MyBP-C binds myosin S2 in mouse heart as revealed by super-resolution microscopy.

Abstract

Supporting the hypothesis that myosin binding protein C (MyBP-C) binds to myosin subfragment-2 (S2) in the heart, a monoclonal antibody (MF30) to S2 was inhibited by the presence of MyBP-C in skinned mouse heart papillary muscle. Using a form of super-resolution microscopy called expansion microscopy, the relative positions of the fluorescent labels attached to a hydrogel could be expanded approximately four-fold in all three dimensions prior to imaging the sample with a Zeiss Airyscan super-resolution microscope. This muscle tissue was prepared for expansion microscopy by labeling with the fluorescently conjugated MF30 and embedding in a hydrogel, followed by crosslinking the fluorescent labels to the hydrogel and thoroughly digesting the proteins within the hydrogel. Images of these samples readily enable measurements of the relative fluorescent intensities of the P-, C-, and D-zones along thick filaments in the A-bands of the sarcomere. The A-bands from wild type mice showed low fluorescent intensities from MF30 in the C-zones relative to the P- and D-zones. Since MyBP-C resides in the C-zones of the thick filament, it was hypothesized that MyBP-C inhibited the MF30 binding in the C-zone. This hypothesis was tested by comparing tissues from MyBP-C knockout mice with the wild type mice using this super-resolution microscopy technique. The overall fluorescence intensity from MF30 was significantly greater in the tissue from MyBP-C knockout mice vs. wild type tissue. Furthermore, the MF30 fluorescence intensity in the C-zone was no longer inhibited in the MyBP-C knockout mice, relative to the D-zone in the wild type. These data support the hypothesis that MyBP-C binds myosin S2 and may compete with the MF30 binding to S2. (Supported by NIH/NHLBI R01HL149164.)