N-terminal extension in cardiac myosin-binding protein C regulates myofilament binding

Abstract

Rationale: Mutations in the gene encoding the sarcomeric protein cardiac myosin-binding protein C (cMyBP-C) are a leading cause of hypertrophic cardiomyopathy (HCM). Mouse models targeting cMyBP-C and use of recombinant proteins have been effective in studying its roles in contractile function and disease. Surprisingly, while the N-terminus of cMyBP-C is important to regulate myofilament binding and contains many HCM mutations, an incorrect sequence, lacking the N-terminal 8 amino acids has been used in many studies.

Objectives: To determine the N-terminal cMyBP-C sequences in ventricles and investigate the roles of species-specific differences in cMyBP-C on myofilament binding.

Methods and results: We determined cMyBP-C sequences in mouse and human by inspecting available sequence databases. N-terminal differences were confirmed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cosedimentation assays with actin or myosin were used to examine binding in mouse, human and chimeric fusion proteins of cMyBP-C. Time-resolved FRET (TR-FRET) with site-directed probes on cMyBP-C was employed to measure structural dynamics. LC-MS/MS supported the sequencing data that mouse cMyBP-C contains an eight-residue N-terminal extension (NTE) not found in human. Cosedimentation assays revealed that cardiac myosin binding was strongly influenced by the presence of the NTE, which reduced binding by 60%. 75% more human C0-C2 than mouse bound to myosin. Actin binding of mouse C0-C2 was not affected by the NTE. 50% more human C0-C2 than mouse bound to actin. TR-FRET indicates that the NTE did not significantly affect structural dynamics across domains C0 and C1.

Conclusions: Our functional results are consistent with the idea that cardiac myosin binding of N-terminal cMyBP-C is reduced in the mouse protein due to the presence of the NTE, which is proposed to interfere with myosin regulatory light chain (RLC) binding. The NTE is a critical component of mouse cMyBP-C, and should be considered in extrapolation of studies to cMyBP-C and HCM mechanisms in human.

Keywords: Cardiac myosin-binding protein C; Contractile proteins; Myofilament; Myosin; N-terminal extension; Regulation.