Thomas A Bunch1, Victoria C Lepak1, Rhye-Samuel Kanassatega1, Brett A Colson2. 1. Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ 85724, United States. 2. Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ 85724, United States. Electronic address: bcolson@email.arizona.edu.
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.
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.
Authors: Michael J Previs; Ji Young Mun; Arthur J Michalek; Samantha Beck Previs; James Gulick; Jeffrey Robbins; David M Warshaw; Roger Craig Journal: Proc Natl Acad Sci U S A Date: 2016-02-23 Impact factor: 11.205
Authors: Matthew R Locher; Maria V Razumova; Julian E Stelzer; Holly S Norman; Richard L Moss Journal: Am J Physiol Heart Circ Physiol Date: 2011-01-07 Impact factor: 4.733
Authors: Matthew R Locher; Maria V Razumova; Julian E Stelzer; Holly S Norman; Jitandrakumar R Patel; Richard L Moss Journal: Am J Physiol Heart Circ Physiol Date: 2009-04-24 Impact factor: 4.733
Authors: Willem J De Lange; Adrian C Grimes; Laura F Hegge; Alexander M Spring; Taylor M Brost; J Carter Ralphe Journal: J Gen Physiol Date: 2013-09 Impact factor: 4.086
Authors: Andrea E Deranek; Anthony P Baldo; Melissa L Lynn; Steven D Schwartz; Jil C Tardiff Journal: Biochemistry Date: 2022-06-13 Impact factor: 3.321
Authors: Rhye-Samuel Kanassatega; Thomas A Bunch; Victoria C Lepak; Christopher Wang; Brett A Colson Journal: J Mol Cell Cardiol Date: 2022-02-25 Impact factor: 5.763