Myosin Binding Protein-C (MyBP-C) comprises a family of thick filament associated proteins that contributes to their assembly and maintenance, and regulates the formation of actomyosin cross-bridges during contraction. Three distinct isoforms have been characterized, including the cardiac (c), slow (s) skeletal and fast (f) skeletal. The expression of the cardiac isoform is confined in the developing and mature heart, whereas the skeletal isoforms can co-exist in the same muscle. The core structure of MyBP-C consists of seven immunoglobulin (Ig) domains and three fibronectin-III (Fn-III) domains, numbered from the NH2-terminus as C1-C10. During the last forty years, numerous studies have focused on elucidating the mechanisms that modulate the activities of cMyBP-C in the formation of actomyosin cross-bridges. On the contrary, the regulation and roles of the skeletal isoforms have remained obscure, and mainly inferred due to the structural similarity they share with cMyBP-C. Our group has been studying the slow skeletal form of MyBP-C aiming to understand its regulation and activities. Using molecular tools, we have shown that the MYBPC1 gene, encoding sMyBP-C, is heavily spliced giving rise to multiple variants that can be co-expressed in the same muscle and myofiber. These share common domains, but also differ by the inclusion or skipping of novel insertions located in the NH2-terminus, the FN-III C7 domain and the COOH-terminus. Both the NH2 and COOH termini can retain native myosin and actin and modulate the sliding velocity of actin filaments past myosin heads, though to different extents and in a variant-specific manner. Moreover, using proteomic tools, we have demonstrated that sMyBP-C undergoes phosphorylation mediated by PKA and PKC. In particular, we have identified four phosphorylation sites in the NH2-terminus of the protein, with one of them located within a unique insertion present only in select variants. We therefore hypothesize that sMyBP-C comprises a multi- faceted family of thick filament accessory proteins whose functions are regulated via complex phosphorylation of its NH2-terminus. Our goals in the current proposal are to examine how phosphorylation affects the biochemical and biophysical properties of the different sMyBP-C variants (Aim 1), and to generate the first phospho-mutant sMyBP-C animal model to assess the role of phosphorylation in vivo (Aim 2). The proposed studies will greatly advance our understanding on the regulation of the multifaceted sMyBP-C subfamily via phosphorylation, which is an outstanding biological question with important and broad implications in muscle pathophysiology.
Contraction of skeletal muscles involves the sliding of thin actin filaments past thick myosin filaments. This process is highly regulated by a number of accessory proteins, including Myosin Binding Protein-C slow (sMyBP-C), which is modulated via phosphorylation. Our goal is to understand how phosphorylation impacts the ability of sMyBP-C to regulate skeletal muscle contractility, which is currently elusive.