Abstract Body (Do not enter title and authors here): Background: Genome-wide association studies have identified over 100 genetic loci associated with dilated cardiomyopathy (DCM). However, functional validation of hundreds of potential candidate genes remains challenging. Contractile dysfunction is a hallmark in DCM that can be modeled and studied in 3D human cardiac microtissues. Here we developed Contrack, a high-throughput contractility assay that allows systematic evaluation of effects of chemical or gene perturbations on cardiac contractions using a 3D human cardioid model in a scalable manner.
Methods: 3D cardioids were generated from self-aggregating hPSC-derived ventricular cardiomyocytes and fibroblasts in 96-well plates, which were electrically paced and recorded at 37C. Videos were analyzed using a custom, automated analysis pipeline that produces displacement and velocity curves, from which the amplitude and rate of contraction and relaxation can be derived and used as proxy readouts for cardioid contractility measurements.
Results: Contrack recapitulates contraction responses to changes in fibroblast ratio, tissue size, pacing rate, and beta-adrenergic stimulation. We performed a pilot screen of 26 cardiovascular drugs including beta blockers, calcium channel blockers, and ACE inhibitors across 384 cardioids. 211 videos of contracting cardioids were acquired and analyzed using Contrack. Reduced cardioid contractility was found with treatments of doxorubicin and verapamil. Interestingly, we found isoxsuprine significantly increased cardioid contractility. Immunostaining isoxsuprine-treated cardiomyocytes showed improved sarcomere content and organization. We also performed a CRISPR-based knockout of genes previously identified to improve cardiac function by the transfer learning model, GENEFORMER. We found ESRRG knockout significantly improved cardioid contractility in a TTN truncating variant model of DCM.
Conclusions: Contrack allows for streamlined analysis that meets the increasing demand for cost-efficient, large-scale functional validation in the study of cardiovascular disease. It provides a powerful translational platform for investigation of disease mechanisms and drug discovery relevant to heart failure and cardiomyopathy.