Striated muscle contraction is driven by cyclical interaction between myosin-containing thick and actin-containing thin filaments and is regulated by the tropomyosin-troponin complex. In the absence of calcium, troponin constrains tropomyosin in a blocking position where it shields myosin binding sites on actin. Calcium binding to troponin enables azimuthal movement of tropomyosin, which exposes myosin binding sites, and relieves contractile inhibition.Previous models indicated that K326 and K328 on actin form electrostatic contacts with E181 of tropomyosin in an inhibitory state (Li, et al. 2011), and that K328 also contacts E286 of myosin S1 during contraction (Behrmann, et al. 2012). A recent proteomic study showed that K326 and K328 are acetylated in guinea pig cardiac thin filaments (Foster, et al. 2013). Since this post-translational modification would negate the positively charged lysines, and potentially ablate vital ionic interactions between actin and tropomyosin or myosin, we predicted K326 and K328 acetylation would alter muscle performance. We tested this hypothesis in vivo by expressing K326Q, K328Q, or K326Q/K328Q acetyl-mimetic actin in Drosophila muscle. Polarized light microscopy of K328Q and K326Q/K328Q indirect flight muscle revealed a severe disturbance to fiber structure, and flight tests confirmed complete loss in flight ability relative to control. Flies harboring only the K326Q mutation, however, had similar fiber structure to control, but nonetheless displayed a significant decrease in flight performance. These results suggest that K326 and K328 play important roles in maintaining proper muscle function. We are currently investigating the effects of the pseudo-acetylated actin residues on Drosophila hearts. Overall, our findings highlight the utility of Drosophila as a model that permits efficient targeted design and assessment of molecular and tissue-specific responses to muscle protein modifications, in the physiological context of muscle.