Gut microbiota-induced elevation of succinate exacerbates diabetic myocardial ischemia/reperfusion injury by promoting macrophage polarization

Figures

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  Figure 1. Schematic of the animal and cell experimental protocols. (A) A T2D mouse model was induced by a HFD and low-dose STZ injection, followed by myocardial IR. (B) Diabetic PGF mice were established by ABX and underwent myocardial IR operation. (C) To deplete macrophages, diabetic mice received an intraperitoneal injection of Cl2MDP-Lipo 3 days prior to myocardial IR injury. (D) Protocol of the CM transfer model. IR, ischemia/reperfusion; T2D, type 2 diabetes; HFD, high-fat diet; ABX: antibiotic mixture; PGF: pseudo germ-free; STZ, streptozocin; HR: hypoxia reoxygenation; CM: Conditioned medium; HG: high glucose.

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  Figure 2. Succinate levels in the diabetic myocardium are elevated during diabetes and are associated with alterations in specific gut microbiota. (A) Succinate levels in the serum, caecal contents, intestine, and myocardium after diabetes induction (n = 6). (B) mRNA expression of the tight junction genes Tjp1 and Ocln (n = 6). (C) Negative correlation between myocardial succinate levels and the mRNA expression of Tjp1 and Ocln (n =10). (D) Altered microbiota composition at the phylum level and an increased Firmicutes/Bacteroidetes (F/B) ratio after diabetes induction (n = 9). (E) Increased relative abundance of succinate-producing bacterial families (Prevotellaceae, Bacteroidaceae, and Veillonellaceae) and decreased abundance of succinate-consuming families (Odoribacteraceae and Clostridaceae) in diabetic mice (n = 9). (F) The ratio of succinate-producing to succinate-consuming bacteria (P+B+V/O+C) at the family level (n = 9). (G) The ratio of succinate producers to consumers was positively correlated with myocardial succinate levels (n = 9). (H) Comparison of the relative abundance of succinate-producing and succinate-consuming bacteria at the genus level between diabetic and nondiabetic…

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  Figure 3. Depletion of the gut microbiota reverses elevated succinate levels and attenuates diabetes-aggravated myocardial IR injury. (A) Body weight and blood glucose levels were monitored after STZ or ABX administration (n=10). (B) Succinate levels in the cecal contents, intestine, serum, and myocardium after ABX treatment (n = 6). (C) Myocardial infarct size assessed by Evans blue/TTC double staining (n = 5). (D) Serum LDH and cTnT levels in diabetic and nodiabetic mice post-IR (n=8). (E) Cardiomyocyte apoptosis determined by TUNEL assay. Scale bar = 50 μm (n=6). (F) Representative images and quantification of LVEF and LVFS (n=6). Data are expressed as mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, #P < 0.05, ##P < 0.01, ####P < 0.0001, $$$P < 0.001, $$$$P < 0.0001, ns, not significant. IR, ischemia/reperfusion; NoDIA: nodiabetes, DIA:diabetes, ABX: antibiotic mixture; LVEF: left ventricular ejection fraction; LVFS: left ventricular shortening fraction.

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  Figure 4. FMT from diabetic mice increases succinate levels and exacerbates myocardial IR injury in diabetic PGF mice. (A) Succinate levels in the cecal contents, intestine, serum and myocardium, (n=6). (B) Serum LDH and cTnT levels post-IR (n=8). (C) Myocardial infarct size assessed by Evans blue/TTC double staining (n = 5). (D) Cardiomyocyte apoptosis was assessed by a TUNEL assay. Scale bar = 50 μm. (n=6). (E) Representative images and quantification of LVEF and LVFS (n=6). Data are expressed as mean ± standard error of the mean. * P < 0.05, ** P < 0.01, ***P < 0.001, ****P < 0.0001. FMT: Faecal microbiota transplantation; IR, ischemia/reperfusion; NoDIA: nodiabetes, DIA:diabetes, ABX: antibiotic mixture; LVEF: left ventricular ejection fraction; LVFS: left ventricular shortening fraction.

