title: Top-down proteomics reveals concerted reductions in myofilament and Z-disc protein phosphorylation after acute myocardial infarction.
abstract: Heart failure (HF) is a leading cause of morbidity and mortality worldwide and is most often precipitated by myocardial infarction. However, the molecular changes driving cardiac dysfunction immediately after myocardial infarction remain poorly understood. Myofilament proteins, responsible for cardiac contraction and relaxation, play critical roles in signal reception and transduction in HF. Post-translational modifications of myofilament proteins afford a mechanism for the beat-to-beat regulation of cardiac function. Thus it is of paramount importance to gain a comprehensive understanding of post-translational modifications of myofilament proteins involved in regulating early molecular events in the post-infarcted myocardium. We have developed a novel liquid chromatography-mass spectrometry-based top-down proteomics strategy to comprehensively assess the modifications of key cardiac proteins in the myofilament subproteome extracted from a minimal amount of myocardial tissue with high reproducibility and throughput. The entire procedure, including tissue homogenization, myofilament extraction, and on-line LC/MS, takes less than three hours. Notably, enabled by this novel top-down proteomics technology, we discovered a concerted significant reduction in the phosphorylation of three crucial cardiac proteins in acutely infarcted swine myocardium: cardiac troponin I and myosin regulatory light chain of the myofilaments and, unexpectedly, enigma homolog isoform 2 (ENH2) of the Z-disc. Furthermore, top-down MS allowed us to comprehensively sequence these proteins and pinpoint their phosphorylation sites. For the first time, we have characterized the sequence of ENH2 and identified it as a phosphoprotein. ENH2 is localized at the Z-disc, which has been increasingly recognized for its role as a nodal point in cardiac signaling. Thus our proteomics discovery opens up new avenues for the investigation of concerted signaling between myofilament and Z-disc in the early molecular events that contribute to cardiac dysfunction and progression to HF.
authors: From the ‡Department of Cell and Regenerative Biology, University of Wisconsin-Madison, 1300 University Ave., Madison, Wisconsin 53706;
authors: Ying
authors: Y
authors: Peng
authors: From the ‡Department of Cell and Regenerative Biology, University of Wisconsin-Madison, 1300 University Ave., Madison, Wisconsin 53706; §Molecular Pharmacology Training Program, University of Wisconsin-Madison, 1300 University Ave., Madison, Wisconsin 53706;
authors: Zachery R
authors: ZR
authors: Gregorich
authors: From the ‡Department of Cell and Regenerative Biology, University of Wisconsin-Madison, 1300 University Ave., Madison, Wisconsin 53706;
authors: Santosh G
authors: SG
authors: Valeja
authors: From the ‡Department of Cell and Regenerative Biology, University of Wisconsin-Madison, 1300 University Ave., Madison, Wisconsin 53706;
authors: Han
authors: H
authors: Zhang
authors: From the ‡Department of Cell and Regenerative Biology, University of Wisconsin-Madison, 1300 University Ave., Madison, Wisconsin 53706; §Molecular Pharmacology Training Program, University of Wisconsin-Madison, 1300 University Ave., Madison, Wisconsin 53706;
authors: Wenxuan
authors: W
authors: Cai
authors: ¶Department of Chemistry, University of Wisconsin-Madison, 1300 University Ave., Madison, Wisconsin 53706;
authors: Yi-Chen
authors: YC
authors: Chen
authors: From the ‡Department of Cell and Regenerative Biology, University of Wisconsin-Madison, 1300 University Ave., Madison, Wisconsin 53706; ‖Human Proteomics Program, University of Wisconsin-Madison, 1300 University Ave., Madison, Wisconsin 53706;
authors: Huseyin
authors: H
authors: Guner
authors: From the ‡Department of Cell and Regenerative Biology, University of Wisconsin-Madison, 1300 University Ave., Madison, Wisconsin 53706;
authors: Albert J
authors: AJ
authors: Chen
authors: From the ‡Department of Cell and Regenerative Biology, University of Wisconsin-Madison, 1300 University Ave., Madison, Wisconsin 53706;
authors: Denise J
authors: DJ
authors: Schwahn
authors: ‡‡Department of Medicine, University of Wisconsin-Madison, 1300 University Ave., Madison, Wisconsin 53706;
authors: Timothy A
authors: TA
authors: Hacker
authors: §§Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, 719 Indiana Ave., Indianapolis, Indiana 46202; ¶¶Center for Computational Biology and Bioinformatics, Indiana University School of Medicine, 410 West 10th Street, Indianapolis, Indiana 46202.
authors: Xiaowen
authors: X
authors: Liu
authors: From the ‡Department of Cell and Regenerative Biology, University of Wisconsin-Madison, 1300 University Ave., Madison, Wisconsin 53706; ¶Department of Chemistry, University of Wisconsin-Madison, 1300 University Ave., Madison, Wisconsin 53706; ‖Human Proteomics Program, University of Wisconsin-Madison, 1300 University Ave., Madison, Wisconsin 53706; ge2@wisc.edu.
authors: Ying
authors: Y
authors: Ge
mesh_terms: D000818:Animals; D001665:Binding Sites; D004195:Disease Models, Animal; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D009203:Myocardial Infarction / Q000378:metabolism* / Q000473:pathology; D009210:Myofibrils / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D013552:Swine
chemical_list: D008840:Microfilament Proteins