Dephosphorylated Cardiac Myosin-Binding Protein C (cMyBP-C) Activates Native Cardiac Thin Filaments within the C-Zone of Native Cardiac Thick Filaments

Abstract

cMyBP-C is a potent modulator of actomyosin motion generation within two distinct ∼350nm regions (C-zones) flanking the myosin thick filament bare zone. cMyBP-C's modulatory capacity is tunable by phosphorylation of 4 serines within the cMyBP-C motif. To determine cMyBP-C's phosphorylation-dependent impact on calcium activation of fully regulated actin thin filaments (i.e. containing troponin and tropomyosin), we observed rhodamine-phalloidin labeled native thin filament shards (∼250nm) moving over native thick filaments isolated from mouse ventricular tissue (Previs et al., 2012). For these experiments, the state of cMyBP-C phosphorylation in native thick filaments was either ∼64% on each of the 4 serines upon isolation (wild-type) or reduced to ∼22% by lambda-phosphatase treatment. Native thin filament movement was observed by TIRF microscopy at 25mM KCl, 100µM ATP, 20°C. At pCa 5, thin filaments landed on wild-type thick filaments and moved for 402±21nm at an initial fast velocity of 2.2±0.1µm/s before slowing abruptly to 0.9±0.1µm/s for 311±19nm within the C-zone. Similar trajectories were observed on phosphatase-treated thick filaments (fast velocity: 2.0±0.1 µm/s, 392±16nm; slow velocity: 0.8±0.1µm/s, 347±15nm within the C-zone). At pCa 7, little to no thin filament motion was observed on wild-type thick filaments but surprisingly, on phosphatase-treated thick filaments, a single slow phase of velocity (0.6±0.1µm/s) with a run length (397±137nm) similar to the C-zone length was observed. We conclude that cMyBP C plays two mechanistic roles within the thick filament C-zone in vivo: 1) allowing for cMyBP-C phosphorylation-dependent activation of the thin filaments at resting calcium levels; 2) providing an internal load that governs the speed of thin filaments once the muscle is calcium activated.