Single-cell multi-omics analysis reveals the mechanism of action of a novel antioxidant polyphenol nanoparticle loaded with STAT3 agonist in mediating cardiomyocyte ferroptosis to ameliorate age-related heart failure

Figures

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  Fig. 1. Cell Type Distribution and Expression Features of scRNA-seq Data. A Visualization of cell annotation results grouped based on UMAP clustering, where each color represents a specific cell type; B Visualization of cell annotation results based on UMAP clustering, with each color representing a distinct cell type, left panel for the Control group (n = 28), right panel for the HF group (n = 17); C Expression patterns of known lineage-specific marker genes across different cell types in Control and HF groups, with darker blue indicating higher average expression levels and larger circles representing more cells expressing the gene; D Proportional distribution of cell types in each sample, with each color representing a specific cell type; E Bar graph comparison of cell numbers for each cell type, where red represents the Control group (n = 28) and blue represents the HF group (n = 17); F Box plot showing changes in cell type proportions, with red indicating the Control group (n = 28) and blue indicating the HF group (n = 17), * denotes statistical significance compared to the Control group, **p < 0.01, ***p < 0.001

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  Fig. 2. Analysis of Key Cell Expression Characteristics. A UMAP clustering results showing the distribution of CM_1 and CM_2 cells, with different colors indicating distinct CM subtypes; B UMAP clustering results displaying the distribution of Macrophages_1 and Macrophages_2 cells, with different colors representing different macrophage subtypes; C UMAP clustering results illustrating the distribution of various T cell subsets, with different colors denoting different T cell subsets; D Total number and interaction strength of cell communications in the Control group (n = 28), where the thickness of lines in the network graph represents the number of pathways and the size of circles in the boxplot indicates the strength of interactions; E Total number and interaction strength of cell communications in the HF group (n = 17), with similar visualization as in D; F Differences in AUC scores for CM subtypes, macrophage subtypes, and T cell subsets between HF group (n = 17) and Control group (n = 28) based on an aging gene set; G Differences in AUC scores for CM subtypes, macrophage subtypes, and T cell subsets between HF group (n = 17) and Control group (n = 28) based on a ferroptosis gene…

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  Fig. 3. Identification of Key Genes Associated with Aging and Ferroptosis in CMs. A Volcano plot illustrating differential gene expression in CM_1 cells from Control and HF groups, where blue dots to the right of the dotted line represent genes with lower expression in the HF group and red dots to the left indicate genes with higher expression in the HF group; B Heatmap showing the expression of the top 20 DEGs in CM_1 cells, with blue indicating downregulation, red indicating upregulation, and darker colors reflecting greater significance of differential expression; C Volcano plot displaying differential gene expression in CM_2 cells from Control and HF groups, using a similar color scheme as in (A); (D) Heatmap presenting the expression of the top 20 DEGs in CM_2 cells, following the color scheme as in (B); (E) Venn diagram depicting the intersection of DEGs in CM_1 and CM_2 cells, aging-related genes, and ferroptosis-related genes; F Bubble plot of GO enrichment analysis for the 17 intersecting genes, with circle color indicating significance of enrichment (from blue to red) and circle size representing the number of enriched genes; G Bubble plot of KEGG enrichment analysis for the 17…

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  Fig. 4. Impact of STAT3 Gene on the Biological Functions of CMs. A Schematic diagram of experimental procedure involving lentivirus transfection for silencing or overexpression of STAT3; B Viability changes of CMs in different intervention groups at 12, 24, 36, and 48 h detected by CCK-8 assay; C Assessment of proliferative capacity of CMs in different intervention groups using EDU experiment, with EDU-positive cells shown in red indicating cells in the proliferative phase, and blue representing DAPI-stained cell nuclei (scale bar = 50 μm); D Statistical analysis of EDU staining results; E Assessment of apoptosis in CMs in different intervention groups using Annexin V/PI double staining flow cytometry, with the bar graph showing the percentage of cells in Q2 and Q3 quadrants indicating apoptotic cells; F Evaluation of migration of CMs in different intervention groups using wound healing assay (scale bar = 100 μm); G Evaluation of migration capacity of CMs in different intervention groups using Transwell assay (scale bar = 50 μm). Quantitative data in the figures are presented as Mean ± SD. Each cell experiment was repeated 3 times. * denotes comparison between two groups, **p < 0.01,…

