Characterization of α-Tropomyosin (TM) Mutants that Cause Hypertrophic Cardiomyopathy (HCM) in Humans: In vitro Motility Assays with a Microscopic Heat Pulse

Abstract

HCM is characterized by thickening of left ventricular wall and interventricular septum, with abnormalities in contractile functions. It has been thought that Tm's flexible association with actin is important for its function. With in vitro motility assays, we investigated α-Tm mutants V95A and D175N (based on human sequence), which are known to cause HCM. Reconstituted thin filaments with rabbit skeletal actin and Tn were placed on HMM-coated glass surface. At the room temperature (24°C), we applied a microscopic heat pulse by focused laser beam, which momentarily generated a concentric temperature gradient with the center reaching 50°C. Because the pulse duration was brief (2 s), this procedure did not denature proteins. This area was viewed in a microscope field, which allowed observations of gliding thin filaments at different temperatures in the same field. We found that the gliding occurred on elevating the temperature even in the absence of Ca (Oyama et al., BBRC, 2012; see also Ishiwata, BBA, 1978). The thermally-activated association of HMM to actin results in a temporary opening of the thin filament, where it is likely that Tm/Tn is partially dissociated from the actin filament, which causes a partial activation of the thin filament. We found that the thin filament reconstituted with V95A was more difficult to become thermally activated at around physiological temperature than that with WT or D175N. This result suggests that the critical temperature for activation is higher for V95A than WT and D175N. These results further suggest that the thermal stability of the association of V95A Tm to the actin filament is higher than that of WT Tm. Our results indicate that extra stability of the actin-Tm association may cause HCM.