Hypertrophic cardiomyopathy (HCM) is a highly prevalent hereditary cardiac disease with over 1400 mutations identified in 11 genes encoding the protein constituents of sarcomere, the basic contractile units of muscle. However, the molecular mechanisms by which mutations in sarcomeric proteins lead to cardiac hypertrophy and failure remain poorly understood. Sarcomeres are composed of myofilaments flanked by dense protein structures called Z-discs, playing critical roles in controlling contraction and mediating signaling pathways. Moreover, recent evidence has suggested that sarcomeric protein post-translational modifications (PTMs) play essential roles in the modulation of contractile function. Our preliminary data convincingly show that the PTMs of sarcomeric proteins, including multiple key myofilament and Z-disc proteins, are altered in the myocardium of HCM patients as compared to donor hearts. Changes in the expression of myofilament protein isoforms were also detected in HCM patient myocardium. Intriguingly, we have observed consistent alterations in sarcomeric protein PTMs and isoform expression regardless of the mutation types. Thus, we hypothesize that different HCM- causing mutations activate convergent hypertrophic signaling pathways, yielding comparable alterations in PTMs and protein isoforms of the sarcomeric proteins that cause abnormal contractile function and pathological hypertrophy. To test our hypothesis, we will employ a highly interdisciplinary approach featuring top-down proteomics, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), cardiac tissue engineering, human patient heart tissue samples, and functional studies to delineate the underlying mechanisms of HCM. Our recently established top-down proteomics platform represents a comprehensive tool to detect and characterize HCM-related changes in the sarcomeric proteome with high reproducibility and throughput. Recently, we have made a major methodologic breakthrough that allows us to perform top-down proteomic analysis of minimal amounts of biopsy tissue, as well as iPSC-CMs cultured in either a 2D monolayer or 3D engineered cardiac tissue (ECT). In addition, biochemical analyses will be performed to assess changes in intracellular signaling pathways. Furthermore, we will perform functional measurements to relate sarcomeric protein PTM alterations and maladaptive signaling to contractile dysfunction.
We aim to determine: 1) the changes in sarcomeric protein PTMs, key hypertrophic signaling pathways, and mechanical properties in myocardial tissue from HCM patients; 2) the early molecular alterations and functional consequences of different sarcomeric protein mutations in HCM patient-specific iPSC-CMs; 3) the specific molecular alterations and functional consequences in iPSC-CM- ECT from multiple HCM patients carrying the same mutation. This application, enabled by cutting-edge technology, is highly significant with a strong scientific premise and a novel hypothesis. The proposed research has direct translational potential and will lead to an in-depth understanding of the underlying mechanisms in HCM.

Public Health Relevance

Hypertrophic cardiomyopathy (HCM) is a highly prevalent hereditary cardiac disease with over 1400 mutations identified in 11 genes encoding the protein constituents of sarcomere, but the molecular mechanisms by which mutations in the sarcomeric proteins lead to cardiac hypertrophy and failure remain poorly understood. In this application, we will employ a highly interdisciplinary approach featuring top-down proteomics, induced pluripotent stem cells derived cardiomyocytes, cardiac tissue engineering, clinical human heart tissues, and functional studies to delineate the underlying mechanisms of HCM. If successful, our studies will lead to an in-depth understanding of the underlying mechanisms in HCM and identify new therapeutic targets to treat this devastating disease. 1

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL096971-09
Application #
10086487
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Adhikari, Bishow B
Project Start
2011-08-05
Project End
2021-11-30
Budget Start
2020-12-01
Budget End
2021-11-30
Support Year
9
Fiscal Year
2021
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Chen, Bifan; Brown, Kyle A; Lin, Ziqing et al. (2018) Top-Down Proteomics: Ready for Prime Time? Anal Chem 90:110-127
Smelter, Dan F; de Lange, Willem J; Cai, Wenxuan et al. (2018) The HCM-linked W792R mutation in cardiac myosin-binding protein C reduces C6 FnIII domain stability. Am J Physiol Heart Circ Physiol 314:H1179-H1191
Cai, Wenxuan; Hite, Zachary L; Lyu, Beini et al. (2018) Temperature-sensitive sarcomeric protein post-translational modifications revealed by top-down proteomics. J Mol Cell Cardiol 122:11-22
Lin, Ziqing; Guo, Fang; Gregorich, Zachery R et al. (2018) Comprehensive Characterization of Swine Cardiac Troponin T Proteoforms by Top-Down Mass Spectrometry. J Am Soc Mass Spectrom 29:1284-1294
Wu, Zhijie; Tiambeng, Timothy N; Cai, Wenxuan et al. (2018) Impact of Phosphorylation on the Mass Spectrometry Quantification of Intact Phosphoproteins. Anal Chem 90:4935-4939
Lam, Maggie P Y; Ge, Ying (2018) Harnessing the Power of Proteomics to Assess Drug Safety and Guide Clinical Trials. Circulation 137:1011-1014
Martin-Garrido, Abel; Biesiadecki, Brandon J; Salhi, Hussam E et al. (2018) Monophosphorylation of cardiac troponin-I at Ser-23/24 is sufficient to regulate cardiac myofibrillar Ca2+ sensitivity and calpain-induced proteolysis. J Biol Chem 293:8588-8599
Chen, Zhilong; Song, Jiangping; Chen, Liang et al. (2018) Characterization of TTN Novex Splicing Variants across Species and the Role of RBM20 in Novex-Specific Exon Splicing. Genes (Basel) 9:
Chen, Bifan; Lin, Ziqing; Alpert, Andrew J et al. (2018) Online Hydrophobic Interaction Chromatography-Mass Spectrometry for the Analysis of Intact Monoclonal Antibodies. Anal Chem 90:7135-7138
Chen, Zhilong; Maimaiti, Rexiati; Zhu, Chaoqun et al. (2018) Z-band and M-band titin splicing and regulation by RNA binding motif 20 in striated muscles. J Cell Biochem 119:9986-9996

Showing the most recent 10 out of 50 publications