Functional Proteomic Analysis of Experimental Autoimmune Myocarditis-Induced Chronic Heart Failure in the Rat

Abstract

Congenital or acquired problems in myocardium cause heart failure. To study congenital heart failure, we have used TO-2 hamsters.1,2) To study the acquired type, we have used experimental autoimmune myocarditis (EAM). The immunization of Lewis rats with pig cardiac myosin results in acute myocarditis-induced heart failure followed around 8 weeks late by a stable chronic phase.3) We have investigated the therapeutic effects of several drugs and conducted etiological studies in the acute and chronic phases of heart failure using the EAM model. [3][4][5][6] We have been interested in proteins that correlate well with the progression of EAM-induced heart failure. In the present study, we conducted a proteomic analysis to specify and identify proteins which correlate well with the changes in cardiac function in the chronic phase of heart failure. Although there have been several reports of proteomic analyses of heart failure, 7-11) none was on EAM-induced heart failure. Moreover, these reports compared only levels of proteins with those in normal heart, and did not examine the correlation between protein expression and cardiac function. A detailed comparison of changes in protein expression with changes in the heart function of the failing heart would provide insights into the mechanisms underlying myocardial remodeling and deterioration and may lead to the discovery of potential drug targets.In this study, we performed a detailed comparison of changes in protein expression in the EAM-induced chronic heart failure model with heart function and pathological parameters. We focused on major proteins in myocardium prefractionated into membranous (M) and soluble (S) fractions.We found several proteins, including glucose regulated protein (GRP)78 and heat shock protein (HSP)90b which showed excellent correlations between their levels and heart functions. MATERIALS AND METHODS Animals and ProceduresNine-week-old male Lewis rats (Charles River Japan Inc., Kanagawa, Japan) were used. Autoimmune myocarditis was induced as described previously.3) Ten immunized rats were used as a heart failure group (HF-group) and 10 age-matched normal Lewis rats were used as a control group (N-group). The morbidity of experimental autoimmune myocarditis was 100%. The acute phase of myocarditis-induced heart failure ceased at around 4 weeks and stable chronic heart failure developed. The rats were used at 8 weeks after immunization (HF-group). The protocol was approved by the Committee on the Guidelines for Animal Experimentation of Niigata University. All animals were treated in accordance with the guidelines for animal experiments as laid out by our institute.Measurement of Hemodynamic Parameters Echo cardiographs and hemodynamic parameters were measured under 2% isoflurane in pure oxygen gas anesthesia with spontaneous respiration.1,3) B-and M-mode echo-cardiograms were obtained using a linear scan probe (13 MHz, EUB6500, Hitachi, Tokyo, Japan). The ejection fraction (EF) and fractional shortening (FS) were calculated. Left ventricular press...

Characterization of the Human Myocardial Proteome in Inflammatory Dilated Cardiomyopathy by Label-free Quantitative Shotgun Proteomics of Heart Biopsies

“…For 174 of the 485 proteins covered, disease-associated changes exceeding a minimum cutoff value of 1.3 ( p < 0.05) were found. This corresponds to about 35% of all proteins, which exceeds the changes described for 2D gel approaches, where often approximately 10−15% of the protein species were found to be affected. , These differences might be caused by two issues: (i) by gel-free mass spectrometry-based approaches, total protein levels are recorded, whereas by 2D gels, proteins are separated into species including post-translational modifications or fragments, (ii) in 2D gel approaches the number of proteins displaying statistically significant changes grossly depends on the p I range covered and the reproducibility of quantification across gels, which is rather low if traditional staining methods (e.g., colloidal coomassie brilliant blue or silver nitrate) were used but significantly improved by application of differential in-gel electrophoresis…”

“…This corresponds to about 35% of all proteins, which exceeds the changes described for 2D gel approaches, where often approximately 10À15% of the protein species were found to be affected. 28,29 These differences might be caused by two issues: (i) by gel-free mass spectrometry-based approaches, total protein levels are recorded, whereas by 2D gels, proteins are separated into species including post-translational modifications or fragments, (ii) in 2D gel approaches the number of proteins displaying statistically significant changes grossly depends on the pI range covered and the reproducibility of quantification across gels, which is rather low if traditional staining methods (e.g., colloidal coomassie brilliant blue or silver nitrate) were used but significantly improved by application of differential in-gel electrophoresis. 30 Functional classification of the proteins displaying iDCM-associated changes in levels not only confirms but extends the results of proteomic analyses of different animal models and some 2DE analyses of human specimen performed in the 1990s and reviewed by Arrell et al 31 and McGregor and Dunn.…”

Nutritional B vitamin deficiency alters the expression of key proteins associated with vascular smooth muscle cell proliferation and migration in the aorta of atherosclerotic apolipoprotein E null mice

“…The HUPO Plasma Proteome Project identified more than 300 human plasma proteins as strong biomarkers of vascular function and/or disease. These include markers of inflammation (transferrin), coagulation (fibrinogen A a-polypetide, fibronectin) and cytoskeletal organisation (actin, myosin, talin and villin) (Sanzen et al 2010). Protein profiling has also been employed to investigate mechanism of vascular dysfunction in animal models.…”

Proteomic analysis of physiological versus pathological cardiac remodeling in animal models expressing mutations in myosin essential light chains

“…Heat shock protein (HSP)-90β and stress-induced phosphoprotein 1 were both found to be downregulated in Δ43 hearts relative to A57G hearts (Tables 1, 4). In an autoimmune myocarditis-induced chronic heart failure rat model HSP-90β was suggested to play a protective role (Sanzen et al 2010). Increased levels of stress-induced phosphoprotein-1 have been associated with cancer (Cho et al 2014).…”