Titin's P-zone domains A164-167 are essential for thick filament structural arrangement.

The sarcomeric protein titin plays a central role in thick filament structure and function through its modular A-band domains, including the understudied P-zone, which links the C-zone to the M-band. To investigate the first four domains of titin's P-zone (A164-A167), we deleted them in a mouse model (TtnΔA164-167). Echocardiography and cardiomyocyte mechanics revealed mild changes to diastolic function and enlargement of the heart, but preserved contractility. The EDL muscle showed contractile deficits at the whole muscle level and increased passive stiffness at the myofiber level. Immunoelectron and super-resolution microscopy revealed altered thick filament architecture, including a ∼40-nm shift of titin and myosin binding protein-C epitopes toward the M-band, disruption of titin's α and β conformations, and shorter thick filaments. The structural changes are consistent with the loss of a myosin helical repeat. These findings establish a key structural role of titin's P-zone domains A164-A167 in templating thick filament protein arrangement, including the importance of titin's α and β conformations.