Fig 1: Validation of the most representatitve protein changes detected in remote myocardium after acute infarction. (a) Western blot analysis and quantitative densitometry of TPM1 confirming the transient increase in thin filament sarcomeric proteins early after reperfusion. Full-length blots are presented in Supplementary Figure S8 as well as their raw integrated densities before and after normalization can be consulted in Supplementary Figure S12. Protein values are normalized relative to GAPDH. Results are shown as the mean ± s.e.m. of values obtained in at least four animals. *Indicates p value < 0.05 versus baseline by Mann-Whitney test. (b) Label free proteomics analysis confirming the decrease in thick filament proteins and an increase in thin filament proteins early after reperfusion (in at least four animals). Results are expressed as in Fig. 4d. Label free individual protein quantifications included in the thick filament and thin filament protein groups are shown in Supplementary Figure S14. (c) Quantitative analysis by PRM targeted proteomics of proteins ANT2, ACO2, ATP5A1, ATP5O, UQCRC1, MDH2, COX6B, GAPDH, MYH7, MYOM1, MYL3 and TPM1, confirming the transient changes in abundance of mitochondrial and contractile sarcomeric proteins after early reperfusion. Protein values are normalized by the total base peak area of the chromatograms. Results are shown as the mean ± s.e.m. of at least four animals at each time point in two technical replicates. *Indicates p value < 0.05; **indicates p value < 0.01, ***indicates p value < 0.001 versus baseline by Mann-Whitney test. iTRAQ quantifications of the proteins from panels a and c are shown in the Supplementary Figure S11b for comparison.
Fig 2: Validation of the most representatitve protein changes detected in ischemic myocardium after acute infarction. (a) Western blot analysis and corresponding quantitative densitometry of ATP5A, UQCRC2, SDHB, NDUFB8, UQCRFS1, COXIV and ATP5B confirming the decrease in mitochondrial protein content 7 days after reperfusion with respect to baseline. (b) Western blot analysis and quantitative densitometry of cardiac Myosin heavy chain, TNNT2 and TPM1 confirming the decrease in sarcomeric contractile proteins 7 days after reperfusion. Each lane of western blot corresponds to a protein extract obtained from one animal. Protein values are normalized relative to GAPDH. In the bar plots, results are shown as the mean ± s.e.m. of five determinations. *Indicates p value < 0.05; **indicates p value < 0.01 versus baseline by Mann-Whitney test. Full-length blots from panels a and b are presented in Supplementary Figure S8 as well as their raw integrated densities before and after normalization can be consulted in Supplementary Figures S9 and S10. iTRAQ quantifications of the proteins from panels a and b are shown in the Supplementary Figure S11a for comparison. (c) Label free individual protein quantifications of ATP5A, ATP5B, UQCRFS1, SDHB, MYH7, TNNT2 and TPM1 confirming the decrease in mitochondrial and contractile protein content. Protein quantifications are represented as log2(fold-change) of iBAQ intensities of individual animals from R-Day 7 compared with pooled average of iBAQ intensities from all Baseline group animals. (d) Label free quantitative proteomics confirming the decrease in mitochondrial and contractile proteins and the increase in acute-phase related proteins, collagens, extracellular matrix proteins (ECM), proteolysis related proteins and gene expression related protein contents, 7 days after reperfusion with respect to the baseline. Protein abundances were expressed as the iBAQ values. Results are shown as the mean ± s.e.m of five determinations performed in individual animals, *indicates FDR < 0.05; **indicates FDR < 0.01, ***indicates FDR < 0.001 versus baseline by Mann-Whitney test after Benjamini–Hochberg correction. Label free individual protein quantifications included in the mitochondrial, contractile, acute-phase, collagens, ECM, proteolysis and gene expression protein groups are shown in Supplementary Figure S13.
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