Discovery of small molecule regulators of atrial cardiomyocyte action potential duration to restore normal cardiac rhythm in atrial fibrillation

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PROJECT SUMMARY Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia, afflicting over 33 million people worldwide and 6 million in the US. AF causes reduced quality of life, stroke and systemic thromboembolism, heart failure, and increased mortality. Treatment of AF and its complications with nonspecific drugs or procedures is characterized by unsatisfactory outcomes and significant cost. Acquired heart disease, cardiac remodeling, neurohormonal factors, aging, and genetic traits have all been correlated with presence of AF. Rapid, uncoordinated atrial chamber activity is due to shortened or prolonged cardiomyocyte action potential durations acting within a vulnerable myocardial substrate, causing persistent arrhythmia that features triggering or sustaining circuit re- entry or early and/or delayed after-depolarizations, respectively. We have recently developed a novel, high throughput kinetic imaging and analysis platform to characterize cardiomyocyte electrophysiological properties at single cell resolution, which can be used to conduct high throughput screening (HTS) on functional human atrial cardiomyocytes derived from Id1-programmed cardiac progenitors created from iPS cells. Our innovation is the use of this and related assays in a phenotypic screening cascade designed to discover previously unknown, atrial-specific modulators of cardiomyocyte electrical properties and rhythm. Our hypothesis is this approach will ultimately generate drug-like starting points for future disease-modifying cardiovascular therapeutics. The primary HTS assay has been fully optimized in a 384-well format, and as a demonstration of assay readiness, 400 compounds have been screened (Kolmogorov-Smirnov D-statistic >0.1). Multiple hits from pilot screens were identified and were confirmed and validated in concentration response experiments. A battery of downstream assays has been developed and piloted to establish a critical path-testing funnel. Several compounds identified from the pilot screen were tested to determine if they affected the action potential duration of atrial cardiomyocytes sensitized by the E375X mutation in KCNA5, and if they had effects on the action potential durations of wild type and primary atrial and ventricular cardiomyocytes. This proposal builds on data from the applicants, an established team from SBP (Drs. Colas and Larson) with basic biology and drug discovery expertise in the field and access to all necessary technologies. The overall goal of this proposal is to generate chemical biology research tools and starting points for new drugs. As the critical path assays are all in place, we anticipate we can rapidly obtain such probe molecules and start to explore their activity. Our future plans are to ultimately determine hits’ suitability for hit-to-lead activities, begin in vivo evaluation of lead compounds in animal models and eventually patients, and determine their cellular mechanism of action. This grant’s work product will serve as preliminary data for hit-to-lead (HTL) grant submissions and parent R01 grant submissions to pursue understanding of their biological mechanisms.