Myosin-based mechanisms are increasingly recognized as supplementing their better-known actin-based counterparts to control the strength and time course of contraction in both skeletal and heart muscle. Here we use synchrotron small-angle X-ray diffraction to determine the structural dynamics of local domains of the myosin filament during contraction of heart muscle. We show that, although myosin motors throughout the filament contribute to force development, only about 10% of the motors in each filament bear the peak force, and these are confined to the filament domain containing myosin binding protein-C, the "C-zone." Myosin motors in domains further from the filament midpoint are likely to be activated and inactivated first in each contraction. Inactivated myosin motors are folded against the filament core, and a subset of folded motors lie on the helical tracks described previously. These helically ordered motors are also likely to be confined to the C-zone, and the associated motor conformation reforms only slowly during relaxation. Myosin filament stress-sensing determines the strength and time course of contraction in conjunction with actin-based regulation. These results establish the fundamental roles of myosin filament domains and the associated motor conformations in controlling the strength and dynamics of contraction in heart muscle, enabling those structures to be targeted to develop new therapies for heart disease.heart muscle | myosin motor | muscle regulation | myosin-binding protein C T he pumping action of the heart is driven by rhythmic contractions of its muscular walls. The healthy heart continuously optimizes the strength and time course of contraction by modulating the calcium transient that triggers the heartbeat and the phosphorylation levels of multiple proteins, including components of the myosin and actin filaments that drive contraction, and by direct mechanical feedback (1-4). These signaling pathways alter contractility by changing the structures of the contractile filaments through downstream effector mechanisms that remain poorly understood. For many years attention was focused on actin filament-based regulation and its link to intracellular calcium signaling (1); more recently it became clear, partly by extrapolation from studies on skeletal muscle (5-9), that myosin filamentbased regulation also plays an important role. Moreover, myosinbased regulation is perturbed in heart disease (10, 11), and has been increasingly targeted for the development of novel therapies to treat the failing heart (12). Such efforts have however been impeded by limited knowledge about the action of myosin-based regulation on the timescale of the heartbeat: that is, about mechanisms that operate much faster than kinase signaling (3,4). Two leading candidate mechanisms of this type emerged from studies of skeletal muscle: direct mechanosensing by the myosin filaments (6, 7), and interfilament signaling by myosin binding protein-C (6,13). Although several studies have suggested that these mechanisms are ...