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  Fig. 5. Influence of STAT3 on CMs Cell Ferroptosis. A Representative fluorescence images of CMs transfected with different lentiviruses for intracellular iron levels identified using FerroOrange (Scale bar = 100 μm); B Statistical analysis of iron content in CMs from different intervention groups using assay kits; C GSH/GSSG ratio in CMs from different intervention groups; D–E Visualization of ROS production in CMs using DCFH-DA under confocal laser scanning microscopy and statistical analysis using flow cytometry (Scale bar = 25 μm); F Measurement of MDA content in different intervention groups; G Representative confocal images of CMs stained with C11-BODIPY 581/591. Red indicates unoxidized lipids, while green represents oxidized lipids (Scale bar = 25 μm); H–I Transcription levels of ferroptosis-related genes SLC7A11 and GPX4 detected by RT-qPCR; J Observation of mitochondrial morphology in cells using TEM, with red arrows pointing to mitochondrial structures (scale bar = 1 μm); K Detection of mitochondrial membrane potential (MMP: mitochondrial membrane potential) in cells from different intervention groups using JC-1 assay. Quantitative data in the figures are presented as Mean ±…

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  Fig. 6. Synthesis and Characterization of Novel Antioxidant PNs. A Schematic diagram of the synthesis steps of PN@Col nanoparticles; B Morphological features of PN (top) and PN@Col (bottom) observed using TEM (scale bar: 100 nm/200 nm); C Morphology of PN (top) and PN@Col (bottom) observed using SEM (scale bar: 5 μm/1 μm); D Particle size distribution of PN (left) and PN@Col (right) analyzed by nanoparticle tracking; E Zeta potential analysis of PN (left) and PN@Col (right) by nanoparticle tracking; F In vitro release of Colivelin from PN@Col nanoparticles. No significant differences were observed between samples at each time point. Data are presented as the mean with a standard deviation from three independent samples prepared under the same conditions; G Fluorescence microscopy images of CMs cells cultured with PN (left) and PN@Col (right) for 24 h, with green fluorescence staining indicating viable cells (scale bar = 25 μm); (H) DPPH and ABTS radical scavenging activities of PN and PN@Col; I Antioxidant capacity assessment of PN and PN@Col. Quantitative data in the figures are presented as Mean ± SD. Each experiment was repeated 3 times. ‘‘ns’’ indicates no statistically significant…

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  Fig. 7. Impact of PN@Col on the Biological Functions of CMs. A Expression changes of p-STAT3 protein in CMs cells from different treatment groups detected by Western blot; B Viability changes of CMs in different treatment groups at 12, 24, 36, and 48 h assessed by CCK-8 assay; C Evaluation of proliferative capacity of CMs in different treatment groups using EDU experiment, with red fluorescence indicating EDU-positive cells in the proliferative phase and blue fluorescence representing DAPI-stained cell nuclei (scale bar = 50 μm); D Assessment of cell migration in different intervention groups of CMs using wound healing assay (scale bar = 100 μm); E Evaluation of migration capacity of CMs in different treatment groups using Transwell assay (scale bar = 50 μm); F Detection of apoptosis in CMs from different treatment groups using Annexin V/PI double staining flow cytometry, with bar graph representing the percentage of cells in Q2 and Q3 quadrants indicating apoptotic cells; G Representative fluorescence images of intracellular iron levels in CMs from different treatment groups identified using FerroOrange (Scale bar = 25 μm); H Visualization of ROS production in CMs using DCFH-DA under…

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  Fig. 8. Impact of PN@Col on Age-related HF Mouse Cardiac Function. A Representative cardiac ultrasound images of mice in different treatment groups; B Representative left ventricular pressure waveforms, with a scale of 40 mmHg vertically and 100 ms horizontally; C Anatomical diagrams of mouse hearts from different treatment groups; D H&E stained images of mouse heart tissues from different treatment groups, with quantification of cross-sectional area of the heart tissues(scale bar: 500 μm/50 μm); E Assessment of CM size with WGA staining results (scale bar: 50 μm); F Evaluation of myocardial interstitial fibrosis deposition in mouse ventricles with Sirius Red staining (left) and Masson staining (right), where CVF (Collage Volume Fraction) quantifies the ratio of Masson-stained blue area to total heart chamber area (scale bar: 500 μm/50 μm), fibrotic area in mice indicated by picro sirius red-positive area (scale bar: 50 μm); G Western blot analysis of BNP, β-MHC, and collagen I protein expression changes in heart tissues from different treatment groups; (H) TUNEL staining detecting apoptosis in mouse myocardial tissue, with red fluorescence representing apoptotic cells and blue…