Flavonifractor Plautii or Its Metabolite Desaminotyrosine as Prophylactic Agents for Alleviating Myocardial Ischemia/Reperfusion Injury

Abstract

Myocardial ischemia/reperfusion (I/R) injury is a major contributor to myocardial damage, leading to adverse cardiac remodeling and dysfunction. Recent studies have highlighted the potential of gut microbiota-derived metabolites in modulating cardiac outcomes. Here, the cardioprotective effects of a commensal bacterium Flavonifractor plautii (F. plautii) and its metabolite desaminotyrosine (DAT) against myocardial I/R injury are investigated. We showed that prophylactic gavage of F. plautii attenuates myocardial I/R injury as evidenced by improved cardiac function and reduced cardiac injury. We also found that its metabolite DAT recapitulates these cardioprotective effects against myocardial I/R injury. Transcriptomic analysis has revealed that DAT preserves cardiac tissue and attenuates immune responses against myocardial I/R injury. Mechanistically, DAT promotes cardiomyocyte survival through the modulation of the nicotinamide adenine dinucleotide phosphate (NADP⁺/NADPH) ratio. Further, DAT suppressed macrophage proinflammatory activities and cardiac inflammation via the reduction in interleukin-6 (IL-6) production. Taken together, our findings indicate that F. plautii and its metabolite DAT exert pleiotropic cardioprotective effects against myocardial I/R injury, suggesting them as potential prophylactic therapeutic options for alleviating myocardial I/R injury.

Keywords: Flavonifractor plautii; cardiac inflammation; cardiomyocyte survival; desaminotyrosine; myocardial ischemia/reperfusion injury.

Figure 1

Prophylactic F. plautii gavage attenuated cardiac dysfunction in mouse hearts after I/R. A) Schematic diagram of the experiment with F. plautii (F.P) gavage. B) The copies of bacterial 16S rDNA detected in the feces collected before and after antibiotics (Abx) treatment. n = 5 for each group. C) The copies of F. plautii rDNA detected in the feces collected after antibiotics (Abx) treatment, after 1 week F. plautii gavage, and 7 d after I/R. n = 5 for each group. D) The ratios of heart weight to body weight (HW/BW) and heart weight to tibial length (HW/TL) in the Sham, PBS‐gavaged I/R (I/R+PBS), and F. plautii‐gavaged I/R (I/R+F.P) groups at 2 weeks after the surgery. n = 7 for each group. E) Representative echocardiographic images at 2 weeks after I/R (left panel). Quantitative data of left ventricular fractional shortening (LVFS), left ventricular ejection fraction (LVEF), left ventricular end‐diastolic dimension (LVEDD), and left ventricular end‐systolic dimension (LVESD) at 2 weeks after I/R (right panels). n = 8 for each group. F) Picrosirius red staining of transverse cardiac sections from hearts at 2 weeks after I/R. Scale bar, 1 mm. Quantifications of fibrotic scar tissues are shown in the right panel. n = 4 for each group. Quantitative data are presented as mean ± SEM. Groups were compared using B) Student's t‐test or C–F) one‐way ANOVA. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2

DAT recapitulated the cardioprotective effects of F. plautii in mouse hearts after I/R. A) Levels of desaminotyrosine (DAT) measured in the serum (left panel) and heart tissues (right panel) by HPLC‐MS after 1 week PBS and F. plautii (F.P) gavage. n = 5 for PBS group; n = 4 for F.P group. B) Schematic diagram of mouse experiment with DAT treatment at a final concentration of 20 mg mL⁻¹ in drinking water and I/R surgery. C) The ratios of HW/BW and HW/TL in the Sham, I/R, and DAT‐treated I/R (I/R+DAT) groups at 1 week after the surgery. n = 7 for each group. D) Representative echocardiographic images at 1 week after I/R are shown in the left panel. n = 7 for each group. Quantitative results of LVFS, LVEF, LVEDD, and LVESD are shown in the right panel. Data are presented as mean ± SEM. Groups were compared using A) Student's t‐test or C,D) one‐way ANOVA. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

