Titin's P-zone domains A164-A167 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 under-studied 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 (Ttn_delta_A164-167). 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 alpha and beta 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 for titin's P-zone domains A164-A167 in templating thick filament protein arrangement, including the importance of titin's alpha and beta conformations.