The proton-sensing G protein-coupled receptor T-cell death-associated gene 8 (TDAG8) shows cardioprotective effects against myocardial infarction

Figures

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  Figure 1. TDAG8 KO mice show high mortality in the early stages after MI. (a) Quantitative analysis of the expression of proton-sensing GPCR mRNAs in intact (n = 3) and infarcted WT hearts (n = 3–7). (b) Quantitative analysis of Tdag8 mRNA levels in infarcted and non-infarcted areas of WT hearts after MI (sham n = 3; MI n = 3–7). Comparisons of group data were assessed with unpaired Student’s t-tests. Data are shown as the mean ± SEM. (c) Kaplan-Meier survival curve analysis in WT mice (sham n = 12, MI n = 35) and TDAG8 KO mice (sham n = 9, MI n = 40) after MI or a sham operation. The difference between WT-MI mice and TDAG8-KO mice was evaluated using the log-rank test. (d) Picrosirius Red staining of cardiac sections in MI- or sham-operated WT mice and TDAG8 KO mice after 28 days. (e) The infarct size was quantitatively estimated. (f) Representative heart sections of infarct areas and non-infarct areas 28 days after MI or sham procedures in WT mice (sham n = 3, MI n = 5) and TDAG8 KO mice (sham n = 3, MI n = 5) stained with Picrosirius Red. Positive staining indicates fibrosis. (g) The fibrotic area was quantified. (h) Echocardiography of WT mice (sham n = 7, MI n = 7) and TDAG8 KO mice…

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  Figure 2. Comparison of TDAG8+ infiltrating cells in infarcted hearts of WT mice and TDAG8 KO mice. (a) Tdag8 mRNA levels in T cells (T cell), neutrophils (Neu), macrophages (Mac), fibroblasts (Fib) and other cell types isolated from hearts of WT mice on post-MI day 3. Three infarcted WT mice hearts were stained with each antibody. After eliminating dead cells and gating singlet live cells, T cells (CD45.2+ Ly6G− CD11b− CD3+), neutrophils (CD45.2+ Ly6G+ CD11b+ CD3−), macrophages (CD45.2+ Ly6G− CD11b+ CD3−), fibroblasts (CD45.2− PDGFRα+) and other cells (CD45.2− PDGFRα−) were sorted. The total mRNA collected from each cell population was used for real-time RT-PCR analysis. (b) Comparison of infiltrating T cells (CD90.2+), macrophages (Ly6G− CD11b+) and neutrophils (Ly6G+ CD11b+) on post-MI day 3 between WT mice (n = 4–7) and TDAG8 KO mice (n = 4) Representative FACS profiles are shown. Comparisons were assessed using unpaired Student’s t-tests. Error bars represent the mean ± SEM (N.S., not significant).

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  Figure 3. Partial regulation of Ccl20 expression by TDAG8 on cardiac macrophages. (a) mRNA expression levels of chemokine genes in infarcted and non-infarcted areas of WT mice (sham n = 3, MI n = 7) and TDAG8 KO mice (sham n = 3, MI n = 5) 3 days after MI. # indicates that Ccl20 mRNA was not detected. (b) Ccl20 mRNA expression levels in cardiac macrophages treated with TNF-α or HMGB1 at pH 6.5 or pH 7.4. Cardiac macrophages were harvested from WT mice or TDAG8 KO mice 3 days after MI and treated with TNF-α (10 ng/ml) or HMGB1 (10 μg/ml) at pH 6.5 or pH 7.4 for 8 h to measure Ccl20 mRNA using real-time RT-PCR. (c) Comparison of infiltrated CCR6+ γδT cells in infarcted hearts of WT and TDAG8 KO mice on post-MI day 3. The tinted histograms indicate the isotype control. Representative FACS profiles are shown. Comparisons were assessed using unpaired Student’s t-test (a,c) or a one-way ANOVA followed by Tukey’s test (b). Error bars represent the mean ± SEM (N.S., not significant).

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  Figure 4. Enhancement of IL-17A-producing γδT cells and left ventricle remodelling in TDAG8 KO mice after MI. (a) mRNA expression levels of the indicated genes in infarcted and non-infarcted areas of WT mice (sham n = 3, MI n = 3–7) and TDAG8 KO mice (sham n = 3, MI n = 3–5) 3 days after MI or sham operation. # indicates that IL-17a mRNA was not detected. (b) All cardiac γδT cells isolated from WT and TDAG8 KO mice were treated with IL-1β (10 ng/ml) and IL-23 (10 ng/ml) on an anti-CD3 antibody-coated plate. Four infarcted hearts were combined for this analysis. After 48 h, the amount of secreted IL-17A in the supernatants was measured using ELISA. ELISA assay was performed in triplicates. (c) Infiltrated IL-17A-producing T cells in the infarcted hearts of WT mice and TDAG8 KO mice 3 days after MI were further analysed for γδTCR and CD4 expression using flow cytometry. Five heart samples were combined as one sample and used for analysis. (d) TUNEL staining of the infarcted area 3 days after MI. Tissue sections from WT mice (sham n = 3, MI n = 3) and TDAG8 KO mice (sham n = 3, MI n = 3) 3 days after MI were stained with TUNEL (red) and DAPI (blue). Arrows indicate apoptotic cells. (e) mRNA…

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  Figure 5. Postulated model showing the cardioprotective effects of TDAG8-expressing cardiac macrophages against MI. MI induces a pH reduction and severe inflammation at infarcted areas. TDAG8 expressed on cardiac macrophages recognises extracellular pH changes and inhibits the Ccl20 transcription stimulated by inflammatory factors (e.g. HMGB1 and TNF-α), which inhibits the recruitment of IL-17A-producing CCR6+ γδT cells. These cells are mediators of cell death and fibrosis, thus explaining the cardioprotective effects of TDAG8.