Abstract Body: Familial dilated cardiomyopathy (DCM) is a genetic heart disorder characterized by enlargement of one or both ventricles, rendering the heart unable to pump blood efficiently. Mutations in cardiac thin filament (CTF) proteins have been associated with characterized clinical manifestations of DCM. Genetic segregation identified a mutation in α-Tropomyosin (Tm) at residue 230 (D230N-Tm) in two unrelated, multigenerational families linked to severe, early onset DCM. To meaningfully link genotype to phenotype our group sought to investigate the effects of D230N-Tm on the biophysical structure of Tm filament. We showed that D230N-Tm caused a compaction of the cardiac Troponin T (cTnT)–Tm overlap region accompanied by a decreased in flexibility of the Tm filament. Given the altered biophysical structure we investigated whether we could target this decrease in flexibility via small molecule intervention and improve function. Computational modeling and biophysical experiments identified small molecule 4-Hydroxy Duloxetine β-D-Glucuronide (Z06) shown to improve compaction of the cTnT-Tm overlap and restore flexibility of the Tm filament. We hypothesized that the decrease in flexibility of Tm impairs its ability to shift through its different conformational states, leading to impaired crossbridge formation. To investigate whether Z06 improves myofilament function, we performed an NADH-coupled ATPase assay using isolated myofibrils from our D230N-Tm transgenic mice and compared them to Non-Transgenic (NTg) littermates. ATPase activity was measured at saturating calcium (100uM) in the presence (250uM) and absence (0uM) of Z06. In the absence of Z06, D230N-Tm exhibited a decrease in ATPase activity (1.793±0.203 umol/min) compared to NTg littermates (5.420±0.562 umol/min), a common trend in DCM models. In the presence of 250uM Z06, D230N-Tm ATPase rates improve to levels comparable to NTg mice (4.816±0.544 umol/min). Moving forward, we will perform calcium titrations, allowing us to investigate if Z06 can restore cooperativity of myofilament activation. Additionally, we will test if the restorative effects of Z06 are present at the myocellular level, and if so, we will optimize delivery to test in vivo effects of Z06 in our D230N-Tm mouse model.