Cardiac myosin binding protein-C (cMyBP-C) regulates muscle contraction and relaxation. Most of the functional effects of cMyBP-C have been attributed to its N’-terminal domains C0-C2. Yet, specific functional roles of its central domains (C3, C4, C5, C6, and C7), if any, are unknown. Structural studies show that C5 contains of a 28 amino acid insertion that is unstructured and hypothesized to contribute to protein-protein interactions. In this study, we aimed to determine the functional effects of C5, C6 and C7 on myofilament force mechanics. We used the novel Spy-C3 “cut and paste” mouse model that allows for the replacement of recombinant cMyBP-C protein within its location in the sarcomere. The homozygous Spy-C3 mouse has a 20 amino acid cassette inserted between C7 and C8 with a tobacco etch virus protease (TEVp) recognition site followed by a “SpyTag.” Detergent-permeabilized cardiomyocytes were attached to a force transducer and exposed to TEVp to cut and wash out the C0-C7 domains. Recombinant C5-C7 domains expressing SpyCatcher (sc) were then ligated to SpyTag at the position of endogenous cMyBP-C. Results showed that cutting cMyBP-C with TEVp led to a significant rightward shift in the tension-pCa curve (repeated measures one-way ANOVA, p < 0.0001), an increase in the rate of cross-bridge detachment determined by stretch activation (Krel) (p = 0.0003), and the presence of spontaneous oscillatory contractions (SPOC) compared to before TEVp treatment. However, replacement with recombinant protein C5C7sc did not rescue the functional effects following loss of C0-C7. These data may indicate that domains C5-C7 alone do not contribute to cMyBP-C's role in sensitizing the myofilament and regulating rates of cross-bridge cycling. This work was supported by NIH HL080367 (SPH), AHA Predoctoral Fellowship (NE), and an AHA Postdoctoral Fellowship (AG).