A cardiac fibroblast-enriched micropeptide regulates inflammation in ischemia/reperfusion injury

Figures

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  Figure 1. Micropeptide IAMP is highly expressed in cardiac fibroblasts. (A) Genomic locus of IAMP in human (KIAA0040) and mouse (4930523C07Rik). The ORF encoding IAMP is highlighted in red. (B) Multiple alignment of amino acid sequences of IAMP proteins across vertebrates. (C) qPCR analysis of IAMP mRNA expression in different tissues of adult mice. n = 3 for each group. (D) Western blotting of IAMP protein and quantification in different tissues of adult mice. n = 3 for each group. (E) qPCR analysis of IAMP mRNA expression in isolated adult cardiomyocytes and noncardiomyocytes. n = 4 for each group. (F) Analysis of IAMP expression in different cell clusters in the human heart using single-cell RNA-Seq data (https://www.proteinatlas.org/). (G) qPCR analysis of IAMP mRNA expression in different cell types. NRC, neonatal rat ventricular cardiomyocytes; NRF, neonatal rat ventricular fibroblasts; NIH-3T3, fibroblast cell line; HUVEC, human umbilical endothelial cells; EA.hy926, endothelial cell line; SMC, human smooth muscle cell; RAW 264.7, macrophage cell line. n = 3 for each group. (H) Immunofluorescence of cardiac fibroblasts infected by indicated adenoviruses. Scale bar: 50 μm. (I)…

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  Figure 2. IAMP represses inflammatory response in cardiac fibroblasts. (A and B) qPCR and Western blot analysis of IAMP expression in IAMP-KD (knockdown) mouse cardiac fibroblasts. n = 4 for each group. (C) Venn diagram showing the number of differentially expressed genes in cardiac fibroblasts upon IAMP knockdown. (D) KEGG pathway enrichment analysis of dysregulated genes. (E) Heatmap showing relative expression of upregulated genes in IL-17 signaling pathway. (F) qPCR analysis of expression of genes in IL-17 signaling pathway in IAMP-KD mouse cardiac fibroblasts. n = 4 for each group. (G) qPCR analysis of mRNA levels of Il6, S100a8, and Mmp3 in IAMP-knockdown (IAMP-KD) mouse cardiac fibroblasts. n = 3 for each group. (H) ELISA detecting protein levels of IL-6, S100A8, and MMP3 in culture medium of IAMP-KD mouse cardiac fibroblasts. n = 6 for each group. (I) qPCR analysis of mRNA levels of Il6, S100a8, and Mmp3 in IAMP-overexpressing (IAMP-OE) mouse cardiac fibroblasts. n = 6 for each group. (J) ELISA detecting protein levels of IL-6, S100A8, and MMP3 in culture medium of IAMP-OE mouse cardiac fibroblasts. n = 4 for each group. (K) qPCR analysis of mRNA levels of Il6, S100a8, and Mmp3 in…

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  Figure 3. IAMP expression is correlated with myocardial inflammation in heart. (A) Relative expression of IAMP in hearts after IR injury from public RNA-Seq datasets. n = 3–4 for each group. (B) qPCR analysis of mRNA level of IAMP in mouse hearts after IR injury. n = 5–10 for each group. (C) Correlation between IAMP and Il6 mRNA levels in hearts mentioned in B. (D) Relative expression of IAMP in patients with different BMI. BMI ≤ 32 (n = 43); BMI > 32 (n = 19). (E and F) Body weights and gene expression in hearts of 8-month-old mice fed with normal (n = 10) or high-fat (n = 19) diets for 5 months. (G) Correlation between IAMP and Il6 expression in hearts mentioned in F. (H) Strategy of generating IAMP-KO mice by CRISPR/Cas9 editing and DNA sequence confirmation. (I) Western blotting of IAMP protein in hearts of control (Ctrl) and KO adult mice. (J) Western blotting of IAMP protein in cardiac fibroblasts isolated from Ctrl and KO adult mice. (K) qPCR analysis of mRNA levels of Il6, S100a8, and Mmp3 mRNA levels in cardiac fibroblasts isolated from Ctrl and KO adult mouse. n = 3 for each group. (L) ELISA detecting protein levels of IL-6, S100A8, and MMP3 in culture medium of cardiac…

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  Figure 4. Loss of IAMP exacerbates ischemia/reperfusion injury in the heart. (A) Schedule for the in vivo study. (B) Evans blue and triphenyltetrazolium chloride (TTC) double staining and quantification of area at risk (AAR) and infarct size (IF) in hearts from Ctrl and IAMP-KO mice 24 hours after I/R surgery. n = 5 for each group. Scale bar: 1 mm. (C) TUNEL staining and quantification of apoptotic cells in cross sections of hearts from Ctrl and IAMP-KO mice 24 hours after I/R or surgery. n = 7 for each group. Scale bar: 100 μm. (D) Fractional shortening (FS), left ventricular posterior wall thickness at end-diastole (LVPW;d), and left ventricular internal dimension at end-diastole (LVID;d) accessed by echocardiography 2 weeks after I/R or sham surgery. n = 7–10 for each group. (E) Sirius red staining and quantifications of fibrotic area in cross sections of hearts from Ctrl and IAMP-KO mice 2 weeks after I/R or sham surgery. Scale bar: 1 mm. (F) qPCR analysis of mRNA levels of fibrosis marker genes. n = 7–8 for each group. (G) Flow cytometry and quantification of percentages of CD45+ cells in the ischemic area of hearts from Ctrl and IAMP-KO mice 24 hours after I/R or sham surgery. n =…

