ABSTRACT: The goal of this project is to understand how cardiac myosin binding protein-C (cMyBP-C) regulates heart muscle contraction and how dysregulation of cMyBP-C causes systolic and diastolic dysfunction. Work from the PI's lab over the past decade firmly established that cMyBP-C binds to thin (actin) filaments and activates contraction in the same way as Ca2+ and strongly bound myosin cross-bridges. These discoveries fundamentally challenged the preconception that cMyBP-C affects contraction exclusively via inhibition of thick (myosin) filaments. Direct interactions of cMyBP-C with the thin filament can also adequately explain profound effects of cMyBP-C to modulate both diastolic and systolic cardiac function. However, until now functional effects due to cMyBP-C interactions with actin were purely hypothetical because there has been no way to distinguish between effects of cMyBP-C binding to actin or myosin in working hearts. An additional problem is a lack of complementary methods to selectively modify cMyBP-C in sarcomeres. Without this combination of tools it has been impossible to target specific interactions with cMyBP-C binding partners in situ. Here we decisively overcome these barriers by creating unique resources that allow us to functionally dissect cMyBP-C interactions with the thin filament. Innovations include 2 new transgenic mouse models, each with a single mutation in a highly conserved actin binding sequence that we identified in the regulatory M-domain. The mutations either increase (L348P) or decrease (E330K) cMyBP-C binding to the thin filament. Preliminary data from the mice suggest that cMyBP-C interactions with actin control fundamental timing of contraction and relaxation because the L348P mutation increased the duration of systolic ejection and slowed diastolic relaxation, while the E330K mutation decreased the duration of systole. Aim 1 of the proposed experiments will use the L348P and E330K mice test the hypothesis that cMyBP-C binding to actin maintains thin filament activation at the end of systole independent of declining activation by Ca2+ or strongly bound cross-bridges. In Aim 2, we created a third unique mouse model, referred to as ?Spy-C? mice, that allows us to replace N'- terminal domains of cMyBP-C in sarcomeres in situ with any desired modification to probe function. In Aim 2 we will use the Spy-C system to test the hypothesis that sarcomere length dynamically regulates cMyBP-C binding interactions with actin and we will further assess the impact of the middle domains (C3-C7) of cMyBP- C and effects of HCM missense mutation hotspots in these domains for the first time. We will identify cMyBP-C interacting partners in the sarcomere by labeling cMyBP-C N'-terminal domains using FRET based sensors. The long-term impact of this work is that we will be able to selectively define the impact of cMyBP-C interactions with the thin filament on systolic and diastolic cardiac function and identify new mechanisms of cMyBP-C regulation.