A ROS-responsive, self-immolative and self-reporting hydrogen sulfide donor with multiple biological activities for the treatment of myocardial infarction

Figures

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  Fig. 1. Schematic illustration of HSD-R for the treatment of myocardial infarction. Based on the COS structure, a novel fluorescent and ROS-responsive H2S donor HSD-R was designed and synthesized for visualization and quantification of H2S release kinetics and distribution profiles, as well as for the treatment of myocardial ischemic injury. The underlying mechanisms responsible for cardioprotective effects of H2S involve its anti-apoptotic, anti-inflammatory, and pro-angiogenic activities.

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  Fig. 2. In vitro characterizations of HSD-R. (A) Fluorescence response of HSD-R (5 μM) to H2O2 (100 μM) and CA (10 μg/mL). Deionized water and HSD-RF (5 μM) were used as controls. (B) Time-dependent fluorescence intensities of HSD-R at 697 nm in the presence of H2O2 (100 μM). (C) Fluorescence responses of HSD-R (5 μM) to various types of ROS at 100 μM. Deionized water was used as a control. (D) Fluorescence intensities at 697 nm at 120 min after addition of ROS. (E) Schematic showing mechanisms responsible for H2O2-triggered release of H2S and generation of fluorescence. COS, carbonyl sulfide; CA, carbonic anhydrase. (F) HPLC traces of HSD-RF (10 μM), HSD-R (10 μM) after reaction with 100 μM H2O2 in PBS for 120 min, and HSD-R (10 μM).

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  Fig. 3. Fluorescence imaging of H2S release from HSD-R in vitro and in vivo. (A) Confocal microscopy images of H9c2 cells after different treatments. Cells were first incubated with HSD-R (5 μM) and WSP-1 (10 μM) for 60 min. After removal of excess HSD-R and WSP-1, PBS (control), low-dose Rosup at 50 μg/mL (Rosup (L)), or high-dose Rosup at 100 μg/mL (Rosup (H)) was added. Fluorescence images were acquired after 30 min. Scale bars, 50 μm. (B) In vivo fluorescence images indicate release of H2S in MI rats after different treatments. MI, untreated MI rats; Sham+HSD-R, healthy rats injected with high dose of HSD-R; MI+HSD-R (L), MI rats treated with low dose of HSD-R; MI+HSD-R (H), MI rats treated with high dose of HSD-R. (C–D) Ex vivo fluorescence images (C) and quantification analysis (D) of H2S release in hearts of MI rats treated with different doses of HSD-R. (E–F) Ex vivo fluorescence images (E) and quantification (F) of H2S release in hearts collected from MI rats at different time points after treatment with the same dose of HSD-R. (G) Ex vivo image shows the distribution of HSD-RF in different organs at 2 h post HSD-R administration. Data in (E–F) are expressed as means ± SD (n = 6).

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  Fig. 4. Improved cardiac function and structure in MI rats by HSD-R treatment. (A) Representative echocardiographic images (M-mode) in different groups post ligation and different treatments for 28 days. (B) Quantified values of left ventricular internal diameter (diastole) (LVIDd), left ventricular internal diameter (systole) (LVIDs), left ventricular end-diastolic volume (EDV), left ventricular end-systolic volume (ESV), left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS) at varied time points during different treatments. (C–D) Representative digital photos of TTC-stained myocardial segments (C) and quantified infract sizes (D) of different groups (at day 7 post-MI). After TTC staining, the infarcted myocardium is white, while the non-infarcted myocardium is red. (E) Representative heart horizontal panoramic views (upper) and microscopic images (lower) of myocardial sections stained with H&E or Masson. Scale bars, 1 mm. (F–G) The LV wall thickness (F) and LV scar size (G) quantified by the midline method based on H&E and Masson sections. Sham, with thoracotomy but without LAD ligation; MI, with LAD ligation and saline injection; HSD-R (L), with…

