Short-Term Blockade of Pro-Inflammatory Alarmin S100A9 Favorably Modulates Left Ventricle Proteome and Related Signaling Pathways Involved in Post-Myocardial Infarction Recovery

Figures

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  Figure 1. Proteomic analysis of left ventricle on day 3 and 7 post-MI. The left ventricle was separated from the heart, harvested and processed for future analysis. (A) At 3 days (3D) post-MI, 1192 proteins were identified in the unoperated control (Ct), Sham, MI, and MI + ABR groups. The relative protein expression is presented as a pairwise comparison between the groups, where deeper blue represents lower abundance and deeper red represents higher abundance. (B) At day 7 post-MI (7D), a total of 1117 proteins were identified. (C) Venn diagram showing the number of common and different up- or downregulated proteins in MI + ABR versus MI control hearts at 3 and 7 days post-MI (D,E). Volcano plots of differential protein abundance, with fold difference between the log2 normalized abundance in the MI + ABR and MI groups plotted versus −log10 adjusted p value, at 3 D post-MI and 7 D post-MI. Each dot represents a protein. Colored dots represent the expression profiles of significantly different proteins with a relative fold change of ≥1.30 or ≤0.77. The calculated p value was obtained using ANOVA hypothesis test and was adjusted using Benjamini-Hochberg correction for the false discovery rate.

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  Figure 2. Associations between the biological processes related to ventricle repair and remodeling and differentially expressed proteins (DEPs) between the treatment groups. The protein–protein interaction networks at 3 days (A) and 7 days post-MI (B) were built using STRING 11.0. The proteins, identified by gene name, are represented as nodes. The thickness of the edge shows the strength of the interaction between two proteins.

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  Figure 3. S100A9 blockade lowers the level of leukocyte cell–cell adhesion molecules in the infarcted myocardium on day 3 post-MI. Normalized spectral abundance of (A) ITGβ1A and (B) MSN in the left ventricle for controls (Ct, Sham), PBS-treated (MI) and ABR-treated (MI + ABR) infarcted mice. Data are presented as mean ± SD; ITGβ1A, integrin beta-1 isoform A; MSN, moesin; ** p < 0.01, *** p < 0.001 vs. Ct; ### p < 0.001 vs. Sham; ^^^ p < 0.001 vs. MI; p values were calculated using Student’s t test.

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  Figure 4. Heatmaps and principal component analysis (PCA) of the relative abundance of DEPs controlling biological processes involved in myocardial repair and remodeling. (A) DEPs involved in the regulation of the muscle cell apoptotic process, (B) regulation of the intrinsic apoptotic signaling pathway and (C) sarcomere organization. Red represents upregulated and blue represents downregulated proteins in the LV collected from the MI + ABR group compared to MI mice. DEPs were considered to be significant at an adjusted p value < 0.05. The calculated p value was obtained using ANOVA hypothesis test and adjusted using Benjamini-Hochberg correction for the false discovery rate (FDR). Colored bars indicate the expression levels. X-marking represents proteins that were not significantly different between the groups. PCA score plot based on the LC-MS/MS data of DEPs involved in the regulation of the muscle cell apoptotic process, regulation of the intrinsic apoptotic signaling pathway, and sarcomere organization GO BP for all experimental groups at 3 days (D) and 7 days (E) post-MI. An ellipse highlights the data points coming from one group samples, which were run in three technical replicates…

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  Figure 5. Impact of 3-day S100A9 inhibition on proteins involved in cardiac hypertrophy. LC MS/MS analysis showing the abundance of ATP2A2 (A), MYH6 (B), TTN (C), NPM1 (D), CSRP3 (E), and NPPA (F) in the experimental groups at day 7 post-MI. Data are presented as mean ± SD. Each sample was run in triplicate; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Ct; # p < 0.05, ### p < 0.001 vs. Sham; ^^ p < 0.01, ^^^ p < 0.001 vs. MI; p values were calculated using Student’s t test; ATP2A2, ATPase 2; MYH6, myosin-6; TTN, titin; NPM1, nucleophosmin; CSRP3, glycine-rich protein 3; NPPA, natriuretic peptide A.

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  Figure 6. Effect of S100A9 blockade on p53 in the LV post-MI. mRNA expression levels of cellular tumor antigen P53 (P53) in the LV of unoperated control Ct, Sham, MI and MI + ABR groups quantified by real-time PCR using Tbp (TATA sequence binding protein) as internal control, at 3 (A) and 7 (B) days post-MI. Interactions of p53 with DEPs implicated in leukocyte cell–cell adhesion, regulation of the muscle cell apoptotic process, regulation of the intrinsic apoptotic signaling pathway, sarcomere organization and cardiac muscle hypertrophy GO biological processes (C). The proteins (identified by gene name) are represented as rectangle nodes. Data are presented as mean ± SD; * p < 0.05 vs. Ct; # p < 0.05 vs. Sham; ^^ p < 0.01 vs. MI; p values were calculated using Student’s t test.

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  Figure 7. Validation of several proteome data in the LV by Western blot analysis and qPCR. Representative Western blots for all experimental groups at 3 and 7 days post-MI (A,B). Semi-quantitative analyses displaying the protein levels of MSN and NPM1 normalized to total protein for MI and MI + ABR groups at 3 days post-MI (C,D). Semi-quantitative analyses showing protein levels of NPM1 and NPPA normalized to total protein for MI and MI + ABR groups at 7 days post-MI (E,F). Heatmap profile of differentially expressed genes at 3 and 7 days post-MI showing mRNA expression levels of Msn, S100a9, S100a8, Npm1, Sfpq, Slc9a3r1, Erp29, Nppa, Bag3, Nol3, Lmod2, Myh6, Atp2a2, Tcap, Ybx3 and Csrp3 in the left ventricle of MI and MI + ABR groups were quantified by real-time PCR, using Tbp as internal control (G). The gene expression data are presented as shades of blue for the log2 ratio of gene expression in MI + ABR vs. MI, depicting different levels of downregulation. Only the ratios for genes with significant differential expression are shown (p < 0.05). The Western blot data represent the mean ± SD; ^ p < 0.05, ^^ p < 0.01 vs. MI using Student’s t test.

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  Figure 8. Schematic diagram of the experimental design. Mice were subjected to myocardial infarction (MI) by permanent left coronary artery ligation. The S100A9 inhibitor ABR-238901 (ABR, 30mg/kg diluted in PBS) or PBS were administered intraperitoneally (i.p.) immediately (0D), at 1 day (1D) and at 2 days (2D) after MI. Echocardiography (ECHO) was performed one day before MI (1D) and at 3 (3D) and 7 days (7D) after MI. The left ventricle of control mice (unoperated and Sham) and mice with induced infarction (MI and MI + ABR) were harvested from the indicated area (yellow circles) at 3 days and 7 days post-MI and homogenized with TRIzol Reagent for RNA and protein isolation. The RNA samples were analyzed by real-time PCR assays. The protein samples were analyzed by Western blot and LC-MS/MS, followed by bioinformatic analysis.