Kinetic Modeling of mant-ATP Turnover to Interpret the Biochemically Defined Myosin Super-Relaxed State

The fluorescent ATP analog mant-ATP has become a valuable tool for quantifying occupancy of the myosin super-relaxed (SRX) state, a biochemically inactive conformation of myosin in striated muscle. Interpretation of mant-ATP fluorescence decay kinetics is confounded by inconsistencies in state definitions and kinetic assumptions. Here, we develop a mass-action kinetic model of myosin cross-bridge cycling and mant-ATP turnover to reconcile these discrepancies and provide a mechanistic framework for interpreting SRX measurements. Our model simulates ATP label-chase experiments and demonstrates that conventional double-exponential fitting methods do not directly quantify SRX occupancy. Instead, we show that slow and fast decay phases of mant-ATP fluorescence arise from label redistribution among kinetically distinct states, not state populations in equilibrium. The model resolves several apparent paradoxes identified in recent studies by reproducing experimental observations without requiring SRX and DRX kinetic isolation or implausible equilibrium constants. Simulations further quantify the impact of experimental factors—such as ADP accumulation, photobleaching, and initial rigor state occupancy—on fluorescence kinetics and SRX estimates. These results support a revised framework for SRX quantification and suggest that label-chase experiments must be interpreted using mechanistic models to accurately assess myosin state distributions and transition kinetics.