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  Fig. 5. RNA-Seq analysis of MI heart tissues with or without HSD-R treatment. (A) Principal component analysis (PCA) of three examined groups (n = 3). Each symbol in the image represents one sample. (B) Correlation analysis of three groups. More saturated red represents higher co-expression interconnection of pair-wise samples. (C) The volcano map of genes detected in the HSD-R and MI groups. Green dots represent 2765 down-regulated genes and red ones denote 2202 up-regulated genes in the HSD-R group as compared to the MI group. (D) The heatmap of 4967 DEGs identified by comparing HSD-R and MI groups according to the cutoff criteria (fold changes ≥ 2, P < 0.05). In the HSD-R group, genes in red represent up-regulated genes compared to those in the MI group, while the blue ones represent down-regulated genes compared to those in the MI group. (E–F) KEGG pathway enrichment analysis of DEGs. Y axis represents the names of enriched KEGG pathways, the dot color represents P values of the enrichment analysis. The dot size and rich factor represent numbers of enriched DEGs. (E) Ten significantly enriched KEGG pathways of 2765 down-regulated DEGs. (F) Ten significantly enriched KEGG pathways of…

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  Fig. 6. In vitro and in vivo anti-apoptotic effects of HSD-R. (A) Representative fluorescent micrographs showing TUNEL+ apoptotic cells (red) in the hearts at day 7 after different treatments. Sham, thoracotomy without LAD ligation; MI, LAD ligation plus saline injection; HSD-R (L), LAD ligation plus HSD-R injection at 25 μg/kg; HSD-R (H), LAD ligation plus HSD-R injection at 100 μg/kg. Scale bar, 200 μm. (B) The quantified numbers of TUNEL+ apoptotic cells. n/HPF, number per high power field. (C–D) Flow cytometry analysis of hypoxia-induced apoptosis of H9c2 cardiomyocytes pretreated with PBS, various doses of HSD-R, or Pro. (E) The heatmap of down-regulated DEGs involved in pro-apoptosis after HSD-R treatment. In the HSD-R group, genes in red represent up-regulated genes compared to those in the MI group, while the blue ones represent down-regulated genes compared to those in the MI group. (F) The protein-protein interaction (PPI) network of down-regulated DEGs involved in pro-apoptosis. The size and color of nodes were defined by the node degree: the larger the node size and the redder the node color, the higher the node degree; lines represent interactions between the nodes. The line…

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  Fig. 7. In vitro and in vivo anti-inflammatory effects of HSD-R. (A) RT-qPCR analysis of the mRNA expression levels of inflammatory factors including TNF-α, Caspase 1, IL-1β, and IL-18 in heart tissues of MI rats. (B) The significantly enriched GO term in GSEA: GO0042116 macrophage activation, with enrichment score of 0.71 and P < 0.001. (C) RT-qPCR analysis of the mRNA expression levels of marker genes of inflammatory M1 macrophages (CD86, IL-12, and iNOS) in heart tissues of MI rats. (D–E) Flow cytometric analysis of the number of M1 (F4/80+CD86+) macrophages. (F–G) Representative immunofluorescence images (F) and quantification (G) of CD86+ cells. For images in (D to G): Control, without IFN-γ and HSD-R treatment; Stimulated, treated with IFN-γ alone; HSD-R (L), simultaneously treated with IFN-γ and HSD-R at 0.2 μM; HSD-R (H), simultaneously treated with IFN-γ and HSD-R at 2 μM. (H–I) Western blot analysis of expression levels of phosphorylated-Stat1 (p-Stat1) and Stat1 in heart tissues of MI rats. Data are expressed as means ± SD from three independent replicates. *P < 0.05, **P < 0.01, ***P < 0.001; ns, no significance.

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  Fig. 8. In vitro and in vivo pro-angiogenic effects of HSD-R. (A) Evaluation of flow velocity and spatial vascular profiles by LASCA. Red polygons show the regions of interest. (B) The quantified mean flux values. (C–D) Representative immunofluorescence images show CD31+ cells (C) and quantitative analysis of the vascularization degree (D). Scale bar, 100 μm. In images of (A to D): Sham, thoracotomy without LAD ligation; MI, LAD ligation plus saline injection; HSD-R (L), LAD ligation and HSD-R injection at 25 μg/kg; HSD-R (H), LAD ligation and HSD-R injection at 100 μg/kg (E–F) Representative images (E) and quantitative analysis (F) of the tube formation by HUVECs. Scale bar, 500 μm. (G–H) Representative images (G) and quantification (H) of migration of HUVECs at various time points after different treatments. Scale bar, 50 μm. (I) RT-qPCR analysis of the mRNA expression levels of typical pro-angiogenesis genes including VEGFa, Fgf1, and Wnt5a. Data are expressed as means ± SD from three independent replicates. For statistical results in (H): ***P < 0.001 versus the control at 12 h; #P < 0.05, ###P < 0.001 versus the control at 24 h. In other cases: *P < 0.05, **P < 0.01, ***P < 0.001;…