Abstract Body: The cardiac thin filament (CTF) is a crucial regulator of contraction and relaxation. It couples the availability of cytoplasmic calcium to mechanical force production. Mutations in CTF proteins have been strongly associated with symptomatic cardiomyopathies. In 2014, a mutation identified at residue 219 of tropomyosin (D219N-Tm) was linked to the development of hypertrophic cardiomyopathy (HCM). Due to the proximity of this mutation to the cardiac troponin T-Tm (cTnT-Tm) overlap region, we investigated the structural effects of D219N-Tm via Time-Resolved Resonance Energy Transfer (TR-FRET). The TR-FRET revealed a compaction at the overlap region in D219N-Tm CTF’s under saturated calcium conditions. To understand how these structural changes affected downstream cardiac function, we generated a D219N-Tm transgenic mouse model. 2-month-old D219N-Tm mice exhibited an increased percent fractional shortening (38.5%) compared to their non-transgenic (NTg) littermates (28.6%). These alterations in systolic function were coupled to changes in cardiac morphology. 2-month-old D219N-Tm hearts showed significantly elevated atrial-weight to heart-weight ratios (0.1504±0.006656) compared to NTg (0.1108±0.01827), suggesting that greater left-ventricular filling pressures may lead to atrial hypertrophy. To assess changes in myofilament activation, we conducted an NADH-coupled ATPase assay with transgenic myofibrils and NTg littermates at varying calcium concentrations. ATPase rates and calcium sensitivity (pCa₅₀) were greater in D219N-Tm myofibrils compared to NTg littermates. We then investigated whether these structural changes affected cellular contractility. Contractility measurements showed reductions in departure velocity time, return velocity time, and time to peak, indicating faster rates of contraction and relaxation in D219N-Tm cardiomyocytes. D219N-Tm cardiomyocytes also exhibited shorter baseline sarcomere lengths compared to NTg controls, which may suggest the presence of residual cross-bridges after diastole. Future studies would treat NTg and D219N-Tm cardiomyocytes with 2,3-butanedione monoxime to deduce whether observed changes in baseline sarcomere length are due to differences in diastolic tension.