Celastrol‐Loaded Conductive Hydrogel Mitigates Myocardial Ischemia‐Reperfusion Injury and Restores Electrophysiological Function

Figures

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  FIGURE 1. Schematic illustration of FGPC hydrogel construction and its therapeutic effects after myocardial ischemia–reperfusion injury. Celastrol was identified through antioxidant compound screening and incorporated into a conductive F127DA/GelMA/PEDOT:PSS hydrogel to form FGPC. Following local epicardial application to the injured myocardium, FGPC attenuates acute oxidative stress, NET formation, and inflammation, while promoting chronic myocardial repair by enhancing revascularization, electrical synchronization, and cardiac functional recovery.

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  FIGURE 2. Antioxidant screening and concentration‐window evaluation of celastrol. (a) Schematic illustration of the antioxidant compound screening workflow. A 96‐well antioxidant compound library was screened in AC16 cells under H2O2‐induced oxidative stress, followed by fluorescence‐based ROS detection and candidate compound ranking. (b) Heatmap showing relative intracellular ROS levels after treatment with library compounds at 1 and 10 µm. The red star indicates celastrol, which was selected from the screening results for further validation (n = 3). (c, d) Top‐ranked antioxidant candidates based on ROS reduction at 1 µm (c) and 10 µm (d). (e) Dose‐dependent effect of celastrol on intracellular ROS levels in AC16 cells (n = 3). (f) Cell viability of AC16 cells treated with different concentrations of celastrol, as determined by CCK‐8 assay (n = 3). (g) ROS levels normalized to cell viability to evaluate antioxidant activity independent of cytotoxicity (n = 3). (h) Representative 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA) fluorescence images and Live/Dead staining images of AC16 cells after different treatments. Green fluorescence in DCFH‐DA staining indicates intracellular ROS;…

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  FIGURE 3. Preparation and characterization of hydrogels. (a) Schematic illustration of FGPC hydrogel fabrication. Celastrol was incorporated into F127DA micelles, followed by integration with GelMA and PEDOT:PSS to form the photo‐crosslinkable conductive hydrogel network. (b) Gross appearance of different hydrogel formulations after photo‐crosslinking. F, F127DA; G, GelMA; FG, F127DA/GelMA; FGP, F127DA/GelMA/PEDOT:PSS; FGPC, F127DA/GelMA/PEDOT:PSS/celastrol. (c) The storage modulus (G′) and loss modulus (G″) of the hydrogels under varying angular frequency. (d) The storage modulus (G′) and loss modulus (G″) of the hydrogels under varying shear strain (γ). (e) Representative SEM images of different hydrogels. Scale bars: 20 µm. (f) In vivo fluorescence imaging of free Cy5‐CLT and FGPC‐delivered Cy5‐CLT over 14 days. (g) In vitro cumulative release profile of Cy5‐CLT from FGPC hydrogel. (h) In vivo degradation of FGPC hydrogel in a subcutaneous implantation model.

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  FIGURE 4. In vitro biocompatibility, antioxidant, and electroconductive properties of different hydrogels. (a) Representative Live/dead staining of AC16 cells cultured on different hydrogels at days 1 and 5. Green: live cells; red: dead cells. Scale bar: 200 µm. (b) CCK‐8 analysis of AC16 cell proliferation on different hydrogels. (c) Representative Ca2 + transient images and traces of neonatal rat ventricular cardiomyocytes (NRVMs) cultured on FG, FGP, and FGPC hydrogels for 7 days (n = 3). (Left) Representative images of calcium transients. (Right) Quantitative analysis of the calcium transient frequency. Scale bar: 50 µm. (d, e) Quantification of Ca2 + transient peak number per 10 s (d) and time to peak (e) (n = 6). (f) Representative immunofluorescence images of α‐actinin and Cx43 in cardiomyocytes cultured on different hydrogels. Green, α‐actinin; red, Cx43; blue, DAPI. Scale bar, 20 µm. (g) Representative DCFH‐DA fluorescence images of AC16 cells cultured on different hydrogels under H2O2‐induced oxidative stress. Scale bar, 200 µm. (h) Quantification of intracellular ROS fluorescence intensity (n = 4). (i, j) Quantification of α‐actinin‐positive area (i) and Cx43‐positive area (j)…

