Aggravated myocardial infarction-induced cardiac remodeling and heart failure in histamine-deficient mice

Figures

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  Figure 1. Histamine expression and HDC-expressing CD11b+ myeloid cells in patients and a murine model with heart failure. (a) The serum histamine levels in heart failure patients and healthy control subjects (Control: 1.22 ± 0.82 ng/ml, heart failure: 2.39 ± 1.30 ng/ml; n = 10–16). (b) The higher serum histamine levels in MI mice than the sham group (Sham: 80.51 ± 11.77 ng/ml, MI 3d: 243.80 ± 38.03 ng/ml, MI 7d: 155.70 ± 15.98 ng/ml, MI 4w: 137.5 ± 19.42 ng/ml; n = 6–8). (c) Representative images of infarcted hearts showed that a large amount of HDC-expressing CD11b+ myeloid cells infiltrated in the hearts of HDC-EGFP mice at MI 3d, MI 7d, and MI 4w compared with the sham group (scale bar = 50 μm; n = 3–5). (d,e) The percentages of EGFP+ cells were analyzed by FACS in the infarcted hearts (d) and peripheral blood (e) of mice at 4 weeks post-MI. FSC indicates Forward Scattering. (n = 6–8). *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 2. Histamine deficiency aggravated cardiac remodeling and dysfunction post-MI. (a) The cumulative survival rate of HDC−/− and WT mice post-MI. (b) H&E staining showed that histamine deficiency caused larger infarct size in HDC−/− mice than WT mice at 4 weeks post-MI (scale bar = 500 μm; n = 8–10). (c) Representative echocardiographic images. (d–f) More serious dilative cardiac remodeling and worse cardiac function in HDC−/− mice than WT mice post-MI (n = 8–10). NS, not statistically significant, *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 3. Histamine deficiency increased fibrosis in the infarcted heart. (a) Masson trichrome staining showed that fibrosis significantly increased in HDC−/− mice post-MI (the top scale bar = 500μm and other scale bars = 50 μm; n = 8–10). (b) Collagen I and III mRNA expression levels in the infarcted heart of HDC−/− mice and WT controls were measured by qRT-PCR (n = 3). (c) MMP-2 and MMP-9 mRNA expression levels in the infarcted hearts of HDC−/− mice and WT controls were measured by qRT-PCR (n = 3). NS, not statistically significant, *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 4. Histamine deficiency increased proinflammatory and profibrotic cytokines levels post-MI. (a,b) IL-6 (a) and TGF-β1 (b) levels in the serum of HDC−/− mice and WT mice at 4 weeks post-MI were measured by ELISA (n = 6). (c) FACS data showed the percentage of CD11b+ myeloid cell numbers in the blood of HDC−/− mice and WT mice at 4 weeks post-MI (n = 8). (d) The neonatal heart fibroblast proliferation rates were measured by the CCK-8 assay (n = 6). (e) TGF-β1 levels were measured by ELISA in the supernatant of CD11b+ myeloid cells (n = 6). NS, not statistically significant, *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 5. Histamine repressed fibroblast proliferation via a histamine receptor-dependent paracrine mechanism. (a,b) The qRT-PCR and western blotting data showed no expression of HDC in the heart fibroblasts (n = 3). (c) No EGFP expression in the heart fibroblasts of HDC-EGFP mice, as measured by fluorescence microscopy (scale bar = 25 μm). (d) The proliferation rates of neonatal heart fibroblasts of HDC−/− and WT mice at baseline or in hypoxic conditions were measured by the CCK8 assay (n = 6). (e) Histamine (10−5 M~10−4 M) had an inhibitive effect on heart fibroblast proliferation (n = 3). (f) The results of H&E staining showed that the larger infarct size observed in HDC−/− mice could be attenuated by histamine treatment. This effect could be blocked by H1R or H2R antagonists (scale bar = 500 μm; n = 6–8). (g) Masson trichrome staining showed that cardiac fibrosis decreased in histamine-treated HDC−/− mice compared with HDC−/− mice and that this effect was reversed by H1R or H2R antagonists (the top scale bar = 500 μm and other scale bars = 50 μm; n = 6–8). NS, not statistically significant, *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 6. STAT6 signaling expression and function in histamine-medicated regulation of cardiac fibrosis post-MI. (a) Western blotting showed that exogenous histamine (10−5 M) could increase phosphorylated STAT6 expression in the heart fibroblasts of WT mice (n = 3). (b) Exogenous histamine (10−5 M) had no inhibitive effect on the heart fibroblasts of STAT6−/− mice (n = 3). (c) The inhibitive effect of histamine on the proliferation of heart fibroblasts of WT mice could be blocked by CP-690550 (n = 3). (d) H&E staining showed that STAT6 knockout caused larger infarct sizes at 4 weeks post-MI (scale bar = 500 μm; n = 8–10). (e–g) Echocardiography showed that STAT6−/− mice had worse cardiac remodeling and function post-MI than WT mice and that exogenous histamine had no beneficial effect on STAT6−/− mice (n = 10–12). (h) Masson trichrome staining showed that cardiac fibrosis was significantly enhanced in STAT6−/− mice compared with WT mice and that exogenous histamine had no beneficial effect on STAT6−/− mice (the top scale bar = 500 μm and other scale bars = 50 μm; n = 6–8). NS, not statistically significant, *p < 0.05, **p < 0.01.