The Effects of Hypoxic Preconditioned Murine Mesenchymal Stem Cells on Post-Infarct Arrhythmias in the Mouse Model

Figure 1. Functional characterization of murine mesenchymal stem cells (MSC). Representative dot plots of the flow cytometric analysis show GFP+ expressing cells (green, B6 eGFP mouse strain), as well as phenotypic alterations in expression of the MSC relevant surface markers over passages: CD44, CD90, CD45 at passage 0 (P0), as well as CD44, Sca-1, CD29, and CD45 at P1, and P2 (A). MSC demonstrate a multipotent differentiation potency proved by immunofluorescence staining and positive expression of collagen II after chondrocytic differentiation (B), osteopontin after osteocytic differentiation (C), and fatty acid binding protein 4 (FABP 4) after adipocytic differentiation (D). Phase contrast images illustrate morphological characteristics following respective differentiations: cytoskeletal structures for osteocytes (C) and lipid drops with visible vacuoles for adipocytes (D).

Figure 2. Antiarrhythmic effects of engrafted mesenchymal stem cells (MSC). Comparison of developed ventricular arrhythmias (VA) between 45 min and 12 h post ligation of the left anterior descending artery. While there were no significant differences in terms of the development of VA after the 1st infarction. (A), stem cell-treated animals showed significantly reduced VA after the 2nd infarction (B). Mean ± SD; * p ≤ 0.05 compared to URI (Mann–Whitney U Test). Detection of GFP+ MSC in heart tissue immediately after transplantation (C) and 9 days after the 1st infarction (D). ES, extrasystoles; BG/TG Bigemini/Trigemini; VT, ventricular tachycardias; URI, untreated re-infarction, n = 7; TRI, stem cell treated re-infarction, n = 6; MIC, myocardial infarction control, n = 7.

Figure 3. Change of myocardial infarct scar: Fast green/Sirius red staining of the murine heart cross sections 10 days after 1st LAD ligation showed a significant decrease of the infarction size in case of stem cell treatment (TRI) in contrast to untreated infarction (URI + MIC). Mean ± SD; * p = 0.05 (t-test); (A). Analysis of the leukocytes infiltration using Hematoxylin/Eosin staining 48 h after a 2nd LAD ligation in animals of URI and TRI group showed significant enlargement of the infarct scar area in contrast to infarction control (MIC) group. Mean ± SD; # p < 0.05 (t-test); (B).

Figure 4. Identification of affected genes. Quality control with high Phred Score for all samples (green-good quality, yellow-mediocre quality, red-bad quality); (A); Venn-diagram with upregulated genes of TRI and URI compared to MIC as well as overlap of genes highly upregulated in TRI and URI (q ≤ 0.05); (B). URI, untreated re-infarction; TRI, stem cell treated re-infarction; MIC, MI control.

Figure 5. Antiarrhythmic pathways and associated genes in TRI-group. High expressed genes (small dots) and pathways (big dots) including macrophage markers, IL-5 signaling pathways, inflammatory and anti-inflammatory pathways (q ≤ 0.05). Node size-number of genes. Edges-relation based on kappa score 0.6 using experimental data.

Figure 6. Antiarrhythmic pathways and associated genes in URI-group. High expressed genes (small dots) and pathways (big dots; q ≤ 0.05). Node size-number of genes. Edges-relation based on kappa score 0.6 using experimental data.

Figure 7. Heatmap of highly upregulated genes. TRI specific upregulated genes compared to MIC and URI using log2 (foldchange) values (q ≤ 0.05). Colors: Red-upregulated; yellow-neutral; blue-downregulated.