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  FIGURE 5. FGPC reduces acute oxidative stress and inflammation in vivo. (a) Experimental timeline of MI/R induction, hydrogel treatment, cardiac functional assessment, electrophysiological examination, and tissue collection. (b) Representative fluorescence images of DHE and MitoSOX staining in heart sections at 24 h after reperfusion. Red, DHE or MitoSOX; blue, DAPI. Scale bars, 200 µm. (c, d) Quantification of DHE (c) and MitoSOX (d) fluorescence intensity. (e) Representative immunofluorescence images of S100a9 in heart sections at 24 h after reperfusion. Green, S100a9; red, α‐actinin; blue, DAPI. Scale bar, 50 µm. (f) Quantification of S100a9 fluorescence intensity. (g) Representative immunofluorescence images of CitH3 and MPO in heart sections at 24 h after reperfusion. Green, CitH3; red, MPO; blue, DAPI. Scale bar, 50 µm. (h, i) Quantification of CitH3 (h) and MPO (i) fluorescence intensity. Data are presented as mean ± SD (n = 5). Statistical significance was determined by one‐way ANOVA followed by Tukey's post hoc test. (* p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant.).

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  FIGURE 6. RNA‐seq reveals inflammatory pathway suppression by FGPC (n = 3). (a) Principal component analysis (PCA) of RNA‐seq samples from the I/R and FGPC groups. (b) Volcano plot identifying differentially expressed genes (DEGs) in myocardial tissue between the FGPC and I/R groups (DEGs were defined as FDR < 0.05 and |log2FC| ≥ 1). (c) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of downregulated differentially expressed genes in the FGPC group compared with the I/R group. (d) Gene ontology (GO) enrichment analysis of downregulated differentially expressed genes, including biological process, cellular component, and molecular function terms. (e) Chord diagram showing representative downregulated genes and their associated enriched GO terms. The color gradient indicates relative log2 fold change. (f) STRING protein–protein interaction (PPI) network of representative downregulated inflammation‐related genes. (g, h) Gene set enrichment analysis (GSEA) plots of cytokine–cytokine receptor interaction (g) and positive regulation of the apoptotic process (h).

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  FIGURE 7. FGPC attenuates myocardial fibrosis and improves cardiac function after myocardial ischemia–reperfusion injury. (a) Representative Masson's trichrome staining images of myocardial sections from the Sham, I/R, FGP, and FGPC groups at 4 weeks after treatment. The lower panels show higher‐magnification views of the boxed regions in the corresponding upper panels. Collagen fibers are stained blue. Scale bars: 500 µm. (b) Quantitative analysis of fibrotic area in each group. (c) Representative M‐mode echocardiographic images of each group at 4 weeks after surgery. The green and red dashed lines indicate the left ventricular internal diameters at end‐diastole and end‐systole, respectively. (d–i) Longitudinal echocardiographic analysis of left ventricular function from baseline to 4 weeks after surgery, including LVEF (d), LVFS (e), LVIDs (f), LVIDd (g), LV systolic volume (Volume;s) (h), and LV diastolic volume (Volume;d) (i). Data are presented as mean ± SD (n = 8). Statistical significance was determined by one‐way ANOVA followed by Tukey's post hoc test for comparisons at 4 weeks. (* p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant).

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  FIGURE 8. FGPC improves electrical stability and neovascularization in vivo. (a) Representative immunofluorescence images of α‐actinin and Cx43 in the infarct border zone at 4 weeks after surgery. Green, α‐actinin; red, Cx43; blue, DAPI. Scale bars: 50 and 10 µm for magnified images. (b, c) Quantification of α‐actinin‐positive area (b) and Cx43‐positive area (c). (d) Representative surface ECG and intracardiac electrograms during programmed electrical stimulation. VT/VF, ventricular tachycardia/ventricular fibrillation. Scale bar, 1 s. (e–g) Quantification of VT/VF incidence (e), VT duration (f), and ventricular effective refractory period (VERP) (g). VT: ventricular tachycardia; VF: ventricular fibrillation. VERP: Ventricular Effective Refractory Period. (h) Representative immunofluorescence images of CD31 and α‐SMA in cardiac sections at 4 weeks after surgery. Red, CD31; green, α‐SMA; blue, DAPI. Scale bars: 100 and 20 µm for magnified images. (i, j) Quantification of CD31‐positive vessel density (i) and α‐SMA‐positive vessel density (j). Except for the data of VT/VF incidence, the other data are presented as mean ± SD (n = 8). Statistical significance was determined by one‐way ANOVA…