Abstract Body: Skeletal muscle actin (ACTA1) is robustly expressed in skeletal muscle and expressed at lower levels in the heart. While ACTA1 mutations are known to cause skeletal myopathies, several case reports indicate that some ACTA1 mutations may also cause cardiomyopathy. We previously reported that R256H ACTA1 is associated with dilated cardiomyopathy without apparent skeletal myopathy. To establish a causative link between R256H ACTA1 and cardiomyopathy, we created an Acta1^R256H/+ knockin mouse. These mice have no baseline cardiac defects consistent with the lack of Acta1 expression in healthy murine hearts. Intriguingly, Acta1 expression increases following cardiac injury and stress. Following transaortic constriction Acta1^R256H/+ knockin mice display a heart failure phenotype. We analyze calcium-dependent force production of single skinned cardiomyocytes and found that despite the lack of baseline cardiac phenotype in Acta1^R256H/+ mice, skinned isolated cardiomyocytes displayed decreased force production. Decreased force production was also observed in ACTA1^R256H/+ human iPSC-derived cardiomyocytes. To dissect how ACTA1 mutations might cause contractile defects, we purified recombinant R256H ACTA1 and observed decreased stability (thermal shift assay) and polymerization (fluorescent actin polymerization) compared to WT ACTA1 protein. Importantly, we found that R256H ACTA1 acts in a dominant fashion where addition of a small amount of mutant protein to WT ACTA1 is sufficient to disrupt contractility (in vitro motility). To understand the structural changes responsible for this phenomenon, we resolved the structure of the R256H ACTA1 filament by cryo-EM. We find a disrupted interaction between ACTA1 K240 and tropomyosin E117, a residue important for maintaining tropomyosin in the open state to allow for molecular contractility. Collectively, we show that an ACTA1 mutation is capable of causing dilated cardiomyopathy, which may be related to multiple defects in actin function. Future studies will be required to decipher whether other ACTA1 mutations reduce contractility through similar mechanisms.