Thin Filament-Mediated Modulation of Mouse Cardiac Cross-Bridge Kinetics by Ca2+-Sensitizing Mutation CTNC-A8V or Bepridil

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Recent genetic and functional data with the Ala8Val missense mutation in cardiac troponin C (cTnC-A8V) suggest its pathogenicity for the development of hypertrophic cardiomyopathy (HCM) in humans and mice. Previous functional studies performed using cTnC-A8V knock-in mice revealed increased Ca2+-sensitivity of contraction and impaired kinetics of cardiac muscle relaxation of skinned and intact cardiomyocytes. This study aims to further understand the molecular mechanisms that underlie the development of HCM caused by the cTnC-A8V mutation by measuring the kinetics of tension redevelopment (kTR) and sinusoidal stiffness at two distinct sarcomere lengths (SL). Papillary muscles were isolated from the left ventricle of cTnC-A8V knock-in homozygote and WT mice. SL of skinned preparations was set to 1.9µm or 2.1µm using HeNe laser diffraction after the ends were chemically fixed with glutaraldehyde to minimize end-compliance. SL dependent changes in mechanical properties were similar in WT and cTnC-A8V preparations: increased pCa50, with increased stiffness and decreased kTR at any given activation level. However, regardless of SL, cTnC-A8V preparations exhibited an increased pCa50, kTR and sinusoidal stiffness at submaximum Ca2+ levels relative to WT. Maximum sinusoidal stiffness increased 2x in WT preparations stretched from SL 1.9µm to 2.1µm, while for the cTnC-A8V preparations the SL-dependent increase in maximum sinusoidal stiffness was blunted. To determine whether the difference in cross-bridge kinetics at SL 2.1µm between cTnC-A8V and WT were due to the increased Ca2+-sensitivity of cTnC, WT preparations were treated with 300μM Bepridil, a Ca2+-sensitizer. As seen with the cTnC-A8V, sensitizing the thin filament with Bepridil resulted in an increased pCa50 and kTR. These data suggest an important role of the thin filament controlling not only the number of cross-bridges during Ca2+-activation, but may also influence cross-bridge kinetics.