Mechano-matching hydrogel patch promotes myocardial infarction repair via YAP-Bcl-2 pathway activation

Figures

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  Fig. 1. Schematic illustration of the synthesis of the mechano-matching hydrogel patch and its effects in reducing infarct size, decreasing myocardial apoptosis, and alleviating myocardial fibrosis after MI (Created with Biorender.com).

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  Fig. 2. Characterization of the SilMA hydrogel. (A) Schematic for preparing SilMA hydrogel. (B) Photographs of SilMA hydrogel before and after UV curing, and the stability of hydrogel was proved by placing in different orientations. (C) 1H NMR spectra of SF and SilMA. (D) The stress-strain curves of three groups of hydrogel patches. (E) The Young’s modulus of three groups of hydrogel patches (n = 3 per group). (F) The conductivity in three groups of SilMA hydrogel (n = 4 per group). (G) Swelling property of three groups of SilMA hydrogels under the PBS (n = 5 per group). SF: Silk fibroin,GMA: Glycidyl methacrylate,AKG: α-Ketoglutaric acid. Data are means ± standard deviation. ∗p < 0.05, ∗∗p < 0.01.

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  Fig. 3. Biocompatibility of three hydrogel patches. (A) Experimental design. (B) The echocardiographic images of healthy rats with and without different cardiac patches for 1 week and 4 weeks. (C) Representative parameters of cardiac function of healthy rats with and without three groups of hydrogel cardiac patches for 1week and 4 weeks (n = 3 per group). (D) Live/dead staining of H9c2 cardiomyocytes after seeding on three groups of hydrogels or regular cell culture plate for 3 days (Scale bar, 100 μm). Data are means ± standard deviation. ∗p < 0.05, ∗∗p < 0.01.

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  Fig. 4. The MMHP improved heart function after MI. (A) Experimental design. (B) The echocardiographic images of rats with MI after patching with or without three groups of hydrogels and sham-operated rats for 1 week and 4 weeks. (C) Echocardiographic assessment of cardiac function in five groups of rats (n = 3 for sham group, n = 9–10 for other four groups). (D) Echocardiographic assessment of cardiac function in five groups of rats at 4 weeks after surgery. Data are means ± standard deviation. ∗p < 0.05, ∗∗p < 0.01.

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  Fig. 5. The MMHP group exhibited a significant reduction in infarct size and a decrease in apoptosis after MI. (A) Representative images of Masson’s trichrome staining of cardiac tissue from five groups of rats after 4 weeks of surgery (Scale bar, 1 mm). Blue and red represent fibrous tissue and myocardium, respectively. (B) Statistical analysis of infarct size in four groups of rats (n = 6 per group). (C) Statistical analysis of Ventricular wall thickness in five groups of rats (n = 6 per group). (D) Representative images of TUNEL staining in cardiac tissue slices from five groups of rats (Scale bar, 100 μm). (E) The quantitative statistics of the number of TUNEL positive cells based on the TUNEL staining (n = 4 per group). (F) Representative images of WGA immunofluorescence in cardiac tissue slices from five groups of rats (Scale bar, 20 μm). (G) The quantitative statistics of cell size based on the WGA immunofluorescence (n = 3 per group). Data are means ± standard deviation. ∗p < 0.05, ∗∗p < 0.01.

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  Fig. 6. The MMHP suppressed the apoptosis after MI. (A) qPCR analysis of Caspase-3 and Bcl-2 mRNA expression in five groups of rats (n = 3 per group). (B) Immunoblots of Cleaved caspase-3 and Bcl-2 in ventricular myocardium from sham-operated rats or infarcted zone of cardiac tissue from other four groups of rats. (C) Immunoblots of Bcl-2 in mitochondria from five groups of rats. (D) Representative images of Cleaved caspase-3 (red), and DAPI (blue) immunofluorescence in ventricular myocardium from sham-operated rats or infarcted zone of cardiac tissue from other four groups of rats (Scale bar, 50 μm). (E) The quantification of mean fluorescence intensity for Cleaved caspase-3 (n = 4 per group). (F) Representative images of Bcl-2 (green), and DAPI (blue) immunofluorescence in ventricular myocardium from sham-operated rats or infarcted zone of cardiac tissue from other four groups of rats (Scale bar, 50 μm). (G) The quantification of mean fluorescence intensity for Bcl-2 (n = 4 per group). Data are means ± standard deviation. ∗p < 0.05, ∗∗p < 0.01. C-caspase-3: Cleaved caspase-3, MFI: Mean Fluorescence Intensity.

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  Fig. 7. MMHP triggered intracellular YAP activation. (A) qPCR assay of YAP mRNA expression in five groups of rats (n = 3 per group). (B) Immunoblots of YAP and pYAP in ventricular myocardium from five groups of rats. (C) The quantification of pYAP/YAP ratio (n = 3 per group). (D) Schematic for cyclic stretching on cardiomyocytes in vitro. (E) Immunoblots of various proteins in the cytoplasm and nucleus of cardiomyocytes following cyclic stretching, with or without MMHP and H2O2 treatment. (F) Quantification of Immunoblots results (n = 3 per group). Data are means ± standard deviation. ∗p < 0.05, ∗∗p < 0.01. C-YAP: Cytoplasmic YAP, C-caspase3: Cleaved caspase-3, N-YAP: Nuclear YAP.

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  Fig. 8. MMHP drive cardiomyocyte protection through mechanical cue-mediated YAP activation. (A) Immunoblots of various proteins in the cytoplasm and nucleus of cardiomyocytes seeded on MMHP following cyclic stretching, with or without ML-7 and H2O2 treatment. (B) Quantification of Immunoblots results (n = 3 per group). (C) Immunoblots of various proteins in the cytoplasm and nucleus of cardiomyocytes following cyclic stretching, with or without TDI and H2O2 treatment. (D) Quantification of Immunoblots results (n = 3 per group). Data are means ± standard deviation. ∗p < 0.05, ∗∗p < 0.01. C-YAP: Cytoplasmic YAP, C-caspase3: Cleaved caspase-3, N-YAP: Nuclear YAP.

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  Fig. 9. Mechanistic schematic of cardiomyocyte protection via activation of theYAP-Bcl-2 pathway triggered by mechanical cues from mechano-matching hydrogel patch (Created with Biorender.com).