In situ self-assembled stimulus-responsive and conductive hydrogel modulate myocardial infarction inflammatory homeostasis via PPARα/NFκB signaling and enhance the therapeutic efficacy of hiPSC-CMs

Figures

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  Scheme 1. Schematic illustration of the synthesis process of the injectable multifunctional supramolecular hydrogel HCPA with ROS-responsive and conductive properties, along with its impact on improving the infarcted microenvironment by modulating PPARα/NFκB pathway, thereby enhancing the therapeutic efficacy of hiPSC-CMs.

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  Fig. 1. Preparation and characterization of HCPA hydrogel. (A) Schematic diagram of the molecular structure and synthesis of HCPA hydrogel. (B) Electronic pictures of HCPA hydrogel generated in situ. (C) Scanning electron microscopy (SEM) images of different hydrogels. Scale bars = 100 μm. (D) Evaluation of the injectability of the different hydrogels. (E) Adhesion testing of HCPA hydrogel. (F) Dynamic strain scan curve of HCPA hydrogel. (G) Oscillation frequency change curve of HCPA hydrogel. (H) Viscosity curve with shear rate of HCPA hydrogel. (I) Self-healing test of HCPA hydrogel.

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  Fig. 2. Properties and functions of HCPA hydrogel. Time-dependent curves of UV-Vis absorption spectra for the clearance of different types of ROS by HCPA hydrogel: (A) ·N; (B) ·OH; (C) O2−·. The clearance rates of HCPA hydrogel for different types of ROS: (D) ·N; (E) ·OH; (F) O2·, n = 4. Mass degradation rates of HCPA hydrogel under different conditions: (G) H2O2 solutions with different concentrations; (H) PBS with different pH; (I) pH 7.4 PBS solution for the first 5 days and 400 μM H2O2 in pH 6.5 PBS solution for the next 5 days. (J) Conductivity of different samples. (K) Schematic diagram and representative image of small bulb luminescence test of HCPA hydrogel.

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  Fig. 3. Construction of directed cardiac differentiation system of hiPSCs and detection of biocompatibility of HCPA hydrogel. (A) Microscopic images of cells at different stages, n = 4. Scale bars = 100 μm. (B) qRT-PCR assay for the expression of cell marker genes at the corresponding stages. hiPSCs: SOX2; Mesoderm: T; Cardiac progenitors: ISL1; Cardiomyocytes: α-Actinin, ∗∗∗P < 0.001 vs. Day2, n = 6; ###P < 0.001 vs. Day5, n = 6; &&&P < 0.001 vs. Day10, n = 6; $$$P < 0.001 vs. Day0, n = 6. (C) Representative immunofluorescence images of cTnT, n = 4. Scale bars = 20 μm. (D) Representative immunofluorescence images of α-Actinin, n = 4. Scale bars = 20 μm. (E) Flow cytometry assay of cTnT-positive cardiomyocytes, ∗∗∗P < 0.001 vs. Blank, n = 4. (F) CCK8 results of cardiomyocytes treated with HCPA hydrogel, n = 5-10. (G) ELISA assays of AST and Cr content in serum of mice injected with HCPA hydrogel intramyocardially, n = 6.

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  Fig. 4. HCPA hydrogel-encapsulated hiPSC-CMs treatment enhanced cardiac function in MI mice and promoted retention and integration of hiPSC-CMs. (A) Schematic diagram of experimental timeline. (B) Representative electrocardiograms of mice. (C) Representative echocardiograms of mice in different groups at day 28. (D) Analyses of echocardiogram related EF, FS, LVIDd and LVIDs, ∗∗∗P < 0.001 vs. Sham, n = 10; ##P < 0.01 vs. MI, ###P < 0.001 vs. MI, n = 10; &P < 0.05 vs. MIH, &&P < 0.01 vs. MIH, &&&P < 0.001 vs. MIH, n = 10; $P < 0.05 vs. MIC, $$P < 0.01 vs. MIC, $$$P < 0.001 vs. MIC, n = 10. (E) Representative immunofluorescence images of transplanted hiPSC-CMs alone and HCPA hydrogel-encapsulated hiPSC-CMs 28 days after MI, n = 4. CM-DiI (Red), α-Actinin (Green), Cx43 (White), DAPI (Blue). Scale bars = 20 μm.

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  Fig. 5. HCPA hydrogel-encapsulated hiPSC-CMs treatment promoted angiogenesis and attenuated cardiac remodeling in MI mice. (A, B) Representative immunofluorescence images and quantification of CD31 (red) 28 days after different treatments, ∗∗P < 0.01 vs. Sham, n = 4; ###P < 0.001 vs. MI, n = 4; &&&P < 0.001 vs. MIH, n = 4; $$$P < 0.001 vs. MIC, n = 4. Scale bars = 20 μm. (C) Representative images of HE staining of heart sections of mice, n = 4-6. Scale bars = 20 μm. (D, E) Representative images of Masson's trichrome staining and quantification analysis of collagen content of heart sections of mice at 28 days after different treatments, ∗∗∗P < 0.001 vs. Sham, n = 4-6; ###P < 0.001 vs. MI, n = 4-6; &&P < 0.01 vs. MIH, n = 4-6; $$P < 0.01 vs. MIC, n = 4-6. Scale bars = 20 μm. (F, G) Representative images of Sirius red staining and quantification analysis of collagen content of heart sections of mice at 28 days after different treatments, ∗∗∗P < 0.001 vs. Sham, n = 4-6; ###P < 0.001 vs. MI, n = 4-6; &&P < 0.01 vs. MIH, n = 4-6; $P < 0.05 vs. MIC, n = 4-6. Scale bars = 20 μm.

