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.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
Project #
Application #
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Adhikari, Bishow B
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Wisconsin Madison
Schools of Medicine
United States
Zip Code
Gregorich, Zachery R; Chang, Ying-Hua; Ge, Ying (2014) Proteomics in heart failure: top-down or bottom-up? Pflugers Arch 466:1199-209
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
Xiu, Lichen; Valeja, Santosh G; Alpert, Andrew J et al. (2014) Effective protein separation by coupling hydrophobic interaction and reverse phase chromatography for top-down proteomics. Anal Chem 86:7899-906
Peng, Ying; Gregorich, Zachery R; Valeja, Santosh G et al. (2014) Top-down proteomics reveals concerted reductions in myofilament and Z-disc protein phosphorylation after acute myocardial infarction. Mol Cell Proteomics 13:2752-64
Guner, Huseyin; Close, Patrick L; Cai, Wenxuan et al. (2014) MASH Suite: a user-friendly and versatile software interface for high-resolution mass spectrometry data interpretation and visualization. J Am Soc Mass Spectrom 25:464-70
Gregorich, Zachery R; Ge, Ying (2014) Top-down proteomics in health and disease: challenges and opportunities. Proteomics 14:1195-210
Peng, Ying; Ayaz-Guner, Serife; Yu, Deyang et al. (2014) Top-down mass spectrometry of cardiac myofilament proteins in health and disease. Proteomics Clin Appl 8:554-68
Valkevich, Ellen M; Sanchez, Nicholas A; Ge, Ying et al. (2014) Middle-down mass spectrometry enables characterization of branched ubiquitin chains. Biochemistry 53:4979-89
Chen, Xin; Ge, Ying (2013) Ultrahigh pressure fast size exclusion chromatography for top-down proteomics. Proteomics 13:2563-6
Peng, Ying; Chen, Xin; Zhang, Han et al. (2013) Top-down targeted proteomics for deep sequencing of tropomyosin isoforms. J Proteome Res 12:187-98

Showing the most recent 10 out of 15 publications