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

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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, Ca2+ 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 Ca2+ handling. Adult cardiomyocytes expressing 40 kDa fragments exhibited similar impairment of Ca2+ 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 Ca2+ 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.

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Abbreviations

cMyBP-C:

Cardiac myosin binding protein-C

Ca2+ :

Calcium ions

h:

Hour(s)

I–R:

Ischemia–reperfusion

Ig:

Immunoglobulin

kDa:

Kilodaltons

LDH:

Lactate dehydrogenase

MI:

Myocardial infarction

M domain:

Myosin binding domain

Min:

Minutes

MOI:

Multiplicity of infection

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

N′:

N-terminal

NRVCM:

Neonatal rat ventricular cardiomyocyte

PBS:

Phosphate buffered saline

PKA:

Protein kinase A

SR:

Sarcoplasmic reticulum

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Acknowledgments

We thank our collaborators, Jody L. Martin, PhD, in the adenoviral core faculty for viral production and NRVCMs preparation, and Aleksey Zima, PhD, for providing us the rabbit adult ventricular cardiomyocytes at the Cell and Molecular Physiology, Loyola University Chicago, Stritch School of Medicine, Maywood, IL 60153, USA. We also thank, Jeffery D. Molkentin, PhD, Cincinnati Children’s Hospital, Cincinnati, OH 45229, USA, for providing us the mouse I–R injury surgery training.

Funding

This study was funded by National Institutes of Health grants HL007692 (Dr. Sarkey), HL101297, HL62426 (Dr. de Tombe) and R01HL105826 (Dr. Sadayappan), American Heart Association—Post-Doctoral Training Grant (10POST4230040 to Dr. Sundaresan) and—Scientist Development Grant (0830311N to Dr. Sadayappan).

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Authors and Affiliations

  1. Department of Cell and Molecular Physiology, Stritch School of Medicine, Loyola University Chicago, 2160 South First Ave., Maywood, IL, 60153, USA

    Suresh Govindan, Jason Sarkey, Xiang Ji, Pieter P. de Tombe & Sakthivel Sadayappan

  2. Department of Surgery, University of Chicago, Chicago, IL, 60637, USA

    Nagalingam R. Sundaresan & Mahesh P. Gupta

Authors
  1. Suresh Govindan
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  2. Jason Sarkey
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  3. Xiang Ji
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  4. Nagalingam R. Sundaresan
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  5. Mahesh P. Gupta
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  6. Pieter P. de Tombe
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  7. Sakthivel Sadayappan
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Corresponding author

Correspondence to Sakthivel Sadayappan.

Additional information

Suresh Govindan and Jason Sarkey contributed equally to this work.

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Govindan, S., Sarkey, J., Ji, X. et al. Pathogenic properties of the N-terminal region of cardiac myosin binding protein-C in vitro. J Muscle Res Cell Motil 33, 17–30 (2012). https://doi.org/10.1007/s10974-012-9292-y

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  • DOI: https://doi.org/10.1007/s10974-012-9292-y

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