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  Fig. 6. HCPA hydrogel-encapsulated hiPSC-CMs treatment improved myocardial electrical conduction, promote gap junction formation in MI mice. (A) Representative electrocardiograms of mice from different treatment groups at 28 days. (B) Electrocardiogram measurement of QRS duration, ∗∗∗P < 0.001 vs. Sham, n = 4; ###P < 0.001 vs. MI, n = 4-5; &P < 0.05 vs. MIH, n = 5; $$$P < 0.001 vs. MIC, n = 4-5. (C) Representative electrical propagation heat maps of mice from different treatment groups at 28 days. (D) Conduction velocity was calculated based on electrical conduction distance and activation time, ∗∗∗P < 0.001 vs. Sham, n = 5; ###P < 0.001 vs. MI, n = 5; &&P < 0.01 vs. MIH, n = 5; $$$P < 0.001 vs. MIC, n = 5. (E-G) Representative immunofluorescence images and quantification of α-Actinin (green) and Cx43 (red) in the hearts of mice from different treatment groups on day 28, ∗∗P < 0.01 vs. Sham, n = 4; ##P < 0.01 vs. MI, ###P < 0.001 vs. MI, n = 4-5; &P < 0.05 vs. MIH, &&&P < 0.001 vs. MIH, n = 4-5; $P < 0.05 vs. MIC, $$$P < 0.001 vs. MIC, n = 4-5. Scale bars = 20 μm; 10 μm (Higher magnification). (H) qRT-PCR analysis to detect the mRNA level of Cx43 in the hearts of mice from different…

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  Fig. 7. HCPA hydrogel modified infarct microenvironment by scavenging ROS and inducing the macrophage polarization from type I to type II. (A) Experimental timeline. (B) Representative fluorescent images and quantification of superoxide anion radical activity (DHE, red) of heart sections 24 h after injection, ∗∗∗P < 0.001 vs. Sham, n = 4; ###P < 0.001 vs. MI, n = 4. Scale bars = 20 μm; 10 μm (Higher magnification). (C) qRT-PCR assays of the expression of CD86 and iNOS of heart, ∗∗∗P < 0.001 vs. Sham, n = 8-9; ##P < 0.01 vs. MI, ###P < 0.001 vs. MI, n = 8-9. (D) qRT-PCR assays of the expression of TNF-α (n = 4), IL1β (n = 9) and IL6 (n = 7) of heart, ∗∗P < 0.01 vs. Sham, ∗∗∗P < 0.001 vs. Sham, n = 4-9; #P < 0.05 vs. MI, ##P < 0.01 vs. MI, ###P < 0.001 vs. MI, n = 4-9. (E) qRT-PCR assays of the expression of MRC1 and Arg1 of heart, ∗∗∗P < 0.001 vs. Sham, n = 8-9; ###P < 0.001 vs. MI, n = 8-9. (F) qRT-PCR assays of the expression of IL10 and TGF-β of heart, ∗∗P < 0.01 vs. Sham, ∗∗∗P < 0.001 vs. Sham, n = 8; ###P < 0.001 vs. MI, n = 8. (G) Representative immunofluorescence images of iNOS of heart sections at different treatment groups for 72 h, n = 4. Scale bars = 20 μm; 10 μm (Higher…

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  Fig. 8. HCPA hydrogel facilitated macrophage polarization by regulating PPARα/NFκB signaling pathway to combat inflammation. (A) Schematic diagram of RNA sequencing and analysis. (B) Heat map of differentially expressed genes (DEGs) of the Sham, MI and MIH groups (red, up-regulated; blue, down-regulated), n = 3. (C) Volcano plot of DEGs identified for MI and MIH groups (red, up-regulated; blue, down-regulated), n = 3. (D) GO enrichment analysis of DEGs. (E) Pathway enrichment analysis of DEGs according to the KEGG. (F) GSEA of MIH versus MI showing global downregulation of inflammatory response genes and NFκB signaling pathway genes. (G) PPI network of PPARα, NFκB and macrophage polarization related genes. (H) Western blot analysis of PPARα protein expression, ∗∗∗P < 0.001 vs. Sham, n = 5; #P < 0.05 vs. MI, n = 5. (I) qRT-PCR assay of the expression of PPARα mRNA, ∗∗∗P < 0.001 vs. Sham, n = 7; #P < 0.05 vs. MI, n = 7. (J) Western blot analysis of IκBα protein expression, ∗∗P < 0.01 vs. Sham, n = 4; ##P < 0.01 vs. MI, n = 4. (K) qRT-PCR assay of the expression of IκBα mRNA, ∗∗∗P < 0.001 vs. Sham, n = 7; ###P < 0.001 vs. MI, n = 7. (L) Western blot analysis of p-NFκB protein expression,…