Sub-sarcomeric regulation of thin and thick filaments in skeletal muscle myofibrils

Muscle contraction relies on the coordinated activation of myosin motors from a folded-OFF state on the thick filament surface and their actin tracks on the thin filaments in response to calcium. Thick filaments contain distinct regulatory zones defined by the presence of myosin-binding protein C (MyBP-C) and titin super-repeats, but the control of myosin OFF/ON states within these zones has not been directly resolved. Here we do so, by fluorescence polarization microscopy (FPM). Using orientation-specific probes on myosin we show that folded-OFF motors are enriched in the MyBP-C-containing C zone in relaxed myofibrils. Under titin-based passive tension or partial calcium activation, active motors are enriched in the D zone at the filament tips, which lacks MyBP-C. Troponin probes further reveal that myosin enhances thin-filament activation in the region of filament overlap and drives activation into adjacent non-overlap regions. These findings uncover zone-specific control of myofilament activation within the sarcomere and establish FPM as a powerful tool for investigating disease-linked myofilament protein variants and therapeutic modulation.