Data from: Robotic manipulation of cardiomyocytes to identify gap junction modifiers for arrhythmogenic cardiomyopathy
Data files
Oct 17, 2024 version files 2.61 MB
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Fig_S5._Error_of_injection_depth.xlsx
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Fig._1D(ii)_cell_height_from_Live_staining_image_.fig
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Fig._1E_cell_height_phase_image.xlsx
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Fig._1F_heights_of_nucleus_and_cytoplasm_region.xlsx
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Fig._2B_Depth_vs._Dye_Transfer.xlsx
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Fig._2C_Temp_vs._Dye_transfer.csv
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Fig._2D_Temp_vs._viability.csv
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Fig._2E_known_gap_junction_modifiers.csv
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Fig._3A_(ii)__PKP2_KD.csv
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Fig._3B_Relative_beat_amplitude.csv
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Fig._3C_Dye_transfer_(PKP2_KD).csv
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Fig._4_Relative_dye_transfer_drug_screening.csv
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Fig._5A_Mean_heart_rate_(Flecainide).csv
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Fig._5B_Mean_heart_rate_(PCO_400).csv
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Fig._5C_Beating_irregularity_(Flecainide).csv
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Fig._5D_Beating_irregularity_(PCO_400).csv
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Fig._5E_QRS_duration_(Flecainide).csv
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Fig._5F_QRS_duration_(PCO400).csv
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Fig._S10_Flecainide.csv
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Fig._S10_PCO_400.csv
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Fig._S10_PQ15.csv
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Fig._S10_Quinine.csv
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Fig._S10_Tolazamide.csv
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Fig._S6_time_dependent_dye_spreading.xlsx
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Fig._S9_Relative_beat_rate.csv
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README.md
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Abstract
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.
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, yu.sun@utoronto.ca, jason.maynes@sickkids.ca
DATA & FILE OVERVIEW:
- 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.
- Fig.1E cell height phase image.xlsx —Cross-sectional cell height of the same cardiomyocytes obtained by DHM phase imaging.
- 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.
- 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.
- 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.
- Fig. 2D Temp vs. viability.csv — Cell viability also increased from 22.0°C to 37.5°C and then decreased at higher temperatures.
- Fig, 2E Gap junction modifiers.csv — Dye diffusion was measured in iPSC-CMs treated with known enhancers of gap junction activity.
- 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.
- 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.
- 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.
- 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.
- 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).
- 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).
- 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).
- 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).
- 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).
- 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).
- Fig. S5. Error of injection depth.
- 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.
- 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.
- Fig. S10 Flecainide.csv — Dose-dependent enhancement of gap junction function by Flecainide, (Effective concentration) EC50=0.01 µM.
- Fig. S10 PCO 400.csv — Dose-dependent enhancement of gap junction function by PCO 400, EC50=0.04 µM.
- Fig. S10 PQ15.csV — Dose-dependent enhancement of gap junction function by PQ15, EC50=0.22 µM.
- Fig. S10 Quinine.csv — Dose-dependent enhancement of gap junction function by Quinine, EC50=1 nM.
- Fig. S10 Tolazamide.csv — Dose-dependent enhancement of gap junction function by Tolazamide, EC50=0.03 µM.
Please refer to the Materials and Methods section in the paper.