Effects of the active parts of Yangxin Tongmai Formula on myocardial injury and spatial metabolic remodeling in rats with myocardial ischemia-reperfusion injury

Figures

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  Figure 1. Experimental workflow and effects of apr-YTF on cardiac contractile function in rats with MIRI-induced blood stasis syndrome. (A) CONSORT-style animal flow diagram and experimental workflow of apr-YTF treatment in rats with MIRI. A total of 55 male Wistar rats were used. After 1 week of acclimatization, 10 rats were allocated to the Control group, and 45 rats underwent MIRI modeling by LAD ligation for 30 min followed by reperfusion for 2 h. During modeling, 15 rats died, and the successfully modeled rats were randomly assigned to the MIRI, Vehicle, and apr-YTF groups. Endpoint assessments were performed after 14 days of intervention. (B–E) Representative echocardiographic images of the Control, MIRI, Vehicle, and apr-YTF groups, respectively. (F) Quantitative analysis of left ventricular ejection fraction (LVEF). (G) Quantitative analysis of left ventricular fractional shortening (LVFS). Data are presented as mean ± SD, n = 3 per group. ***P < 0.001 vs. Control group; ##P < 0.01 vs. MIRI group.

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  Figure 2. Effects of apr-YTF on myocardial injury, histopathological changes, myocardial fibrosis, and infarct size in rats with MIRI-induced blood stasis syndrome. (A–D) Serum levels of myocardial injury-related enzymes, including creatine kinase (CK), creatine kinase-MB isoenzyme (CK-MB), lactate dehydrogenase (LDH), and lactate dehydrogenase 1 (LDH1). (E–H) Representative hematoxylin and eosin (HE)-stained myocardial sections from the Control, MIRI, Vehicle, and apr-YTF groups, respectively. (I) Quantitative analysis of myocardial collagen fiber area. (J–M) Representative Masson's trichrome-stained myocardial sections from the Control, MIRI, Vehicle, and apr-YTF groups, respectively. Collagen fibers are stained blue, and cardiomyocytes are stained red. (N–Q) Representative triphenyltetrazolium chloride (TTC)-stained myocardial sections from the Control, MIRI, Vehicle, and apr-YTF groups, respectively. Red areas indicate viable myocardium, whereas white areas indicate infarcted myocardium. (R) Quantitative analysis of myocardial infarct area. Data are presented as mean ± SD, n = 3 per group. *P < 0.05, ***P < 0.001 vs. Control group; #P < 0.05, ###P < 0.001 vs. MIRI group.

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  Figure 3. MALDI-MSI-based spatial metabolic profiling of myocardial tissues in rats with MIRI-induced blood stasis syndrome. (A) Classification distribution of metabolites detected by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in positive-ion mode. (B) Representative ion images showing the spatial distribution of selected metabolic features in rat myocardial tissue. The color scale indicates relative ion intensity from low to high. (C) Principal component analysis (PCA) score plot of myocardial metabolic profiles among the Control, MIRI, and apr-YTF groups. Each point represents one biological sample, and the ellipse indicates the distribution tendency of each group. MALDI-MSI analysis was performed using myocardial tissues from 4 rats per group.

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  Figure 4. Multivariate and univariate analyses of differential metabolic features in myocardial tissues. (A,C,E) S-plot, volcano plot, and hierarchical clustering heatmap for the comparison between the apr-YTF group and the MIRI group. (B,D,F) S-plot, volcano plot, and hierarchical clustering heatmap for the comparison between the MIRI group and the Control group. In the S-plots, red points represent metabolic features with variable importance in projection (VIP) > 1. In the volcano plots, red and green points represent upregulated and downregulated differential metabolic features, respectively, whereas gray points represent features that did not meet the differential screening criteria. In the heatmaps, red indicates relatively high abundance and green indicates relatively low abundance. Differential metabolic features were screened based on fold change, P value, and VIP value.

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  Figure 5. Pathway enrichment analysis and spatial distribution of reversed differential metabolic features after apr-YTF intervention. (A) Metabolic pathway enrichment analysis of differential metabolic features between the MIRI group and the Control group. (B) Metabolic pathway enrichment analysis of differential metabolic features between the apr-YTF group and the MIRI group. The bubble size represents the number of enriched metabolic features, and the color indicates the P value. (C) Spatial ion images of 17 reversed differential metabolic features in myocardial tissues from the Control, MIRI, and apr-YTF groups. These features showed marked changes after MIRI modeling and an opposite or recovery trend after apr-YTF intervention. The statistical and annotation information for the 17 reversed differential metabolic features, including fold change, VIP values, P values, FDR-adjusted q values, MS/MS support status, and annotation confidence, is summarized in Table 2. The color scale indicates relative ion intensity from low to high.

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  Figure 6. On-tissue MS/MS spectra of nine representative reversed differential metabolic features with MS/MS support. The MS/MS spectra are shown for nine reversed differential metabolic features that were improved after apr-YTF intervention and supported by on-tissue tandem mass spectrometry (MS/MS) spectral matching: (A) dopamine, m/z 136.0618; (B) benzyl cinnamate, m/z 221.0185; (C) 3-(3,4,5-trimethoxyphenyl)propanoic acid, m/z 241.1547; (D) 2-O-(α-D-glucopyranosyl)-D-glyceric acid, m/z 291.0692; (E) methylene blue, m/z 320.0896; (F) Glu-Arg-Phe-Asp, m/z 566.3221; (G) Ile-Arg-Ile-Ile-Val, m/z 613.4816; (H) arachidonoyl platelet-activating factor C-16, m/z 790.4860; and (I) 1,2-distearoyl-sn-glycero-3-phosphocholine, m/z 790.5350.