Abstract Body: Introduction: Mutations in the myofilament protein TNNI3 can perturb cardiomyocyte function leading to hypertrophic, restrictive, or dilated cardiomyopathy. Though multiple pathogenic mutations have been identified, the precise molecular and physiological mechanisms leading to these different pathologies, as well as different severities of disease, are not fully understood. Further, the significance of more than 200 of the TNNI3 variants identified in patients remains uncertain, as their effect on cardiomyocyte function remains unclear.

Methods: We have developed a high-throughput functional assessment platform that simultaneously measures cardiomyocyte contractility and calcium handling to interrogate the physiological effect of TNNI3 variants on cardiomyocyte function. We use this platform to analyze the effect of a library of TNNI3 variants and compare these in vitro measurements to clinical data from Stanford patients carrying these mutations.

Results: Our platform was able to segregate pathogenic from benign and synonymous variants with high accuracy using the measured relationship between calcium handling and contractility within the cardiomyocytes. Unbiased clustering of variants by multiple functional variables identified putative subgroups of disease arising from different patterns of disrupted cardiomyocyte physiology. Additionally, by linking in vitro functional measurements with patient data, we show that the measured diastolic function of variants predicts both the age of onset and diastolic pressures from cardiac catheterization in patients with HCM or RCM carrying those mutations. Lastly, we use this platform to provide evidence of pathogenicity for two different variants of uncertain significance identified in patients at Stanford.

Conclusion: Overall, our functional measurement platform provides a powerful new approach for not only distinguishing between disease-causing and benign variants, but also for predicting the severity of the clinical presentation caused by TNNI3 variants based on the measured cardiomyocyte function