DAT preserved cardiac tissue and attenuated immune response in mouse hearts after I/R. A) Principal component analysis (PCA) on the transcriptome data obtained from injured heart tissues of mice in the I/R and DAT‐treated I/R (I/R+DAT) groups at 1 week after the surgery, along with the transcriptome data obtained from corresponding tissue samples from the Sham group at 1 week after the surgery. B) Volcano plot of genes differentially regulated in I/R versus Sham groups (left panel) and I/R+DAT versus I/R groups (right panel). The blue dots denote downregulated genes, the red dots denote upregulated genes, and the grey dots denote genes without significantly differential expression. C) KEGG analysis of pathways associated with genes downregulated in I/R versus Sham groups and upregulated in I/R+DAT versus I/R groups (indicated in red, left panel) and pathways associated with genes upregulated in I/R versus Sham groups and downregulated in I/R+DAT versus I/R groups (indicated in blue, right panel). D) GSEA plots showing the enrichment of gene sets related to cardiac muscle cell contraction (upper panel) and inflammatory response (lower panel) in I/R+DAT versus I/R groups. E) Picrosirius red staining of transverse cardiac sections from hearts at 1 week after I/R. Scale bar, 1 mm. Quantifications of fibrotic scar tissues are shown in the right panel. n = 8 for Sham and I/R groups; n = 6 for I/R+DAT group. F) Immunohistochemical staining for IL‐6 (upper panel), TNF‐α (middle panel), and IL‐1β (lower panel) at 1 week after I/R. Scale bar, 10 µm. Quantitative data are shown to the right. n = 6 for Sham group; n = 8 for I/R group; n = 7 for I/R+DAT group. Data are presented as mean ± SEM. E,F) Groups were compared using one‐way ANOVA. **p < 0.01; ***p < 0.001.

Figure 4

DAT promoted cardiomyocyte survival by modulating the NADP⁺/NADPH ratio. A) Triphenyltetrazolium chloride (TTC) staining of heart sections collected from Sham, I/R, and DAT‐treated I/R (I/R+DAT) groups at 1 d after the surgery. n = 8 mice for each group. B) Representative images and C) quantification of TUNEL staining in heart sections from Sham, I/R, and I/R+DAT groups at 1 d after the surgery. TUNEL (green) and DAPI (blue). Scale bar, 20 µm. n = 4 for Sham and I/R groups; n = 3 for I/R+DAT group. D) Schematic diagram illustrating the in vitro experiments with neonatal rat ventricular cardiomyocytes (NRVMs) subjected to DAT pretreatment at 100 µm followed by oxygen glucose deprivation/reoxygenation (OGD/R) that simulated myocardial I/R injury. E) Cell viability measured by Alamar blue staining of NRVMs (left panel) and LDH (lactate dehydrogenase) release from NRVMs (right panel) in the presence or absence of DAT under normoxic and OGD/R conditions. n = 6 for each group. F) DHE staining in heart sections from Sham, I/R, and I/R+DAT groups. Scale bar, 20 µm. n = 5 for Sham group; n = 3 for I/R group; n = 4 for I/R+DAT group. DHE (red) and DAPI (blue). G) DHE staining of NRVMs in the presence or absence of DAT under normoxic and OGD/R conditions. n = 6 for each group. H) The ratio of NADP⁺/NADPH calculated in the presence or absence of DAT under OGD/R condition. n = 4 for each group. I) Schematic diagram illustrating the in vitro experiments with NRVM subjected to DAT pretreatment at 100 µm followed by OGD/R in the presence or absence of oxidized NADP⁺. J) Cell viability measured by Alamar blue staining of NRVMs (left panel) and LDH release from NRVMs (right panel). n = 8 for each group. Quantitative data are presented as mean ± SEM. Groups were compared using A,C,F) one‐way ANOVA, E,F) two‐way ANOVA or H,J) Student′s t‐test. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

