Cardiac Myosin Binding Protein-C (MYBP-C) Impedes Actin Filament Motility on Native Mouse Ventricular Thick Filaments Only Within the C-Zone

Abstract

MyBP-C is a potent modulator of cardiac contractility and is localized to two distinct regions (C-zones) flanking the myosin thick filament (TF) bare zone. To determine whether MyBP-C modulates actomyosin motion generation over the entire length of the TF or only within the C-zones, we isolated native mouse ventricular TFs by mechanical and proteolytic dispersion. Negatively-stained EM images showed 1.6µm long TFs with defined central bare zones. TFs contained intact MyBP-C, confirmed by western blotting with domain specific antibodies. In a total internal reflectance microscopy based motility assay, TFs were imaged using fluorescent ATP and the velocity of fluorescent shards of actin filaments was measured over the entire length of TFs at 100 µM ATP and 20°C. Four distinct velocities were observed, presumably corresponding to motion toward and away from the bare zone, over regions with and without MyBP-C, the average velocity of which was similar to that generated by monomeric myosin in the motility assay (1.0±0.1µm/s). Limiting our analysis to bare zone directed motility, actin moved at 1.8±0.1µm/s starting from the TF end and slowed abruptly to 1.0±0.1µm/s at 285±8nm from the bare zone. In contrast, bare zone directed motility on TFs from MyBP-C knockout mice demonstrated only one velocity (1.7±0.1µm/s), equal to the faster velocity on wildtype (WT) TFs, suggesting the slow velocity on the WT TFs is due to MyBP-C. Similarly, only one bare zone directed velocity (1.9±0.1µm/s) was observed on WT TFs after N-terminal proteolytic truncation of the MyBP-C with calpain to within the MyBP-C motif (C1-C2 linker). We conclude that the N-terminal domains of MyBP-C directly modulate actomyosin motion generation only in a ∼285nm region of the thick filament, presumably the C-zone, where MyBP-C is localized.