Pseudo-acetylation of Lysine 326 and 328 on cardiac actin impairs relaxation and enhances force production in murine cardiomyocytes

Thin filament-mediated contractile regulation involves a pivoting of tropomyosin (Tpm) along F-actin from the ‘Blocked’ (B)- to the ‘Closed’ (C)-state as Ca 2+ binds to troponin C, followed by its displacement to the 'Open' (M)-state during myosin binding to actin. In the B-state Tpm sterically blocks actomyosin associations; a fundamental requirement for proper relaxation. F-actin-Tpm interactions are largely electrostatic in nature and involve a cluster of highly-conserved, positively-charged residues on actin including lysines 326 and 328. Proteomic analyses have revealed that these residues can be acetylated in vivo, in mammalian hearts. Since lysine acetylation neutralizes its positive charge, this post translational modification may play an important role in modulating F-actin-Tpm interactions and thus, thin filament-mediated contractile regulation. We therefore tested this hypothesis in both mouse and in silico models. A myocardial-targeting adeno-associated virus (AAV) vector was used to deliver a Myc-tagged mActc1 wildtype (WT) gene or a mutant construct that encoded Myc-tagged mActc1 with K326 and K328 replaced by polar, but uncharged glutamines. Therefore, our animals expressed either WT ( Myc-mActc1 WT ) or ‘acetyl-mimetic’ ( Myc-mActc1 K326,328Q ) actin exclusively in the heart. Epifluorescence imaging revealed efficient cardiomyocyte (CM) transfection throughout all chambers. Immunohistochemistry of isolated CMs with anti-Myc antibodies showed that the virally-expressed WT and acetyl-mimetic actins discretely incorporated into the CM myofibrillar apparatus, and co-polymerized with endogenous actin. CMs were isolated, subjected to electrical pacing, and their Ca 2+ and contractility dynamics were examined. Ca 2+ transients from individual AAV-Myc-mActc1 WT (control) vs. AAV-Myc-mActc1 K326,328Q (test) CMs showed no significant differences. However, myocytes from test hearts showed significantly slower relaxation kinetics when compared to controls. We are currently using molecular dynamics simulations to examine the movement of Tpm back to its B-state configuration following Ca 2+ removal, to determine if re-establishing the thin filament B-state is compromised by K326/328 acetylation, and thus retarded, in silico. Additionally, single myocyte, steady-state force measurements showed that the test CMs generated significantly higher maximum tension, with no change in Ca 2+ sensitivity vs. controls. Thus, acetylation of K326 and K328 of cardiac actin could destabilize the inhibitory B-state of Tpm along the thin filaments, impairing force inhibition, and leading to a hypercontractile CM phenotype. Further testing of this hypothesis in a CRISPR mouse model is ongoing. National Institutes of Health (R01HL124091) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.