ABSTRACT Cardiomyopathies, including hypertrophic (HCM) and dilated (DCM) cardiomyopathy, are conditions in which heart muscle dysfunction may lead to arrhythmias and heart failure. Cardiomyopathies are most commonly caused by variants in sarcomere genes that encode contractile proteins. The immediate effect of these genetic variants is perturbation of contractile function. However, a clear understanding of how the thousands of different variants in individual sarcomere genes differentially affect contractile function to cause HCM and DCM has not been attained. Furthermore, traditional systems have not been able to efficiently study the interaction between genetic variants affecting contractile function and varying levels of biomechanical workload that models the in vivo state. Cardiomyocytes differentiated from induced pluripotent stem cells (iPSC-CMs) are a promising model system that allow the study of HCM- and DCM-causing mutations in a human cell context, but the capacity of this model system for contractile analysis has been limited because of technical and biologic hurdles. My preliminary data shows that an optimized bioengineered platform enables generation of contracting micrometer- scale 2-dimensional heart muscle tissues (referred to as M2D) on an elastomer substrate. M2D tissues exhibit coordinated, uniaxial contraction, robust myofibrillar alignment, and expected responses to contractile agonists/antagonists. In addition, my preliminary data shows that the M2D tissues are amenable to modified RNA transfection, enabling >90% mutant replacement of contractile proteins. I hypothesize that the M2D technology will enable mechanistic determination of dysregulated contractile velocity and workload relationships in cardiomyopathy patient iPSCMs compared to controls, and, moreover, that these analyses will enable subclassification of contractile defects due to thick vs. thin filament mutations that will predict responses to pharmacologic modulation of contractile function. The first aim tests the capacity of the M2D system to discriminate contractile dysregulation in patient iPSCM muscle tissues with thick (MYH7, MYBPC3) vs thin (TNNT2) filament sarcomere gene variants in a total of 10 patient iPSC lines, as compared to controls. Modified RNA transfections will be used as additional models since we are able to achieve very high transfection efficiencies in the M2D system. Both myofibrillar alignment and contractile function will be quantified using custom analysis tools. Sensitivity of contractile function to calcium concentration will also be assessed in both patient and control muscle tissues. The second aim will test whether thick vs. thin filament variant iPSCMs have a differential reversal of contractile dysregulation with the myosin inhibitor Myk-461. The implementation of the M2D technology to interrogate contractile function in the presence of sarcomere gene variants will be transformative for precision analysis of patient-specific he...