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  Figure 5. IAMP regulates HIF-1α protein expression. (A) qPCR analysis of mRNA levels of IAMP, Il6, Mmp3, and Cxcl1 in control and IAMP-KD cardiac fibroblasts with or without PDTC (10 �M) treatment. n = 3 for each group. (B) Venn diagram showing the numbers of differentially expressed gene in IL-17–stimulated IAMP-KD cardiac fibroblasts. (C) Pathway enrichment analysis of the upregulated genes using the NCI pathway interaction database. (D) STRING interaction network of HIF-1α and NF-κB with proteins of upregulated genes. The yellow and blue boxes represent HIF-1α– and NF-κB–interacting proteins, respectively. (E) Western blotting and quantification of HIF-1α protein (n = 3) and qPCR analysis of Hif1a mRNA (n = 4) in the IAMP-KD cardiac fibroblasts. (F) Western blotting and quantification of HIF-1α protein (n = 3) and qPCR analysis of Hif1a mRNA (n = 4) in the IAMP-OE cardiac fibroblasts. (G) Western blotting and quantification of HIF-1α protein (n = 3) and qPCR analysis of Hif1a mRNA (n = 3) in the cardiac fibroblasts isolated from IAMP-KO and Ctrl mice. *P < 0.05; **P < 0.01; ***P < 0.001, by 2-tailed Student’s t test (F and G) or ANOVA with Tukey’s correction (A and E).

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  Figure 6. IAMP regulates proinflammatory gene expression through HIF-1α. (A) Western blotting and quantification of HIF-1α protein in cardiac fibroblasts with or without NiCl2 treatment. n = 3 for each group. (B) qPCR analysis of mRNA levels of Il6, S100a8, and Mmp3 in cardiac fibroblasts with or without NiCl2 treatment. n = 3 for each group. (C) Western blotting and quantification of HIF-1α protein in cardiac fibroblasts with or without hypoxia treatment. n = 3 for each group. (D) qPCR analysis of mRNA levels of Il6, S100a8, and Mmp3 in cardiac fibroblasts with or without hypoxia treatment. n = 3 for each group. (E) qPCR analysis of mRNA levels of Il6, S100a8, and Mmp3 in cardiac fibroblasts transfected with indicated siRNAs. n = 3 for each group. (F) ELISA detecting protein levels of IL-6 and MMP3 in culture medium of cardiac fibroblasts transfected with indicated siRNAs. n = 3 for each group. (G) qPCR analysis of mRNA levels of Hif1a, Il6, S100a8, Mmp3, and Cxcl1 in cardiac fibroblasts treated with indicated siRNA and adenoviruses. n = 4 for each group. (H) ELISA detecting protein levels of IL-6, S100A8, and MMP-3 in culture medium of cardiac fibroblasts treated with indicated siRNA and…

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  Figure 7. IAMP destabilizes HIF-1α proteins through interacting with HSP90. (A–C) HIF-1α stability in cardiac fibroblasts transfected with IAMP siRNAs (A), isolated from IAMP-KO and Ctrl mice (B), or infected by Ad-FLAG-IAMP (C) were exposed to NiCl2 (50 mM) for 24 hours. Cells were cultured with cycloheximide (CHX) (50 μg/mL) with indicated time before harvest. Western blotting and quantification of HIF-1α were shown. n = 3–4 for each group. (D and E) Coimmunoprecipitation and Western blotting showing the interaction of exogenous HA-HSP90 and FLAG-IAMP (D) and endogenous HSP90 and exogenous FLAG-IAMP in HEK293 cells (E). (F) Coimmunoprecipitation and Western blotting showing the interaction of endogenous HSP90 and exogenous FLAG-IAMP in cardiac fibroblasts. (G) Coimmunoprecipitation and Western blotting showing the interaction of endogenous HSP90 and IAMP in cardiac fibroblasts isolated from IAMP-KO or Ctrl mice. (H) Western blotting and quantification of HIF-1α protein in HEK293 transfected with indicated plasmids for 12, 24, and 48 hours. n = 4 for each group. (I) Western blotting and quantification of HIF-1α protein in HEK293 transfected with indicated plasmids. n = 3 for each group.…

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  Figure 8. A proposed working model for the regulation of inflammation in cardiac fibroblasts and cardiac ischemia/reperfusion injury by IAMP