DAT suppressed the proinflammatory activities of macrophages. A) Schematics of the experiment in mice with macrophage depletion. Clodronate liposomes (Cl₂MDP) were intravenously injected to deplete macrophages in vivo before the mice were subjected to I/R surgery. PBS liposomes (PBS‐L) was used as control for Cl₂MDP. B) Representative echocardiographic images of mouse hearts from Sham, PBS‐L‐injected I/R (I/R+PBS‐L), DAT‐treated PBS‐L‐injected I/R (I/R+DAT+PBS‐L), and DAT‐treated Cl₂MDP‐injected I/R (I/R+DAT+Cl₂MDP) groups at 1 week after I/R (left panel). Quantitative data on LVFS and LVEF are shown to the right. n = 8 for each group. C) Representative images and quantification of IL‐6 (left panel), TNF‐α (middle), and IL‐1β (right panel) signals by immunostaining at 1 week after I/R. Scale bar, 10 µm. n = 4 for each group. D) Schematic diagram illustrating the in vitro experiments with bone marrow‐derived macrophages (BMDMs) stimulated with lysates from injured cardiac tissues of I/R mouse hearts 1 d after the surgery (I/R lysate), with lysates from the same region of cardiac tissues of mouse hearts subjected to Sham surgery (Sham lysate) as controls in the presence or absence of DAT pretreatment at 100 µm. E) qRT‐PCR analysis showing the mRNA levels of Il6 and Tnf. n = 3 for each group. Quantitative data are presented as mean ± SEM. Groups were compared using B,C) one‐way ANOVA or E) two‐way ANOVA. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6

DAT inhibited proinflammatory IL‐6 production by macrophages. A) Schematic diagram (left panel) and flow cytometry analysis of DHE‐positive BMDMs stimulated with Sham lysate and I/R lysate for 24 h with or without DAT pretreatment at 100 µm (right panel). n = 3 for each group. B) Schematic diagram illustrating the in vitro experiments with BMDMs stimulated with 4 h H₂O₂ in the presence or absence of DAT pretreatment at 100 µm. C) qRT‐PCR analysis showing the mRNA levels of Il6 and Tnf in BMDMs. n = 3 for each group. D) ELISA showing the secretion of IL‐6 and TNF‐α by BMDMs. n = 3 for each group. E) Schematic diagram illustrating the in vitro experiments with H₂O₂‐stimulated BMDMs with or without IL‐6 supplementation or Olamkicept treatment (Olam) (IL‐6 receptor antagonist) in the presence or absence of DAT‐pretreatment (100 µm). F) qRT‐PCR analysis of Tnf mRNA levels in BMDMs (left panel) and ELISA of TNF‐α secretion by BMDMs (right panel). n = 4 for each group. G) Serum IL‐6 levels in mice from the Sham, I/R, and DAT‐treated I/R (I/R+DAT) groups at 1 d after the surgery. n = 4 for Sham group; n = 5 for I/R and I/R+DAT groups. H) Schematic diagram illustrating the experiments in mice from Sham, PBS‐injected I/R (I/R+PBS), DAT‐treated PBS‐injected I/R (I/R+DAT+PBS), and DAT‐treated Olamkicept‐injected I/R (I/R+DAT+Olam) groups at 1 week after the surgery. I) Representative echocardiographic images of mice from Sham, I/R+PBS, I/R+DAT+PBS, and I/R+DAT+Olam groups at 1 week after the surgery. J) Quantitative data of LVFS and LVEF. n = 6 for each group. K–M) Representative images and quantification of K) IL‐6, L) TNF‐α, M) and IL‐1β signals by immunostaining at 1 week after I/R. n = 6 for each group. Scale bar, 10 µm. Quantitative data are presented as mean ± SEM. Groups were compared using G,J,K,L,M) one‐way ANOVA, A, C, D, and F‐right panel) two‐way ANOVA or F‐left panel) Student's t‐test. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.