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  Figure 5. Succinate exacerbates diabetic myocardial IR injury and promotes macrophage polarization towards the M1 phenotype. (A) Experimental protocol: diabetic mice received daily intraperitoneal injection of succinate for 7 days before myocardial IR injury. (B) Serum LDH and cTnT levels in diabetic and nodiabetic mice post-IR (n=8). (C) Myocardial infarct size assessed by Evans blue/TTC double staining (n = 5). (D) Cardiomyocyte apoptosis determined by TUNEL assay. Scale bar = 50 μm (n=6). (E) Representative images and quantification of LVEF and LVFS (n=6). (F) Representative flow cytometry plots showing iNOS+CD206- (M1) and iNOS-CD206+ (M2) macrophages (n=3). (G) quantitative analysis of M1 macrophages (iNOS+CD206-) and M2 macrophages (iNOS-CD206+) in the myocardium (n = 3). (H) mRNA expression of M1-related genes (Tnf, Il1, Il6, and Nos2) in cardiac tissue (n = 6). (I) mRNA expression of M2-related genes (Arg1 and Il10) in cardiac tissue (n = 6). Data are expressed as mean ± standard error of the mean. * P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. IR, ischemia/reperfusion; NoDIA: nodiabetes, DIA:diabetes; LVEF: left ventricular ejection fraction; LVFS: left ventricular…

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  Figure 6. Macrophage activation is essential for succinate-mediated diabetic myocardial IR injury. (A) Flow cytometry analysis of cardiac macrophages (F4/80+CD11b+) post myocardial IR (n=3). (B) Serum LDH and cTnT levels post-IR (n=8). (C) Myocardial infarct size assessed by Evans blue/TTC double staining (n = 5). (D) Cardiomyocyte apoptosis determined by TUNEL assay. Scale bar = 50 μm (n=6). (E) Representative images and quantification of LVEF and LVFS (n=6). (F) Representative flow cytometry plots of H9c2 cardiomyocyte apoptosis in the indicated groups (n = 3). (G) LDH release and cell viability of H9c2 cells in the indicated groups (n = 6). Data are expressed as mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. IR: ischemia/reperfusion; NoDIA: nodiabetes; DIA: diabetes; Veh: Vehicle. HR: hypoxia reoxygenation; CM: Conditioned medium; HG: high glucose. Group definitions: HR+HG group: H9c2 cells cultured in high-glucose (25 mM) serum-free DMEM under HR conditions; HR+HG+CM (Control) group: H9c2 cells cultured in HG DMEM under HR with CM from unstimulated control macrophages; HR+HG+CM group: H9c2 cells cultured in HG DMEM under HR with CM collected…

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  Figure 7. Succinate-induced macrophage polarization aggravated diabetic myocardial IR injury via SUCNR1. (A) Representative immunofluorescence images showing SUCNR1 and F4/80 colocalization in the infarct zone at 24 h post-I/R. Scale bar=50 µm, (n=3). (B) Sucnr1 mRNA levels in cardiac tissue lysates collected from the infarct zone at 24 h post-I/R (n=6). (C) Western blot analysis of SUCNR1 protein levels in the infarct zone at 24 h post-I/R (n=3). (D) Serum LDH and cTnT levels post-IR (n=8). (E) Myocardial infarct size assessed by Evans blue/TTC double staining (n = 5). (F) Cardiomyocyte apoptosis determined by TUNEL assay. Scale bar = 50 μm (n=6). (G) mRNA expression of M1-related genes in whole cardiac tissue collected from the infarct zone at 24 h post-I/R. (n = 6). Data are expressed as mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. IR, ischemia/reperfusion; NoDIA: nodiabetes, DIA:diabetes.

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  Figure 8. The succinate–SUCNR1 axis is required for macrophage polarization and exacerbates diabetic myocardial IR injury. (A) Western blot analysis of p-AKT and HIF-1α in the infarcted area (IA) and non-infarcted area (NIA) of the left ventricle collected at 24 h post-I/R from diabetic WT and Sucnr1-/- mice. Succinate treatment increased HIF-1α and p-AKT levels, whereas these effects were reversed in Sucnr1−/− diabetic mice (n=3). (B) Western blot analysis of p-AKT and HIF-1α in cultured RAW 264.7 macrophages. The upregulation of p-AKT and HIF-1α induced by succinate was abolished in shSucnr1-transfected macrophages (n=3). (C) mRNA expression of M1-related genes in RAW 264.7 macrophages treated with the HIF-1α inhibitor 2MeOE2 or the PI3K inhibitor LY294002 (n=6). (D) Flow cytometry analysis of H9c2 cardiomyocyte apoptosis in the indicated groups (n=6). (E) LDH release and cell viability of H9c2 cells in the indicated groups (n = 6). Data are expressed as mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. IR, ischemia/reperfusion; NoDIA: nodiabetes, DIA:diabetes, CM: Conditioned medium, HG: high glucose, HR: hypoxia reoxygenation. NIA: non-infarcted…

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  Figure 9. Gut microbiota-derived succinate exacerbates diabetic myocardial ischemia/reperfusion (IR) injury by promoting M1 macrophage polarization via the SUCNR1–PI3K/AKT–HIF-1α pathway.