Proteomic analysis of mitral valve in Lewis rat with acute rheumatic heart disease

Abstract

Rheumatic heart disease (RHD) makes a heavy burden in human lives and economy. The proteomic analysis of acute rheumatic heart disease (ARHD) can provide precious data to study RHD at the early stages, but no one has looked into. So based on our early research we applied the method of continuous GAS stimulation on Lewis rats to duplicate the animal model of ARHD. And the mitral valves of rats in control group (n=10) and ARHD group (n=10) were selected for proteomic analysis of ARHD with the iTRAQ labeling based 2D LC-ESI-MS/MS quantitative technology. We identified 3931 proteins in valve tissue out of which we obtained 395 differentially expressed proteins containing 176 up-regulated proteins and 119 down-regulated proteins. Changes in levels of GAPDH (6.793 times higher than the control group) and CD9 (2.63 times higher than the control group) were confirmed by Western blot or immunohistochemistry. The differentially expressed proteins such as GAPDH, CD9, myosin, collagen and RAC1 may be potential biomarkers for ARHD. Moreover, the mitral valve protein profile shed light on further understanding and investigating ARHD.

Keywords: Acute rheumatic heart disease; ITRAQ quantitative proteomics; animal model; mitral valve protein profile; mitral valve tissue.

Figure 1

Hematoxylin and Eosin (H&E) staining and Masson staining of myocardial and valvular tissue. A. Normal myocardial and valve tissue in control group. Magnifications, ×200; B. Diffuse inflammatory cells infiltration in myocardium (arrows). Magnification, ×100; C. Diffuse inflammatory cells infiltration in valve (arrows). Magnification, ×100; D. Anitschkow cells (arrows). Magnification, ×400; E, F. Valve fibrosis (arrow) from 24 weeks of rats. Magnification, ×100 and ×400; G. Masson staining of valve in control group at 24 weeks. Magnification, ×40; H. Masson staining of valve in RHD group at 24 weeks. Magnification, ×40.

Figure 2

Validation of CD9 using immunohistochemical staining. A. CD9 was almost nil expressed in control group; B. CD9 overexpressed in cytoplasmic membrane and cytoplasm in ARHD group. magnification ×400.

Figure 3

Validation of GAPDH on pooled lysates by Western blotting. GAPDH was up-regulated in ARHD groups and the gray scale in ARHD group is 4.5 of that of the control group, which is consistent with the iTRAQ result.

Figure 4

Gene ontology for molecular functions of the identified 3931 proteins between the valve of ARHD and control group. The function about binding accounts for the highest ratio of 48.78 percent.

Figure 5

Focal adhesion pathway analysis based on the KEGG database. Ras-related C3 botulinum toxin substrate 1(RAC1), Ras-related protein Rap-1A, Chondroadherin, Myl9 protein, Protein Mylk and Collagen I increased in ARHD group, Talin increased in control group.