µM. 24. Fig. S10 Quinine.csv ---
 Dose-dependent enhancement of gap junction function by Quinine, EC50=1 nM.
 25. Fig. S10 Tolazamide.csv --- Dose-dependent enhancement of gap junction
 function by Tolazamide, EC50=0.03 µM.