Project Summary:The cardiac thin filament is the essential regulator of cardiac contractility and relaxation at the molecular level.It is comprised of five discrete proteins: cTnC cTnI cTnT actin and tropomyosin that have co-evolved tosustain efficient cardiac performance at rest during exercise and importantly to respond to pathologicstressors. Mutations in genes encoding each of these proteins have been definitively linked to the developmentof a range of human genetic cardiomyopathies including hypertrophic (HCM) and dilated (DCM) forms.Despite 25 years of study by many groups including ours to define the direct link(s) between the biophysicalinsult and the resultant complex cardiomyopathy many questions remain and significantly limit our ability touse genotype to prognosticate and eventually even treat individuals with genetic cardiomyopathies. The recentdevelopment of Mavacamten a first-in-class targeted myosin inhibitor is a game-changing advance that waspredicated on decades of basic research into the fundamental biology of the sarcomere. Thus the question isno longer if we can target the sarcomere but for the thin filament the question is what function to target andeventually when to treat. The cardiac thin filament is a highly dynamic allosteric machine where most of thecomponent proteins are comprised of a-helices connected by variably sized unstructured linkers wheredynamic flexibility is the rule not the exception and this has limited the availability of high resolution structurefor these regions. Most of the known pathogenic mutations in cTnI and cTnT are clustered within these highlyflexible domains where there is likely a distribution of tolerance whereby mutations impair function (enoughto cause disease) but do not break it. We thus propose that by examining the range of these dynamicperturbations within these domains we can identify new structural and dynamic disease mechanisms that canbe functionally binned studied and modulated. We provide proof-of-principle preliminary data in this proposal.Over the recent funding period we expanded our structural methodologies to include Time-Resolved FRETwith a Single Donor Dual Acceptor approach that allows us to use actin as an anchor to refine highly flexiblestructures. We will next use known highly divergent (HCM vs DCM) mutations within each flexible domain toprobe both structure and dynamics with the premise that these mutations will define the limits of tolerability ineither direction and use spectroscopy and measurements of Ca2+ dissociation and association kinetics coupledto computation to define and test these hypotheses. Finally we will close the loop by utilizing our existingextensively characterized transgenic animal models based on the same mutations used to set our limits andperform 3-timepoint RNA-Seq to discover unique early transcriptional signatures to help link theseperturbations to the resultant early remodeling cascade that eventually leads to distinct patterns of ventricularremodeling. The long term goal is to use this coupled structural dynamic transcriptomic platform to identifynew targets both primary and secondary for future therapeutics and even biomarker discovery.