Mechanisms of Regenerative Potential Activation in Cardiac Mesenchymal Cells

Figures

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  Figure 1. (a) Principal component analysis (PCA) displays variability in gene expression in healthy (green dots, n = 3) and postinfarction (orange dots, n = 3) myocardial tissues using the Phantasus Web Tool. Major component 1 (PC1) on the x-axis and PC2 on the y-axis accounted for 41.4% and 25.2% of the total variability in gene expression, respectively. PC1 covers a significant part (41.4%) of the experimental variability and largely reflects the difference between the two states of the heart muscle. PC2 represents experimental variability (25.2%) associated with the difference in gene profile between various biological repeats (laboratory animals). The samples are visually divided into two main groups. (b) Volcano plots showing differentially expressed genes (adjusted p-value < 0.05) in post-infarction samples compared to controls, performed using Bioconductor software in R. The y-axis corresponds to the mean expression value of −log10 (p-value), and the x-axis displays the log2 (fold-change) value. The red dots represent significantly differentially expressed genes.

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  Figure 2. The dynamics of the expression of the Notch signaling pathway and Bmp2/Runx2 in tissues by quantitative PCR analysis. SH—healthy myocardium from sham-operated rats (n = 3); 8 h-MI—tissue from the post-infarction zone of the myocardium from rats 8 h after induction (n = 4); 24 h-MI—tissue from the post-infarction zone of the myocardium from rats 24 h after induction (n = 9). Vertical—the relative amount of mRNA in each group, measured by the 2−ΔΔCT method; box plots with whiskers at min to the max are presented. * The asterisk shows significant differences between SH and MI groups at p < 0.05 (unpaired nonparametric Mann–Whitney test).

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  Figure 3. (a) Cardiac mesenchymal cells (CMCs) derived from healthy (SH-CMC) and postinfarct (MI-CMC) myocardium of rat during 5-day cultivation. CMCs were isolated 24 h after myocardial infarction. SH-CMC were obtained from rat hearts after sham operation. The primary cell culture of CMCs demonstrated the ability to form fibroblast colony-forming units (CFU-F) and had adhesion to a culture plastic. (b) Immunophenotyping of primary cultures of CMC using flow cytometry. The histograms show the expression levels of surface markers CD31, CD34, C45, CD90, and CD166 (red lines) concerning isotype (gray lines).

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  Figure 4. The proliferation rate of postinfarction cardiac mesenchymal cells (MI-CMC, orange curve, n = 9) and healthy cardiac mesenchymal cells (SH-CMC, blue curve, n = 3). Each curve represents the average values between the samples and the standard error of the mean (vertical lines). Horizontal—the time during which the measurements of proliferative activity were taken every 15 min. The experiment lasted for 72 h. Vertical—the value of the cell index, which reflects the quantitative information about the biological status of cells, including their number and viability. The seeding density was 5000 cells per well. The significance of differences between the SH-CMC and the MI-CMC is p < 0.05 with D’Agostino and Pearson omnibus normality test.

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  Figure 5. (a) Principal component analysis (PCA) displays variability in gene expression in healthy (green dots, n = 3) and postinfarction (orange dots, n = 3) CMCs using the Phantasus Web Tool. Major component 1 (PC4) on the x-axis and PC5 on the y-axis accounted for 14.5% and 8.9% of the total variability in gene expression, respectively. The samples are visually divided into two main groups. (b) Volcano plots showing differentially expressed genes (p-value < 0.05) in post-infarction samples compared to controls, performed using Bioconductor software in R. The y-axis corresponds to the mean expression value of -log10 (p-value), and the x-axis displays the log2 (fold-change) value. The red dots represent significantly differentially expressed genes.

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  Figure 6. Dysregulated signaling pathways in postinfarction CMC samples identified by IPA analysis. The histogram represents the dysregulated canonical signaling pathways in a stacked bar chart by Fisher’s Exact Test p-value (p-value > 0.05, z-score > 1). The ratio plot shows the number of significant genes expressed in the data versus the total number of genes in that particular signaling pathway.

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  Figure 7. Dynamics of expression of Notch signaling pathway components and Bmp2/Runx2 genes in cardiac mesenchymal cells using quantitative PCR analysis. SH—cells from a healthy myocardium of sham-operated rats (n = 3); 8 h-MI—post-infarction cells from rats 8 h after induction (n = 4); 24 h-MI—post-infarction cells from rats 24 h after induction (n = 9). Vertical—the relative amount of the mRNA in each group, measured by the 2-ΔΔCT method; box plots with whiskers at min to the max are presented. The asterisk shows significant differences between the SH and MI groups at p < 0.05 (unpaired nonparametric Mann–Whitney test).

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  Figure 8. Dynamics of expression of Notch signaling pathway components, Bmp2/Runx2 factors, and hypoxic stress markers in cardiac mesenchymal cells obtained from healthy myocardium of sham-operated rats by quantitative PCR analysis. NX—healthy cells in a state of normoxia (n = 3); 5% HX—healthy cells in a state of hypoxia with an oxygen level of 5% (n = 3); 1% HX—healthy cells in a state of hypoxia with an oxygen level of 1% (n = 3). Horizontal—the time at which the cells were in hypoxic conditions. Vertical—the relative amount of mRNA, measured by the 2-ΔΔCT method; box plots with whiskers at min to the max are presented. * The asterisk shows significant differences between the control and the 5% HX and 1% HX groups at p < 0.05 (unpaired nonparametric Mann–Whitney test).

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  Figure 9. The dynamics of the expression of Bmp2/Runx2 factors and Notch signaling pathways in cardiac mesenchymal cells obtained from healthy myocardium of sham-operated rats by quantitative PCR analysis. CTRL—healthy cells as a negative control (n = 3); 3 μL-NICD—healthy cells that had 3 μL of the virus with NICD added (n = 3); 15 μL-NICD—healthy cells that had 15 μL of the virus with NICD added (n = 3). Vertical—the relative amount of mRNA, measured by the 2−ΔΔCT method; box plots with whiskers at min to the max are presented. * The asterisk shows significant differences between CTRL and the 3 μL-NICD and 15 μL-NICD groups at p < 0.05 (unpaired nonparametric Mann–Whitney test).