Pilot study identifying myosin heavy chain 7, desmin, insulin-like growth factor 7, and annexin A2 as circulating biomarkers of human heart failure

Abstract

In-depth proteomic analyses offer a systematic way to investigate protein alterations in disease and, as such, can be a powerful tool for the identification of novel biomarkers. Here, we analyzed proteomic data from a transgenic mouse model with cardiac-specific overexpression of activated calcineurin (CnA), which results in severe cardiac hypertrophy. We applied statistically filtering and false discovery rate correction methods to identify 52 proteins that were significantly different in the CnA hearts compared to controls. Subsequent informatic analysis consisted of comparison of these 52 CnA proteins to another proteomic dataset of heart failure, three available independent microarray datasets, and correlation of their expression with the human plasma and urine proteome. Following this filtering strategy, four proteins passed these selection criteria, including myosin heavy chain 7, insulin-like growth factor-binding protein 7, annexin A2, and desmin. We assessed expression levels of these proteins in mouse plasma by immunoblotting, and observed significantly different levels of expression between healthy and failing mice for all four proteins. We verified antibody cross-reactivity by examining human cardiac explant tissue by immunoblotting. Finally, we assessed protein levels in plasma samples obtained from four unaffected and four heart failure patients and demonstrated that all four proteins increased between twofold and 150-fold in heart failure. We conclude that MYH7, IGFBP7, ANXA2, and DESM are all excellent candidate plasma biomarkers of heart failure in mouse and human.

Keywords: Animal proteomics; Bioinformatics; Plasma; Secreted protein.

Figure 1. Correlation of Proteomic Dataset with Other Models of Cardiomyopathy

Proteomics data for the top 52 protein candidates in the CnA model was compared to the R9C model of dilated cardiomyopathy. Twenty five proteins showed concordance with up-regulation in both CNA and R9C, which were further compared to GEO microarrays: isoproterenol-induced cardiomyopathy and exercise-induced cardiac hypertrophy (GSE18801), left ventricular hypertrophy model (GSE2459), and microarray analysis of gene expression during mild and severe cardiac hypertrophy (GSE4678).

Figure 2. Schematic Diagram Describing Protein Target Selection

Protein expression from the CnA dataset was mapped against the PLN R9C dataset, a model of dilated cardiomyopathy. Forty-five proteins were present in both datasets. Of these 45 proteins, 25 were upregulated in both conditions, and these proteins were compared subsequently to 3 independent microarray datasets of heart failure. Eleven proteins showed up-regulation in at least one of the microarrays, and these were further mapped to the urinary proteome and high-confidence plasma proteome. Seven proteins were present in either the urinary or plasma proteome. Intensity of the bands is indicated by the color bar, N/D indicates the gene/protein was not detected in the dataset.

Figure 3. Assessment of Protein Expression in Mouse Plasma

Expression of ANXA2, IGFBP7, MYH7, and DESM was evaluated in mouse plasma. (A) Three control samples and three CnA samples were assessed by immunoblot. As a positive control, CnA heart lysate was included (not shown). Molecular weights are indicated on the left. (B) Following immunoblot analysis, quantification of signals at the appropriate molecular weights was. Shown are densities obtained in the WT and CnA samples and are represented in arbitrary units. Mean and standard error of the mean (SEM) are reported; *, p<0.05, t-test.

Figure 4. Antibody Cross-Reactivity in Human Tissue

The cross-reactivity of antibodies used in mouse samples was tested in human explants obtained from dilated cardiomyopathic patients. Shown are representative immunoblots of these human cardiac tissues along with ventricular tissues obtained from wild-type and CnA mice.

Figure 5. Protein Expression in Human Plasma Samples

(A) Immunoblots of ANXA2, IGFBP7, MYH7 and DESM levels were assessed in unaffected and heart failure patient samples. (B) Quantification of proteins targeted in human plasma was performed to evaluate expression levels. Shown are band densities represented in arbitrary units. Mean and SEM are reported; *, p<0.05, t-test.

Figure 6. Protein Expression in Human Cardiac Ventricular Sections

Immunohistochemistry validation using the Human Protein Atlas database was performed and representative images were obtained for MYH7 (antibody HPA001239), DESM (antibody CAB000034), IGFBP7 (antibodies HPA002196 and CAB020668), and ANXA2 (antibody CAB004311). Patient IDs of #2424, #2423 and #3732 are shown. Inset, higher magnification images for sections. Scale bar is 100 μm.