Muscle contraction is regulated by both Ca2+ dependent thin filament activation and mechanosensing-based thick filament activation. Evidence is growing for the involvement in thick filament regulation of the cytoskeleton protein titin, which runs from the midpoint of the thick filament to the end of the sarcomere, and the myosin binding protein C (MyBP-C), which is bound to the backbone of the thick filament in the central one-third of either half filament and bridges thin and thick filaments. We use X-ray diffraction to investigate MyBP-C role in inter-filament communication and titin role in thick filament mechanosensing. The experiments, conducted in intact frog skeletal muscle fibres, exploit the finding that X-ray fine structure of myosin based reflections reports the footprints of both MyBP-C and titin and represent a testbed for future investigation on skinned fibres and whole muscles of rodent models with mutations in specific proteins.