Direct Troponin-Myosin Interaction Enhances ATPase Activity of Cardiac HMM

Abstract

Phenomenological evidence has long suggested that the strongly bound cross-bridges exert a positive feedback on myofilament regulation, which increases the sensitivity of thin filament to Ca2+ and achieves full activation. This positive feedback mechanism is uniquely important to the beat-to-beat regulation of cardiac output. Despite its importance, the molecular mechanism remains elusive. The current wisdom implies that the positive feedback mechanism is mediated by specific movement of tropomyosin on actin surface caused by actin-myosin interaction. Since the Ca2+-regulated interactions between cardiac thin filament and myosin can be considered as an allosteric system which has long-range coupling between distinct components of the system, multiple protein-protein interactions at the interface between thin and thick filament may provide alternative mechanism for the feedback effects on myofilament activation. In this study, we investigated the potential interaction between troponin and myosin, known as troponin-bridge, and how the interaction affects function of myosin and thin filament regulation. The direct interaction between myosin and cardiac troponin was monitored using a sedimentation assay. Different reconstituted troponin complexes were incubated with myosin followed by ultra-centrifugation to pull down the myosin and the bound proteins. Western blot was used to identify the troponin subunits that were bound to myosin. Our results suggest that both N-cTnI(1-129) and C-cTnT (T2) bind to myosin, suggesting a possible direct interaction between myosin and IT arm of troponin. ATPase assay showed that the HMM ATPase activity was significantly enhanced by the presence of troponin regardless of the presence of actin and/or tropomyosin. In addition, truncation of the C-domain (residues 129-212) of cTnI also increased the HMM ATPase activity. The results of our study indicate that troponin-bridge may be a crucial component of thin filament regulation and play an important role in actomyosin interaction and muscle function.