Cardiac Myosin-Binding Protein C N-Terminal Interactions with Myosin: Binding Insights from Fluorescence Lifetime Studies

Abstract

Cardiac myosin-binding protein C (cMyBP-C), a sarcomeric protein associated with the thick filament in the C-zone, finely tunes contractility and relaxation of the heart through its interactions with myosin and actin. These binding interactions are regulated by PKA-mediated phosphorylation, a downstream effect triggered by the β-adrenergic pathway. Mutations in the gene encoding for cMyBP-C are the leading cause for hypertrophic cardiomyopathy (HCM), a cardiac disease characterized by abnormal thickening of the left ventricle and diastolic dysfunction. These mutations result in haploinsufficiency or cause missense mutations, both of which disrupt the normal phosphorylation-dependent interactions of cMyBP-C with myosin and actin. Furthermore, decreased levels of cMyBP-C phosphorylation have been observed in patients with HCM or heart failure (HF), underscoring the importance of phosphorylation in maintaining normal cardiac function. Despite this importance, the binding dynamics of cMyBP-C under both normal and disease conditions remain unknown. Such insights could aid in the development of small-molecule drugs that correct altered binding interactions due to mutations or mimic cardioprotective effects of phosphorylation, including targeting cMyBP-C for HCM therapies. The binding interactions of cMyBP-C with both myosin and actin are widely accepted in the field. However, the details of these interactions, such as whether cMyBP-C N-terminal domains bind to one or the other under certain conditions of contraction, relaxation and phosphorylation, cycle between these partners, exist in different binding populations, or adopts static or dynamic binding, have yet to be fully understood. These open questions highlight the complexity of cMyBP-C’s role in regulating cardiac contractility and the need for further investigation. Conventional methods used to study these interactions, such as isothermal titration calorimetry, cryo-electron microscopy, nuclear magnetic resonance, and cosedimentation assays, while reliable, are labor-intensive, time-consuming, and yield low throughput data. To address this limitation, the Colson Lab utilizes fluorescence lifetime techniques (FLT) instead to study these critical binding interactions of cMyBP-C. We have established actin and cMyBP-C binding assays using FLT and FLT-based Förster Resonance Energy Transfer (FLT-FRET). Focusing on the N-terminal cMyBP-C fragment C0-C2, the recent establishment of actin-C0-C2 FLT assays in the Colson lab have proven valuable for drug screening by identifying numerous compounds that target cMyBP-C. My project focused on developing a complementary myosin assay that uses FLT techniques to detect the phosphorylation-regulated interactions with C0-C2. These assays provide our lab the unique ability to elucidate the intricate interactions of cMyBP-C with myosin and actin, enabling us to investigate the impact of HCM mutations and potential drug compounds on these myofilament interactions. My dissertation is organized into five chapters, starting with an introduction and background in Chapter 1. Chapter 2 details the development and optimization of a myosin-C0-C2 FLT assay. In Chapter 3, this assay is used alongside an actin-C0-C2 FLT assay to investigate the effects of cMyBP-C phosphorylation and HCM mutations. Chapter 4 presents a pilot study, examining 13 compounds identified as potential myosin modulators or through actin-C0-C2 screens. This dissertation concludes in Chapter 5 with a summary of findings, discusses limitations, and outlines future research directions.