Abstract Body: Tropomyosin (Tpm) extends along the F-actin surface of cardiac thin filaments. Its azimuthal location regulates myosin accessibility and force production. Highly-favorable, interfacial electrostatic contacts between K326 and K328 of actin and Tpm promote binding and confine Tpm to an energetically stable position, where it inherently blocks myosin attachment sites. K326 and K328 of vertebrate cardiac actin (ACTC) are acetylatable, which neutralizes lysine’s positive charge. We tested the hypothesis that replacing K326 and K328 with non-polar glutamines, to mimic acetylation, would enhance cardiac contractility in vivo, and reduce Tpm’s innate ability to hinder actomyosin activity in vitro. We first overexpressed Drosophila wildtype (WT) or K326Q/K328Q acetyl-mimetic (AcM) actin in the technically amenable fruit fly heart. Relative to WT, AcM actin prolonged systole under baseline conditions (WT 161±0.01 ms; AcM 211±0.01 ms; p<0.0001; n=24, 17) and increased myocardial shortening speeds at elevated loads (WT 543±0.04 μm/s; AcM 707±0.05 μm/s; p=0.02; n=21), consistent with cardiac hypercontractility. Next, human recombinant WT and AcM ACTCs were produced via baculovirus-based transfection of Sf21cells. Cryo-EM reconstructions of human AcM F-actin-Tpm at ~3.0 Å resolution confirmed no differences in protein ultrastructure or Tpm positioning compared to control. Despite indistinguishable molecular models, in silico assessment revealed highly reduced AcM F-actin-Tpm interaction energy vs. WT (WT -2116±16.4 kcal/mol; AcM -778±12.6 kcal/mol; p<0.0001; n=320, 220), implying that myosin may more readily overcome Tpm’s innate inhibitory bias, facilitating crossbridge cycling and activity upon K326/328 acetylation. Finally, human WT and AcM F-actin-Tpms were differentially labeled with unique fluorescent conjugates and their sliding speeds over the same bed of myosin were simultaneously determined using multiplexed in vitro motility assays. AcM F-actin-Tpm was propelled faster (i.e., was less inhibited), than WT F-actin-Tpm (WT 0.58±0.08 μm/s; AcM 0.95±0.1 μm/s; p=0.018; n=17, 20). Collectively, our findings underscore a potential role for actin K326/328 acetylation in modulating Tpm-based inhibition of cardiac contraction.