Abstract Body: Cardiac myosin binding protein-C (cMyBP-C) possesses 11 domains termed C0 through C10 and are generally divided into N-terminal (C0-C2), middle (C3-C7), and C-terminal domains (C8-C10). N-terminal domains are known to interact with actin and myosin to regulate contraction and relaxation. While C-terminal domains anchor cMyBP-C to the thick filament, less is known about the middle domains, particularly the linker between C4 and C5 and the cardiac-specific C5 loop. In silico and in vitro results indicate the C4-C5 linker and C5 loop mediate cMyBP-C conformational changes. Other results using permeabilized cardiomyocytes suggest that the C4-C5 linker but not the C5 loop regulate contractile function. Another linker between C9 and C10 has been suggested to stabilize cMyBP-C to the thick filament and HCM-associated mutation E1179K lies at the C-terminal end of this linker, possibly highlighting the clinical significance of this linker. However, the in vivo function of the C4-C5 linker, C5 loop, and C9-C10 linker motifs remain unknown. We hypothesize removing these motifs will result in aberrant cardiac function and cellular and gross morphologic changes associated with hypertrophic cardiomyopathy. Our aim is to investigate the physiological effects of the 11-amino acid linker between C4 and C5, the 28-residue loop in C5, and the 17-residue linker between C9 and C10 in mice by removing them individually from cMyBP-C. To accomplish this, we generated three different knock-out mouse models (deltaC4-C5linker, deltaC5loop, and deltaC9-C10linker) using CRISPR-cas9 mediated deletion of these regions. To determine the physiological effects of cMyBP-C lacking these motifs on cardiac function and morphology, echocardiography was performed at 12 weeks of age with heterozygous and homozygous mice. We hypothesized that ablating these critical linkers impacts actomyosin interactions and crossbridge kinetics, leading to sarcomere dysfunction and reduced contractility and cardiac function, and identify possible regulatory mechanisms of these motifs in sarcomere structure, regulation, and function in vivo. Determining the physiological effects of cMyBP-C structural motifs will improve our understanding of cMyBP-C modulatory effects on the thin and thick filament and cardiac function and also identify possible therapeutic targets.