Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates sarcomeric structure, as well as myocardial contractility, and confers cardioprotection. My long-term goal is to define the role(s) of cMyBP-C phosphorylation in contractile function in order to understand the molecular mechanisms that underlie cardioprotection. We recently showed that (1) cMyBP-C is an easily releasable and soluble myofilament, (2) dephosphorylation of cMyBP-C results in its degradation and (3) release into the blood post-myocardial infarction (MI) and (4) its N'-fragments appear within 30 minutes of ischemia-reperfusion injury. In addition, we showed that plasma cMyBP-C levels are significantly increased in animal models and patients with MI. Strikingly, the level of plasma cMyBP-C is significantly higher than the gold standard plasma cardiac troponin I (2.0-fold molar). However, verification and validation of the precise amount of plasma cMyBP-C is the next critical step. Therefore, using selective and specific proteomic approaches, the short-term goal is to develop an assay that precisely quantitates the levels of plasma cMyBP-C. My central hypothesis is that cMyBP-C is a bone fide early, selective and measurable cardiac-specific biomarker, which appears within 30 minutes of ischemia. Thus, the overall objectives of the proposal are as follows: determine a cMyBP-C-specific amino acid region in order to develop a selective proteomics-based assay using the liquid chromatography-tandem-mass spectrometry (LC-MS-MS) and selective reaction monitoring (SRM) approaches (Specific Aim 1);verify the accuracy of the SRM approach for quantifying plasma cMyBP-C levels in both animal and human plasma samples (Specific Aim 2);and cross-validate the SRM assay with the conventional sandwich ELISA assay (Specific Aim 3). Plasma samples taken from ischemia- reperfusion-injured mice and patients with MI will be used in these analyses, compared to naive and sham operated mice and normal healthy controls. Validating the proteomic approaches will result in an assay that can accurately measure the level of cMyBP-C in the circulatory system, as well as define its presence as an early-released, cardiac-specific and selective marker of early-onset MI.

Public Health Relevance

This proposal aims to confirm the efficacy of cardiac binding protein-C (cMyBP-C), a sarcomeric cardiac- specific thick filament assembly protein, as an early circulatory biomarker for myocardial infarction. Specifically, the proposed studies will verify and validate cMyBP-C plasma levels through advanced proteomic approaches, leading to the development of diagnostic assays that advance cardiac care.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Scientist Development Award - Research (K02)
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Special Emphasis Panel (ZHL1-CSR-K (M2))
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Carlson, Drew E
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Loyola University Chicago
Schools of Medicine
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Lee, Kyounghwan; Harris, Samantha P; Sadayappan, Sakthivel et al. (2015) Orientation of myosin binding protein C in the cardiac muscle sarcomere determined by domain-specific immuno-EM. J Mol Biol 427:274-86
Gao, Xianlong; Lin, Brian; Sadayappan, Sakthivel et al. (2014) Interactions between the regulatory subunit of type I protein kinase A and p90 ribosomal S6 kinase1 regulate cardiomyocyte apoptosis. Mol Pharmacol 85:357-67
Kuster, Diederik W D; Cardenas-Ospina, Adriana; Miller, Lawson et al. (2014) Release kinetics of circulating cardiac myosin binding protein-C following cardiac injury. Am J Physiol Heart Circ Physiol 306:H547-56
Sadayappan, Sakthivel; de Tombe, Pieter P (2014) Cardiac myosin binding protein-C as a central target of cardiac sarcomere signaling: a special mini review series. Pflugers Arch 466:195-200
Barefield, David; Kumar, Mohit; de Tombe, Pieter P et al. (2014) Contractile dysfunction in a mouse model expressing a heterozygous MYBPC3 mutation associated with hypertrophic cardiomyopathy. Am J Physiol Heart Circ Physiol 306:H807-15
Witayavanitkul, Namthip; Ait Mou, Younss; Kuster, Diederik W D et al. (2014) Myocardial infarction-induced N-terminal fragment of cardiac myosin-binding protein C (cMyBP-C) impairs myofilament function in human myocardium. J Biol Chem 289:8818-27
Kuster, Diederik W D; Sadayappan, Sakthivel (2014) MYBPC3's alternate ending: consequences and therapeutic implications of a highly prevalent 25 bp deletion mutation. Pflugers Arch 466:207-13
Wang, Li; Ji, Xiang; Barefield, David et al. (2014) Phosphorylation of cMyBP-C affects contractile mechanisms in a site-specific manner. Biophys J 106:1112-22
Simon, Jillian N; Chowdhury, Shamim A K; Warren, Chad M et al. (2014) Ceramide-mediated depression in cardiomyocyte contractility through PKC activation and modulation of myofilament protein phosphorylation. Basic Res Cardiol 109:445
Lynch, Thomas L; Sadayappan, Sakthivel (2014) Surviving the infarct: A profile of cardiac myosin binding protein-C pathogenicity, diagnostic utility, and proteomics in the ischemic myocardium. Proteomics Clin Appl 8:569-77

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