Exploring the super-relaxed state of myosin in myofibrils from fast-twitch, slow-twitch, and cardiac muscle

Muscle myosin heads, in the absence of actin, have been shown to exist in two states, the relaxed (turnover similar to 0.05 s(-1)) and super-relaxed states (SRX, 0.005 s(-1)) using a simple fluo-rescent ATP chase assay (Hooijman, P. et al (2011) Biophys. J. 100, 1969-1976). Studies have normally used purified proteins, myosin filaments, or muscle fibers. Here we use muscle myofibrils, which retain most of the ancillary proteins and 3-D architecture of muscle and can be used with rapid mixing methods. Recording timescales from 0.1 to 1000 s provides a precise measure of the two populations of myosin heads present in relaxed myofibrils. We demonstrate that the population of SRX states is formed from rigor cross bridges within 0.2 s of relaxing with fluorescently labeled ATP, and the population of SRX states is relatively constant over the temperature range of 5 & nbsp;C-30 & nbsp;C. The SRX population is enhanced in the presence of mavacamten and reduced in the presence of deoxy-ATP. Compared with myofibrils from fast-twitch muscle, slow twitch muscle, and cardiac muscles, myofibrils require a tenfold lower concentration of mavacamten to be effective, and mavacamten induced a larger increase in the population of the SRX state. Mavacamten is less effective, however, at stabilizing the SRX state at physiological temperatures than at 5 & nbsp;C. These assays require small quantities of myofibrils, making them suitable for studies of model organism muscles, human biopsies, or human-derived iPSCs.

Automated summary

Muscle myosin heads have been found to exist in relaxed and super-relaxed states. The population of super-relaxed states is formed from rigor cross bridges and is relatively stable over a range of temperatures. The drug mavacamten affects the super-relaxed state differently in different types of myofibrils and is less effective at physiological temperatures.