Myosin-binding protein C stabilizes, but is not the sole determinant of SRX myosin in cardiac muscle

Striated muscle functional regulation occurs through the sequestration of myosin into the super-relaxed (SRX) state. Observing the turnover of single ATP molecules within cardiac sarcomeres defines the spatial arrangement of SRX myosin and their regulation by myosin-binding protein C. The myosin super-relaxed (SRX) state is central to striated muscle metabolic and functional regulation. In skeletal muscle, SRX myosin are predominantly colocalized with myosin-binding protein C (MyBP-C) in the sarcomere C-zone. To define how cardiac MyBP-C (cMyBP-C) and its specific domains contribute to stabilizing the SRX state in cardiac muscle, we took advantage of transgenic cMyBP-C null mice and those expressing cMyBP-C with a 271-residue N-terminal truncation. Utilizing super-resolution microscopy, we determined the lifetime and subsarcomeric location of individual fluorescent-ATP turnover events within isolated cardiac myofibrils. The proportion of SRX myosin demonstrated a gradient along the half-thick filament, highest in the P- and C-zones (72 +/- 9% and 71 +/- 6%, respectively) and lower in the D-zone (45 +/- 10%), which lies farther from the sarcomere center and lacks cMyBP-C, suggesting a possible role for cMyBP-C in stabilizing the SRX. However, myofibrils from cMyBP-C null mice demonstrated an similar to 40% SRX reduction, not only within the now cMyBP-C-free C-zone (49 +/- 9% SRX), but also within the D-zone (22 +/- 5% SRX). These data suggest that the influence of cMyBP-C on the SRX state is not limited to the C-zone but extends along the thick filament. Interestingly, myofibrils with N-terminal truncated cMyBP-C had an SRX content and spatial gradient similar to the cMyBP-C null, indicating that the N terminus of cMyBP-C is necessary for cMyBP-C's role in enhancing the SRX gradient along the entire thick filament. Given that SRX myosin exist as a gradient along the thick filament that is highest in the C-zone, even in the absence of cMyBP-C or its N-terminus, an inherent bias must exist in the structure of the thick filament to stabilize the SRX state.

Automated summary

Striated muscle is regulated by the super-relaxed state (SRX) of myosin, and the turnover of ATP molecules in cardiac sarcomeres provides insights into the spatial arrangement and regulation of SRX myosin by myosin-binding protein C. The SRX state plays a central role in the metabolic and functional regulation of striated muscle.