Epicardial placement of human placental membrane protects from heart injury in a swine model of myocardial infarction

Figures

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  FIGURE 1. HPAC membrane preserved cardiac function. Echocardiographic parameters were measured Pre‐IRI and Post‐IRI and on postoperative day 14 (POD 14) in MI only (black circle; n = 3) and MI + HPAC (red square; n = 3) groups. (a) EF decreased in both groups post‐IRI with nearly restored EF in MI + HPAC group by POD 14 (*p = 0.014). (b) Preserved ESV (**p = 0.0036) and (c) EDV (*p = 0.011) are noted in MI + HPAC group by POD 14. (d) PWTd was significantly increased (*p = 0.0442) in MI only group. EDV, end diastolic volume; EF, ejection fraction; ESV, end systolic volume; PWTd, posterior wall thickness. Data is presented as mean ± S.E.M.

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  FIGURE 2. Infarct size was attenuated by HPAC membrane. After sacrifice, hearts were excised, and 0.5 cm thick short‐axis segments were stained with 1% 2,3,4‐triphenyl tetrazolium chloride (TTC) to determine infarct size. (a) Representative images of transverse slices between cardiac apex and base from the three groups (n = 3 per group) are shown with viable myocardium stained red and infarcted tissue remaining white. Scale bars = 1 cm. (b) Quantitative analysis demonstrated significant (*p = 0.039) decrease in infarct size to left ventricle (LV) area with HPAC membrane application. Data is presented as mean ± S.E.M.

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  FIGURE 3. Cardiac remodeling was evident in swine infarct and border zone. Punch biopsies from remote (nonischemic), border, and infarct zone were excised, fixed, and stained with picrosirius red (PSR) 14 days post ischemia reperfusion injury. Fibrosis is visualized as red fluorescence in PSR‐stained hearts using a Texas red fluorescence filter, and viable, non‐fibrotic tissue is visualized as green. (a) Representative images at merged 5X magnification (scale bar = 2 mm) of PSR and enlarged epicardial section at 20X (scale bar = 10um) are displayed. Robust cardiac remodeling and increased fibrosis is more evident in ischemic areas compared to nonischemic and border zone regions. (b) Quantification of collagen content demonstrated significant difference in groups (*p = 0.042) and zones (*p = 0.017), significant difference between remote and infarct zone in MI only swine hearts (**p = 0.0086), and significant difference between border zone in control and MI + HPAC (*p = 0.042). Data is presented as mean ± S.E.M.

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  FIGURE 4. H&E staining revealed changes in swine cellular architecture. (a) LV tissue from infarct, border, and remote zones were stained with Hematoxylin and Eosin (H&E) (n = 3 per group), and representative images at merged 5X magnification (scale bar = 2 mm) and 20X (scale bar = 10um) are displayed. Tissue from MI + HPAC infarct zone revealed (b) interstitial hemorrhage (white arrow), (c) granulation tissue with giant cells, (d) disordered cardiomyocytes, and (e) proliferation of lymphocytes and dendritic cells.

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  FIGURE 5. Transcriptome profile of punch biopsies from MI + HPAC and MI only hearts. (a) Heatmap representation of the transcriptome analysis with differentially expressed genes. High to low expression is shown by a degradation color from red to blue, respectively. The scale bar shows Z‐score values for the heatmap. (b) Volcano plot of ‐log10 (p value) versus log2 fold change. The ‐log10 (p values) represents the level of significance of each gene while log2 fold change represents the difference between the levels of expression for each gene between MI + HPAC and MI only groups. (c) KEGG pathway network analysis of upregulated genes (p < 0.05) in MI + HPAC versus MI only hearts. The functionally grouped network was visualized based on the degree of connectivity (node size) to extracellular matrix (ECM) pathways. (d) KEGG pathway enrichment bubble plot. The ratio of the number of different proteins in the corresponding pathways to the total number of proteins identified in the graph is greater, indicating the higher difference in protein concentration in the pathway. The size of the dots represents the number of different proteins in the corresponding pathway and the greater the difference…

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  FIGURE 6. Immune and ECM profiles alter with HPA treatment. (a) Heatmap of differential expressed immunomodulatory genes between MI only IZ versus control IZ, MI + HPAC IZ versus control IZ, and MI + HPAC IZ versus MI only IZ. (b) Heatmap of differential expressed ECM genes between MI only IZ versus control IZ, MI + HPAC IZ versus control IZ, and MI + HPA IZ versus MI only IZ. HPAC use demonstrates wound healing response. N = 3 for each group.

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  FIGURE 7. Proteomic analysis of infarct zone. (a) Volcano plot of MI + HPA infarct zone (IZ) to MI only IZ show 13 upregulated [pink] and 14 downregulated [teal] proteins as ‐log10(p‐value) and log2(fold change). (b) Principal component analysis of IZ of all three groups with PC1 of 56.9% and PC2 of 20.2%. (c) Statistical clustering and visualization using Hierarchical clustering (heat map) of IZ from MI + HPAC and MI only hearts. Expression levels were color coded, and clusters highlighted in the dendrogram and Z‐score weighted clusters showed distinct expression patterns visualized as a heat trace. N = 3 per group. p < 0.05 using one‐way ANOVA.

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  FIGURE 8. Proteomic analysis of infarct zone versus non‐infarct tissue. (a) Mini‐heatmaps of differentially expressed proteins between MI only IZ versus control IZ (non‐infarct) and MI + HPAC IZ versus control IZ. (b) GO and KEGG analysis of top pathways of the differentially expressed proteins in IZ between all three groups.

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  FIGURE 9. Immunoblot Analysis of Heat Shock Proteins in Control, IZ, and RZ with and without HPAC. Top Panel: Western blot depicting protein expression of total HSP40, HSP70, HSP90, and β‐tubulin. Bottom Panel: Bar graph summary of HSP40 (left), HSP70 (middle), and HSP90 (right) comparing protein levels in CONTROL, MI only IZ, MI + HPAC IZ, MI only RZ, and MI + HPAC RZ. Total HSP40, HSP70, and HSP90 were normalized to β‐tubulin after adjusting for total protein determined from Ponceau S. Data presented as mean ± S.E.M; n = 3 for each group. HSP40 and HSP90 levels were significantly different (p = 0.0159 and p = 0.0264, respectively, by ordinary one‐way ANOVA and the Tukey post hoc test; *p = 0.0103, **p = 0.0320 by post‐hoc analysis).