Pathogenic properties of the N-terminal region of cardiac myosin binding protein-C in vitro. Journal Article

Authors: Govindan, S.; Sarkey, J.; Ji, X.; Sundaresan, N. R.; Gupta, M. P.; de Tombe, P. P.; Sadayappan, S
Article Title: Pathogenic properties of the N-terminal region of cardiac myosin binding protein-C in vitro.
Abstract: Cardiac myosin binding protein-C (cMyBP-C) plays a role in sarcomeric structure and stability, as well as modulating heart muscle contraction. The 150 kDa full-length (FL) cMyBP-C has been shown to undergo proteolytic cleavage during ischemia-reperfusion injury, producing an N-terminal 40 kDa fragment (mass 29 kDa) that is predominantly associated with post-ischemic contractile dysfunction. Thus far, the pathogenic properties of such truncated cMyBP-C proteins have not been elucidated. In the present study, we hypothesized that the presence of these 40 kDa fragments is toxic to cardiomyocytes, compared to the 110 kDa C-terminal fragment and FL cMyBP-C. To test this hypothesis, we infected neonatal rat ventricular cardiomyocytes and adult rabbit ventricular cardiomyocytes with adenoviruses expressing the FL, 110 and 40 kDa fragments of cMyBP-C, and measured cytotoxicity, Ca(2+) transients, contractility, and protein-protein interactions. Here we show that expression of 40 kDa fragments in neonatal rat ventricular cardiomyocytes significantly increases LDH release and caspase 3 activity, significantly reduces cell viability, and impairs Ca(2+) handling. Adult cardiomyocytes expressing 40 kDa fragments exhibited similar impairment of Ca(2+) handling along with a significant reduction of sarcomere length shortening, relaxation velocity, and contraction velocity. Pull-down assays using recombinant proteins showed that the 40 kDa fragment binds significantly to sarcomeric actin, comparable to C0-C2 domains. In addition, we discovered several acetylation sites within the 40 kDa fragment that could potentially affect actomyosin function. Altogether, our data demonstrate that the 40 kDa cleavage fragments of cMyBP-C are toxic to cardiomyocytes and significantly impair contractility and Ca(2+) handling via inhibition of actomyosin function. By elucidating the deleterious effects of endogenously expressed cMyBP-C N-terminal fragments on sarcomere function, these data contribute to the understanding of contractile dysfunction following myocardial injury.
Journal Title: Journal of Muscle Research Cell Motility
Volume: 33
Issue: 1
ISSN: 1573-2657
Publisher: Unknown  
Journal Place: Netherlands
Date Published: 2012
Start Page: 17
End Page: 30
Language: English
Notes: ID: 12529; Record Owner: From MEDLINE, a database of the U.S. National Library of Medicine.; Status: MEDLINE; Publishing Model: Journal available in: Print-Electronic Citation processed from: Internet; NLM Journal Code: hsn, 8006298; Other ID: Source: NLM. NIHMS376772 Source: NLM. PMC3368277; CAS Registry/EC Number/Name of Substance: 0 (Actins). 0 (Carrier Proteins). 0 (Peptide Fragments). 0 (myosin-binding protein C). 7440-70-2 (Calcium). 9013-26-7 (Actomyosin). EC 1-1-1-27 (L-Lactate Dehydrogenase). EC 3-6-1 (Cardiac Myosins).; Grant Number: HL007692 (United States NHLBI NIH HHS), HL101297 (United States NHLBI NIH HHS), HL62426 (United States NHLBI NIH HHS), R01HL105826 (United States NHLBI NIH HHS); Electronic Date of Publication: 20120417; Entry Date: 20120910