H4K5 lactylation exacerbates acute myocarditis by driving a positive feedback loop between inflammation and metabolic dysfunction

Figures

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  Figure 1. Myocardial damage in acute myocarditis is exacerbated by lactate production. (A) Cardiac function was assessed by transthoracic echocardiography at 1 day after acute myocarditis. Left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) were calculated in each group (n=15 each group). (B) Relative gene expression of IL-1β, IL-6 and TNF-α mRNA was normalized to β-actin mRNA in the cardiac tissues from each group (n=4 each group). (C) Quantification of lactate content in the heart tissues (n=6 each group). (D) Representative images of HE staining of heart tissues from mice. Scale bar=1 cm. (E) Representative immunofluorescence images of cardiac sections stained with myocyte membrane (WGA, green) and nuclei (DAPI, blue) from mice. Scale bar=50 μm. (F) Representative immunoblots and the corresponding analysis of glycolysis associated proteins in the cardiac tissues from each group. β-actin was shown as the loading control (n=6 each group). (G) Representative immunoblots of Panla protein in the cardiac tissues from each group. β-actin was shown as the loading control. (H) Representative immunofluorescence images of cardiac sections stained with…

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  Figure 2. Lactate drives macrophage polarization toward a pro-inflammatory M1 phenotype. (A) Representative flow cytometry and statistical analysis of total macrophages, M1 macrophages and M2 macrophages in the cardiac tissues (n=4 each group). (B) Representative immunohistochemical images and the corresponding analysis of cardiac tissues staining with macrophage marker CD68, M1 subtype macrophage marker iNOS and M2 subtype macrophage marker CD206 (n=6 each group). Scale bar=100 μm. (C) Representative immunoblots and the corresponding analysis of M1 macrophage marker iNOS and M2 macrophage marker Arg1 proteins in the BMDMs treated with LPS and FX-11. β-actin was shown as the loading control. (D) Representative immunoblots and the corresponding analysis of glycolysis associated proteins in the BMDMs treated with LPS and FX-11. β-actin was shown as the loading control. (E) The oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) in the BMDMs treated with LPS and FX-11 were detected using a Seahorse XFp analyzer (n=4 biological replicates). For OCR measurement, cells were treated with oligomycin (O), FCCP (F) and antimycin A/rotenone (A+R). For ECAR measurement, cells…

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  Figure 3. Deficiency in lactate production within macrophages mitigates myocardial injury in myocarditis. (A) Representative immunofluorescence images of cardiac sections stained with macrophage (CD68, yellow), protein lactylation (Panla, pink), myocyte (cTNT, green) and nuclei (DAPI, blue) from LDHAfl/fl and LDHAcko mice after LPS treatment. Quantifications of CD68+ intensity and Panla+CD68+ intensity were shown (n=6 each group). Scale bar=50 μm. (B) Representative immunoblots of Panla protein in the cardiac tissues from LDHAfl/fl and LDHAcko mice. β-actin was shown as the loading control. C-D. Representative immunoblots and the corresponding analysis of glycolysis associated proteins in the cardiac tissues from LDHAfl/fl and LDHAcko mice. β-actin was shown as the loading control (n=6 each group). (E) Echocardiography measurements of cardiac function of LDHAfl/f and LDHAcko mice. LVEF and LVFS were calculated in each group (n=6 each group). (F–H) Representative images and the corresponding analysis of WGA (G) and TUNEL (H) staining of cardiac sections from LDHAfl/fl and LDHAcko mice (n=6 each group). Scale bar were shown as indicated in each images. (I) Relative gene expression of IL-1β,…

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  Figure 4. H4K5 lactylation in macrophages directly activates Rap1 transcription via promoter recruitment. (A) Representative immunofluorescence images of cardiac sections stained with macrophage (CD68, yellow), H4K5 lactylation (H4K5la, pink), myocyte (cTNT, green) and nuclei (DAPI, blue) from mice. Quantifications of H4K5la+CD68+ intensity were shown on right (n=6 each group). Scale bar were shown as indicated. (B) Heatmaps showed the H4K5la binding density (CUT&Tag tag counts) at individual peaks in LPS-treated macrophages, with peaks sorted by signal strength. (C) The genomic landscape of upregulated H4K5la binding peaks was characterized in macrophages. (D) The KEGG analysis of the elevated H4K5la binding peaks at candidate target pathway. (E) The KEGG enrichment of RNA sequencing in macrophages were presented and ranked. The TNF and NF-κB signaling pathways were both elevated in the CUT&Tag sequencing and RNA sequencing upon LPS activation. (F) Representative immunoblots of TNF and NF-kB pathway proteins in the BMDMs treated with LPS and FX-11. β-actin was shown as the loading control. (G) The intersection of the up-regulated gene expression in the three datasets. (H) The…

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  Figure 5. Knockdown of Rap1 reduces macrophage-mediated inflammatory damage. (A) Representative flow cytometric and statistical analysis of M1 macrophages and M2 macrophages in the BMDMs treated with LPS and siRap1 (n=4 biological replicates). (B) Relative gene expression of M1-macrophage markers and M2-macrophage markers mRNA was normalized to 18S mRNA in the BMDMs treated with LPS and siRap1 (n=4 biological replicates). (C) Relative gene expression of glycolysis markers mRNA was normalized to 18S mRNA in the BMDMs treated with LPS and siRap1 (n=4 biological replicates). (D) Representative immunoblots of Panla and H4K5la in the BMDMs treated with LPS and siRap1. H4 was shown as the loading control. (E) Representative immunoblots of P-p65, T-p65 and TNF-a in the BMDMs treated with LPS and siRap1. β-actin was shown as the loading control. (F) Representative flow cytometric and statistical analysis of apoptotic cells in the BMDMs treated with LPS and siRap1 (n=4 biological replicates). Data were analyzed by Two-way ANOVA followed by Bonferroni test. *p <0.05;**p < 0.01; ***p < 0.005 and ****p < 0.001.

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  Figure 6. Inhibition of Rap1 in macrophage rescues cardiac function in acute myocarditis. (A) Echocardiography measurements of cardiac function of mice. LVEF and LVFS were shown at day 1 after LPS injection (n=6 each group). (B) Representative immunofluorescence images of cardiac sections stained with myocyte membrane (WGA, green) and nuclei (DAPI, blue) from mice. Scale bar=50 μm. (C) Representative immunofluorescence images of cardiac sections stained with apoptotic cells (TUNEL, pink) and nuclei (DAPI, blue) from mice. Scale bar=20 μm. (D) Quantification of cross-sectional cell area identified by WGA staining in B (n=6 each group). (E) Quantifications of TUNEL+ cells identified by TUNEL staining in C (n=6 each group). (F) ELISA analysis of inflammatory factor IL-1, IL-6 and TNF-α in murine plasma (n=6 each group). (G) Flow cytometric statistical analysis of total macrophages, M1 macrophages and M2 macrophages in the cardiac tissues (n=6 each group). (H) Quantification of lactate content in the heart tissues and serum (n=6 each group). (I) Representative immunoblots and the corresponding analysis of glycolysis associated proteins in the cardiac tissues from mice. β-actin was shown as the…

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  Figure 7. H4K5 lactylation in macrophages exacerbates acute myocarditis via a Rap1/TNF–NF-κB-driven pro-inflammatory feedback loop.