Abstract P1096: Computational Motion Artifact Removal Acts As Virtual Blebbistatin In Ipsc-derived Cardiomyocytes And Micro-tissues

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Introduction: Drugs like blebbistatin that paralyze tissues are critical for accurate optocardiography but directly affect sarcomeres, making it challenging to study structure-function relationships. To overcome this, we applied Direct Deformation Estimation (DDE), a robust strain estimation tool, to enable computational motion removal from videos of beating monolayers and engineered heart tissues formed from human Induced Pluripotent Stem Cell derived cardiomyocytes (hiPSC-CM). Methods: hiPSC-CM and miniaturized engineered heart muscles were stained with the far-red voltage sensitive dye, BeRST-1. Videos of overall motion (bright-field), Ca 2+ (endogenous GCamP6f reporter) and voltage were captured at 100 Hz. Temporal median filtering and Contrast-Limited Adaptive Histogram Equalization were applied as pre-processing steps to improve convergence. The inverse of conformal mappings estimated from DDE were used to “unwarp” videos to artificially reduce motion. Removal of motion was assessed in bright-field videos by performing optical flow analysis. Improvements in optical mapping accuracy were estimated by calculating the spatial distribution of APD 80 (voltage) and time to 75% decay from peak (τ 75 ; Ca 2+ ). Results: Virtual blebbistatin removed >90% of motion from monolayers and engineered tissues without affecting sarcomere architecture (sarcomeric α-Actinin staining), in contrast to chemical blebbistatin. Virtual blebbistatin further reduced spatial dispersion in both APD 80 and τ 75, leading to overall distributions that were less skewed by artificially low repolarization times. Interestingly, when directly assessing Ca 2+ -contraction coupling in plots of motion-corrected Ca 2+ vs. contraction magnitude, we observed that engineered tissues had more uniform and consistent coupling compared to monolayers. Conclusions: Superior uniformity in calcium-contraction coupling of engineered tissues as opposed to monolayers is consistent with observations that engineered tissues are more likely to recapitulate genotype-phenotype relationships and drug responsiveness. Virtual blebbistatin is a promising tool for non-invasive analysis of Ca 2+ -contraction coupling and robust optocardiography.

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