Abstract 3849: HNO Induces a Disulfide Bond Between Cysteine Residues on Actin (Cys 257) and Tropomyosin (Cys 190) Increasing Cardiac Force Development: A Novel, Redox-based Mechanism for Contractile Regulation
Background: Nitroxyl (HNO) donors increase cardiac inotropy via combined enhancement of SR Ca2+ cycling and myofilament sensitization to Ca2+. HNO reacts with thiols, but the critical -SH targets on the myofilaments are currently unknown.
Aims: Using rat cardiac trabeculae and a new mass spectrometry capture technique based on a modified biotin switch assay, we have identified the sites and the nature of the myofilament modification induced by the novel 1-nitrosocyclohexylacetate (NCA) (a pure HNO releaser) and for comparison the prototypic Angeli’s (AS).
Results: In steady state activations, NCA (25 μM) increased maximal Ca2+ activated force (Fmax) and decreased [Ca2+]i required for 50% of activation (Ca50): Fmax was 123±18 vs. 95±5 mN/mm2 (p<0.05) and Ca50 0.42±0.01 vs. 0.57±0.03 μmol/L (p<0.004) without affecting cooperativity (Hill, 4.92±0.84 vs. 3.94±0.18, p=NS), confirming and expanding upon previous data obtained with AS. NCA action persisted after skinning, proving that NCA/HNO acts directly on the myofilaments. As expected, HNO action was reversed by the thiol reducing agent dithiotreitol (DTT, 5mM). Isolated rat cardiac myofibrils were exposed to NCA (25 μM), AS (500 μM) or controls; modified peptides were then captured and identified by LC/MS/MS. The analysis revealed sites of modification on tropomyosin (Cys 190) and actin (Cys 257) that were specific to NCA treatment (n=3). Western blot analysis of NCA-treated samples on non-reducing gels revealed higher molecular weight forms of tropomyosin and actin that were intermediate to their homodimeric forms which were lost with DTT treatment, indicating a heterodimer formation via disulphide bond.
Conclusions: We identified a disulfide bond between cysteine residues in actin and tropomyosin that is specifically induced by HNO. This likely correlates with HNO mediated increase in force development in skinned fibers. Here we propose a novel redox-based model in which HNO induced crosslinking of tropomyosin to the inner domain of actin imposes restraints on tropomyosin movement that bias its equilibrium position toward the Ca2+-activated state. This effect allows greater access for myosin binding after activation, thereby providing a mechanism for HNO enhanced myofilament response to Ca2+.
This research has received full or partial funding support from the American Heart Association, Mid-Atlantic Affiliate (Maryland, North Carolina, South Carolina, Virginia & Washington, DC).