Sun, Zeyu, authorReardon, Kenneth, advisorHamilton, Karyn, committee memberOrton, Christopher, committee memberReisfeld, Bradley, committee member2007-01-032007-01-032011http://hdl.handle.net/10217/52111The soy-derived phytoestrogen genistein (GEN) has received attention for its potential to benefit the cardiovascular system by providing protection to cardiomyocytes against pathophysiological stresses. Although GEN is a well-known estrogen receptor (ER) agonist and a non-specific tyrosine kinase inhibitor, current understanding of the complex cellular and molecular effects of GEN in cardiomyocytes is still incomplete. The overall goal of this dissertation is to use high throughput proteomics methodologies to better understand the molecular action of GEN in cardiomyocytes and to identify proteins and pathways that respond to GEN treatment. The first study of this project focused on the concentration-dependent proteome changes in cultured HL-1 cardiomyocytes due to GEN treatments. Proteins from HL-1 cardiomyocytes treated with 1 μM and 50 μM GEN were prefractionated into hydrophilic and hydrophobic protein fractions and were analyzed by two-dimensional electrophoresis followed by protein identification using tandem mass spectrometry (MS). In total, 25 and 62 differential expressed proteins were identified in response to 1 μM and 50 μM of GEN treatment, respectively. These results suggest that 1 μM GEN enhanced the expression of heat shock proteins and anti-apoptotic proteins, while 50 μM GEN down-regulated glycolytic and antioxidant enzymes, potentially making cardiomyocytes more susceptible to energy depletion and apoptosis. The second study, employing a two-dimensional liquid chromatography and tandem MS shotgun proteomics workflow, was carried out to dissect the cellular functions changed in cardiomyocytes by ER-dependent or ER-independent actions of GEN. In this study, primary cardiomyocytes isolated from male adult SD rats were treated with 10 μM GEN without or with 10 μM ER antagonist ICI 182,780 (ERA) before proteomics comparison. A total of 14 and 15 proteins were found differentially expressed in response to the GEN, and the GEN+ERA treatment, respectively. Cellular functions such as glucose and fatty acid metabolism and cardioprotection were found to be modulated by GEN in an ER-dependent fashion, while proteins involved with steroidogenesis and estrogen signaling were identified as novel effectors of GEN via ER-independent actions. In this study, a consensus-iterative searching strategy was also developed to enhance the sensitivity of the shotgun proteomic approach. In the last study, an attempt to explore the response to a GEN stimulus in the signaling pathways, we developed a phosphopeptide enrichment method to assist the detection of protein phosphorylation in a complex peptide mixture. The quantitative performance of a sequential immobilized metal affinity chromatography (SIMAC) protocol was evaluated. We further conducted a preliminary application of this protocol in a large-scale, quantitative, label-free phosphoproteomics study to explore the alterations of protein phosphorylation patterns due to ER-independent GEN action in the SD rat cardiomyocytes. This project demonstrates the usefulness of proteomics methodologies to screen novel molecular targets influenced by GEN in cardiomyocytes. This is also the first investigation of the complex cellular impact of this soy-derived phytoestrogen in cardiomyocytes via a systems biology perspective.born digitaldoctoral dissertationsengCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.cardiomyocyteproteomicsphytoestrogensgenisteinText