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.
authors.affiliation: Department of Cell and Molecular Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA.
authors.forename: Suresh
authors.initials: S
authors.lastname: Govindan
authors.forename: Jason
authors.initials: J
authors.lastname: Sarkey
authors.forename: Xiang
authors.initials: X
authors.lastname: Ji
authors.forename: Nagalingam R
authors.initials: NR
authors.lastname: Sundaresan
authors.forename: Mahesh P
authors.initials: MP
authors.lastname: Gupta
authors.forename: Pieter P
authors.initials: PP
authors.lastname: de Tombe
authors.forename: Sakthivel
authors.initials: S
authors.lastname: Sadayappan
mesh_terms: D000107:Acetylation; D000199:Actins / Q000378:metabolism; D000205:Actomyosin / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D024722:Cardiac Myosins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D016923:Cell Death; D002478:Cells, Cultured; D006352:Heart Ventricles / Q000378:metabolism / Q000473:pathology; D047468:Immunoprecipitation; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009119:Muscle Contraction; D009206:Myocardium / Q000378:metabolism / Q000473:pathology*; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000473:pathology*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D059748:Proteolysis; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000378:metabolism / Q000473:pathology; D012518:Sarcomeres / Q000378:metabolism / Q000473:pathology; D000099163:Myosin Binding Protein C
chemical_list: D000199:Actins; D000205:Actomyosin; D002118:Calcium; D024722:Cardiac Myosins; D002352:Carrier Proteins; D007770:L-Lactate Dehydrogenase; D010446:Peptide Fragments; D000099163:Myosin Binding Protein C