Txnip/ PDK4 ‐Mediated Hyperdependence on Fatty‐Acid Oxidation Amplifies Susceptibility of Diabetic Myocardium to Ischemia–Reperfusion Damage

Figures

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  FIGURE 1. The number of C6‐CMs cells decreased after myocardial I/RI in diabetic patients. Enhanced fatty acid metabolism is the material basis of increased myocardial vulnerability. (A) t‐SNE plot shows 11 cell subgroups, NM: Healthy C57BL/6J mice group; NMI: Healthy C57BL/6J I/RI mice group; DM: Diabetic mice group; DMI: Diabetic db/db I/RI mice group; the red in the plots is the cardiomyocyte subgroups; the labeled numbers are the proportions of cardiomyocytes. (B) Histogram showing the proportions of the five cardiomyocyte subgroups under different conditions. (C) Correlation heat map of the five cell subtypes. (D) GSEA heatmap of the five cardiomyocyte subtypes. (E) Violin plot of genes related to fatty acid metabolism. (F) Violin plot of genes related to glucose metabolism. (G) Violin plot of genes related to energy synthesis. (H) Violin plot of genes related to hypoxia resistance. (I) Heatmap of gene transcription levels in the C6‐CMs. (J) Trends of key glucose metabolism genes in the C6‐CMs. (K) Trends of genes related to fatty acid ꞵ‐oxidation in the C6‐CMs. (L) Trends of key energy synthesis genes in the C6‐CMs. (M) Trends of hypoxia response genes in the four C6‐CMs.

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  FIGURE 2. C6‐CMs were mainly localized in ventricular myocytes and varied in number between groups. n = 3 in each group; original magnification ×7.5; NM: Heart tissue of a C57BL/6J mice; DM: Heart tissue of a db/db mouse; (A) blood vessels; (B) ventricular tissue; (C) atrial tissue; nuclei stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) are blue under excitation with ultraviolet (UV) light; positive expression is fluorescein fluorescence. Xirp2 is green, Ttn is red, and Myl3 is purple; (D–H) FISH images of atrial and ventricular tissue from the NM group and DM group; original magnification ×63; (I–K). Comparative q‐PCR analysis of Ttn, Myl3, and Xirp2 in the different groups (*p < 0.05, **p < 0.01, ***p < 0.001).

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  FIGURE 3. Diabetic myocardial I/RI is mainly dependent on fatty acid metabolism. n = 5 in each group. (A) Scatter plot (PCA) of the 3D model. (B) Cluster heatmap of differential metabolites of glucose and fatty acids between in NM and DM. (C) Cluster heatmap of glucose and fatty acids between in NMI and DMI. (D) Experimental procedures. (E) Cluster heatmap of differential metabolites between in NMI and DMI. (F) Comparative analysis of 13C glucose‐labeled metabolites in the tricarboxylic acid cycle between four groups.

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  FIGURE 4. After the intervention of TXNIP/PDK4 inhibitors, the glucose metabolic capacity of healthy and diabetic myocardial I/RI increased, and the fatty acid metabolism of diabetic myocardial decreased, alleviating the myocardial injury after I/RI. n = 3 in each group. (A) Experimental process. (B, C) Q‐PCR analysis of TXNIP and PDK4 mRNA expression at different groups. (D–F) Western blot analysis of TXNIP and PDK4 protein expression at different groups. (G) Western blot analysis of proteins associated with glucose metabolism at different groups. (H–K) The histogram shows the levels of HK, LDH, PFK, and PKM proteins in different groups. (L–P) Western blot analysis of proteins associated with fatty acid metabolism at different groups. D + DMI: Dichloroacetic acid intervention in db/db ischemia–reperfusion group mice; V + DMI: Verapamil intervention in db/db ischemia–reperfusion group mice; D/V + DMI: Dichloroacetic acid and Verapamil combined intervention in db/db ischemia–reperfusion group mice. ^: vs. NM, &: vs. NMI, *: vs. DMI, #: vs. DM.

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  FIGURE 5. After TXNIP/PDK4 inhibitor intervention, diabetic myocardial I/RI showed enhanced cardiac function, decreased infarct area, and reduced apoptosis. n = 6 in each group. (A) Echocardiography in each group. (B–F) The box diagram shows the indicators of hypercardiography in each group. HR: Heart rate, SV: Cardiac volume, CO: Output per minute, EF: Ejection fraction, FS: Left ventricular brachialis shortening rate. (G) Cleaved caspase‐3 western blot bands of each group. (H) The histogram shows the levels of cleaved caspase‐3 proteins in different groups. (I) Representative TTC‐stained myocardial sections from each group (white areas indicate infarcted tissue, red areas indicate viable tissue). (J) Quantitative analysis of myocardial infarct size.