Bioceramics-enhanced patch activates epicardial epithelial-to-mesenchymal transition via Notch pathway for cardiac repair

Figures

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  Fig. 1.. Preparation and characterization of the LMS-containing hydrogel scaffolds. (A) Digital photos and scanning electron microscopy (SEM) images of the scaffolds with different concentrations of LMS. Scale bar, 100 μm. (B) C, N, O, and Si elements distribution and content in the GelMA and GelMA-4LMS scaffolds. Scale bars, 100 μm. (C) The cumulative ionic release curves of the scaffolds containing different concentrations of LMS. n = 3. (D) Live/dead staining images of the CMs seeded on different scaffolds (green, live cells; red, dead cells). Scale bars, 200 μm. (E) Statistics of the viability of CMs cultured on different scaffolds. n = 5. (F) The absorbance at 450 nm of the CMs cultured on different scaffolds after cell counting kit–8 incubation at days 1 and 3. n = 4. OD, optical density; n.s., not significant. (G) The SEM images of the CMs cultured on different scaffolds. Scale bar, 10 μm.

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  Fig. 2.. The functionalization and electrocoupling of naive CMs after cultured on the LMS-containing hydrogel scaffolds. (A) Immunostaining for CX43 (red) and α-actinin (green) proteins expression of CMs cultured on different scaffolds. The enlarged images of CX43 and α-actinin staining in white squares were shown on the lower row. Scale bars, 25 μm. (B) The quantitative statistics of α-actinin– and CX43-positive area and Z-band width. n = 6. Quantitative data of CX43 were presented with median and interquartile range values and compared using the rank sum test. (C) Calcium transients (top) and corresponding frequency signals of Ca2+ (bottom) of CMs on different scaffolds after 7 days of culture. Scale bar, 40 μm. (D and E) Quantitative analysis of calcium transient parameters [time to peak (D) and peak numbers/5 s (E)]. n = 3. (F) Photographs of CMs cultured on different scaffolds for 7 days exhibited contraction behavior under a microscope field (top) and beating signal graphs (bottom). White arrows indicated the direction of contraction. (G) Heatmaps of contraction behavior; the red area was the diastolic scaffolds area minus the systolic scaffolds area. (H) Quantified contraction…

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  Fig. 3.. The GelMA-4LMS scaffolds activated epicardial cell EMT. (A) The morphological changes of epicardial cells were observed under optical microscope (top) and cytoskeletal immunofluorescence staining (bottom) at day 3. Scale bars, 25 μm. (B) The scratch test of epicardial cells at different time points. Scale bars, 250 μm. (C) Immunofluorescence staining of EMT-associated proteins, including epithelial E-cadherin protein (green) and mesenchymal N-cadherin protein (red) at day 1 and 3. Scale bars, 50 μm. (D) Quantitative analysis of E-cadherin and N-cadherin protein expression. n = 3. (E) qRT-PCR validation of the genes related to the Notch pathway at day 3. Quantitative data of Jag2 was presented with median and interquartile range values and compared using the rank sum test. (F) qRT-PCR analysis of EMT-related genes at day 3; DAPT was used to inhibit the Notch pathway. n = 3. (G) Schematic diagram of the potential signal pathway of epicardial cells induced EMT by GelMA-4LMS scaffolds.

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  Fig. 4.. Bioceramics-enhanced scaffolds repaired damaged CMs by inducing epicardial cell EMT and paracrine action. (A) The transwell coculture experiment of epicardial cells cultured with scaffolds and damaged primary CMs. (B) Immunofluorescence staining of cardiac-specific proteins from damaged CMs in the transwell lower chamber. Undamaged (UN) group: the CMs cultured without injury. LPS/negative control (LPS/NC) group: not treated after CMs were damaged for 24 hours. LPS/epicardial cell group: The damaged CMs were cocultured with epicardial cells. LPS/epicardial cell/GelMA group: The damaged CMs were cocultured with epicardial cells inoculated on the GelMA scaffold. LPS/GelMA-4LMS: The damaged CMs were cocultured with the GelMA-4LMS hydrogel scaffold. LPS/Conditioned medium: The damaged CMs were treated with the medium in which epicardial cells were inoculated on the GelMA-4LMS scaffold. LPS/epicardial cell/GelMA-4LMS group: The damaged CMs were cocultured with epicardial cells inoculated on the GelMA-4LMS scaffold. Scale bars, 50 μm. (C) Quantitative statistics of α-actinin– and CX43-positive area. n = 3. (D) Photos of CMs under the microscope during 7 days of culture in different…

