Heart failure is the leading cause of death for both men and women in the United States. The underlying molecular and cellular mechanisms of heart failure are very complex and poorly understood. Key Myofilament Regulatory Proteins (KMRPs), which include cardiac troponin (cTn), tropomyosin (Tm), myosin regulatory light chain 2 (RLC2) and cardiac myosin binding protein C (cMyBP-C), play essential roles in cardiac contractility. The hypothesis is that both extrinsic and intrinsic stresses trigger the molecular signaling processes that result in altered modifications to KMRPs leading to contractile dysfunction and eventually heart failure. Recent studies show convincingly that altered modifications in cTnI and cMyBP-C are directly linked to cardiac dysfunction. An unbiased and systematic analysis of the KMRPs to globally detect the changes in protein modifications, identify which sites are modified or altered, and elucidate how these alterations act in concert during the transition to the heart failure is of paramount importance for the understanding of the underlying molecular mechanisms. However, this remains a major challenge. To address this challenge, we propose to establish a simple and robust top-down mass spectrometry (MS)-based disease proteomics platform to examine KMRPs extracted from both normal and diseased tissues to establish a correlation between altered modifications of KMRPs and cardiac dysfunction. Top-down MS directly analyzes intact proteins providing a """"""""bird's eye view"""""""" to observe all possible modifications simultaneously in one spectrum, which is much more reliable than measuring the proteolytically-digested peptides in the conventional bottom-up approach. The integrated top-down proteomics platform will provide a comprehensive tool to effectively separate the intact KMRPs extracted from myocardial tissues, globally detect all modifications that reflect extrinsic and intrinsic stresses, 3) identify and quantify (novel) modifications, and identify multiple concerted alterations in KMRPs and the changes in the distribution of PTMs among multiple targeted sites during the transition to the end- stage heart failure.
The specific aims i nclude: 1) Establish an integrated top-down disease proteomics technology for the separation and characterization of intact KMRPs with high efficiency, sensitivity and simplicity. 2) Determine altered protein modifications in KMRPs from hypertrophied and failing swine myocardium. 3) Determine the functional effects of protein kinase A (PKA) and protein kinase A (PKC)- mediated phosphorylation in KMRPs of normal and diseased swine myocardium. 4) Determine the functional consequence of one novel alteration in KMRPs, e.g. cTn, in regulating cardiac contractility. The success of this research project, which integrates proteomics and functional studies, will provide a global map of protein modifications occurring to the KMRPs under normal and diseased conditions and shed new insights into the molecular mechanism of contractile dysfunction in heart failure.

Public Health Relevance

Heart failure remains a leading cause of mortality and morbidity in the United States and is approaching epidemic levels in the aging population. This proposal aims to provide the new insights into the molecular mechanism of cardiac dysfunction in heart failure through an integrated proteomics and functional study. The research discoveries could foster the development of new therapeutic targets for better diagnosis and treatment of heart diseases.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL096971-02
Application #
8311648
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Adhikari, Bishow B
Project Start
2011-08-05
Project End
2016-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
2
Fiscal Year
2012
Total Cost
$372,210
Indirect Cost
$122,210
Name
University of Wisconsin Madison
Department
Physiology
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Gregorich, Zachery R; Peng, Ying; Cai, Wenxuan et al. (2016) Top-Down Targeted Proteomics Reveals Decrease in Myosin Regulatory Light-Chain Phosphorylation That Contributes to Sarcopenic Muscle Dysfunction. J Proteome Res 15:2706-16
Cai, Wenxuan; Guner, Huseyin; Gregorich, Zachery R et al. (2016) MASH Suite Pro: A Comprehensive Software Tool for Top-Down Proteomics. Mol Cell Proteomics 15:703-14
Cai, Wenxuan; Tucholski, Trisha M; Gregorich, Zachery R et al. (2016) Top-down Proteomics: Technology Advancements and Applications to Heart Diseases. Expert Rev Proteomics 13:717-30
Chen, Bifan; Peng, Ying; Valeja, Santosh G et al. (2016) Online Hydrophobic Interaction Chromatography-Mass Spectrometry for Top-Down Proteomics. Anal Chem 88:1885-91
Yu, Deyang; Peng, Ying; Ayaz-Guner, Serife et al. (2016) Comprehensive Characterization of AMP-Activated Protein Kinase Catalytic Domain by Top-Down Mass Spectrometry. J Am Soc Mass Spectrom 27:220-32
Jin, Yutong; Peng, Ying; Lin, Ziqing et al. (2016) Comprehensive analysis of tropomyosin isoforms in skeletal muscles by top-down proteomics. J Muscle Res Cell Motil 37:41-52
Gregorich, Zachery R; Peng, Ying; Lane, Nicole M et al. (2015) Comprehensive assessment of chamber-specific and transmural heterogeneity in myofilament protein phosphorylation by top-down mass spectrometry. J Mol Cell Cardiol 87:102-12
Chen, Yi-Chen; Sumandea, Marius P; Larsson, Lars et al. (2015) Dissecting human skeletal muscle troponin proteoforms by top-down mass spectrometry. J Muscle Res Cell Motil 36:169-81
Valeja, Santosh G; Xiu, Lichen; Gregorich, Zachery R et al. (2015) Three dimensional liquid chromatography coupling ion exchange chromatography/hydrophobic interaction chromatography/reverse phase chromatography for effective protein separation in top-down proteomics. Anal Chem 87:5363-71
Chang, Ying-Hua; Ye, Lei; Cai, Wenxuan et al. (2015) Quantitative proteomics reveals differential regulation of protein expression in recipient myocardium after trilineage cardiovascular cell transplantation. Proteomics 15:2560-7

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