Upregulating Compliant Titin in the Heart Attenuates Left Ventricular Stiffness in a Mouse Model with Diastolic Dysfunction

Abstract

indicating a slower myosin cross-bridge turnover rate of the mutant. Likewise, stopped flow kinetics of the ATP induced dissociation of the acto-myosin complex showed a significantly reduced slope of the kobs-[MgATP] relationship for IVS6-1 reconstituted myosin (4.350.02Â105 M-1 s-1, n=5), depicting slower second-order MgATP binding rates compared with WT (5.350.02Â105 M-1 s-1, n=5). Steady-state fluorescence binding experiments of mutant vs. WT reconstituted myosin to pyrene labeled F-actin under rigor conditions demonstrated significantly reduced binding affinity of IVS6-1 (Kd=16.752.8 nM, n=3) compared with WT (Kd=4.651.3 nM, n=3). In skinned porcine cardiac muscles, a significant decrease in maximal force was observed for IVS6-1 reconstituted fibers compared with WT (27.151.0 kN/m2 vs. 34.952.4 kN/m2, n=6). The Ca 2þ sensitivity and cooperativity were significantly different in IVS6-1 (pCa50=5.6650.01, nH=2.950.15) vs. WT (pCa50=5.4850.01, nH=2.250.14). In conclusion, we demonstrate that IVS6-1 may lead to cardiac dysfunction by disrupting the acto-myosin interaction (steady-state and kinetics) and compromising the ability of the mutant myosin to develop contractile force.