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  Fig. 5.. Histological immunofluorescence staining showed that the GelMA-4LMS ECP improved the repair of damaged myocardium. (A) Wheat germ agglutinin (WGA) staining showed CM boundaries in the infarction area and the quantitative statistics of cell size. n = 6. Scale bar, 40 μm. (B) vWF (red) and α-SMA (green) expression in the infarct region in different groups. Scale bars, 25 μm. (C) Wt1 (red) and CD105 (green) immunofluorescence staining of tissue sections. Scale bar, 50 μm. (D) Statistical analysis of arteriole number based on the immunostaining images. n = 5. (E) Immunofluorescence staining of tissue sections for CD86 (green) and CD206 (red). Scale bar, 100 μm. (F) Quantitative analysis of the ratio of M2/M1 macrophages in cardiac sections based on immunostaining images. n = 4. These variance-independent measurement data were presented as median and interquartile range.

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  Fig. 6.. The GelMA-4LMS patch reconstructed myocardial electrical conduction in vivo. (A) The schematic illustration of the electrical propagation captured on a Langendorff perfusion heart using a 64-channel electrode array mapping system. Signals were obtained using no electrical stimulation and 4-mV and 5-Hz pulsed electrical stimulation. The stimulating electrode was positioned inferior to the atrial appendage, and the 64-channel electrode was placed at the border region. (B) The epicardial activation maps for each group using 4-mV and 5-Hz pulsed electrical stimulation. Red, the earliest activation; blue, the latest activation. IR means infarcted region. The dark lines demarcated the boundary between the non-infarcted and infarcted myocardium. Numbers indicated activation time (milliseconds). (C) Voltage amplitude of myocardial infarction area in each group without stimulation. (D) CX43 immunostaining (red) and α-actinin immunostaining (green) in the infarct area for each group. Scale bars, 100 μm. (E) The conduction velocities from healthy myocardium to infarcted myocardium were calculated. n = 4. (F) Quantified the average voltage amplitude of the different groups. n = 4. (G and H)…

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  Fig. 7.. Evaluation of cardiac function through echocardiography and histological analysis. (A) Representative images of Masson’s trichrome staining for heart sections in all groups. Red color, myocardium tissue; blue color, fibrosis tissue. Scale bars, 1 mm (top row) and 250 μm (bottom row). (B and C) Quantitative analysis of infarct area (B) and wall thickness (C) of left ventricular anterior wall based on the images of Masson’s trichrome staining. n = 5. (D) Representative M-mode echocardiographic images of 1 and 4 weeks after surgery. (E) The changes of EF, FS, LVIDd, and LVIDs for each group were observed by trajectories 1 to 4 weeks after surgery. (F) Quantitative analysis of left ventricular function-related parameters after 4 weeks of surgery. These variance-independent measurement data were presented as median and interquartile range.

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  Fig. 8.. Gene expression pattern analysis of rats in different groups at 4 weeks after transplantation by RNA-seq. (A) The PCA of CM RNA-seq. PC, principal component. (B) The number of differentially expressed genes (DEGs) in various groups following comparison. DEGs with |log2 fold change| ≥ 1 and false discovery rate < 0.05 were selected as significant differential genes. (C to E) Hierarchical clustering analysis was performed for DEGs in each group, and K-means algorithm was used to divide the DEGs into two sets [(C) to (D)] with similar expression patterns. A heatmap of DEGs for all groups (E). Blue, low expression; red, high expression. The color bar symbolized the absolute quantification of the normalized z-score intensity. (F to I) Volcano plots showing DEGs for each group versus the MI group. Each dot represented a specific gene, with red and blue dots indicating notably up-regulated and down-regulated genes, respectively. (J) The PCA of RNA-seq showed clustered groups for the GelMA-4LMS ECP group versus the GelMA ECP group. (K) Volcano plot showing DEGs for the GelMA-4LMS ECP group versus the GelMA patch group. (L) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway…