Hyperhomocysteinemia Promotes Cardiac Hypertrophy in Hypertension

Figure 1. HHcy exacerbated AngII-induced cardiac hypertrophy in mice. (a) Schematic illustration of the experimental design. (b) Systolic blood pressure (SBP) collected before and at days 3, 10, 17, and 21 after AngII infusion, n = 12 − 13 per group. (c) Representative M-mode echocardiography at the end of the experiment and calculation of EF and FS, n = 12 − 13 per group. (d) Representative H&E staining of the heart sections and ratios of heart weight/body weight and heart weight/tibial length, n = 12 − 13 per group. (e) Representative WGA staining of the heart sections and quantitation of myocyte cross-sectional area, n = 5 − 6 per group. (f) Relative mRNA levels of ANF and BNP in the hearts, n = 5 − 6 per group. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; ns: not significant.

Figure 2. HHcy exacerbated AngII-induced cardiac inflammation and fibrosis in mice. (a) Representative H&E and CD68 immunohistochemical staining of the heart sections and quantitation of CD68-positive fraction, n = 5 per group. (b) Relative mRNA levels of IL-6, IL-1β, and TNF-α, n = 5 per group. (c) Representative α-SMA immunohistochemical staining of the heart sections and quantitation of α-SMA-positive fraction, n = 5 − 7 per group. (d) Relative mRNA levels of Col 1, Col 3, and α-SMA, n = 5 per group. (e) Representative Masson staining of the heart sections and quantitation of fibrotic fraction, n = 5 − 6 per group. (f) Representative Western blots and quantitation of NF-κB p-p65/p65 and TGF-β, n = 4 per group. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; ns: not significant.

Figure 3. FA supplement attenuated HHcy-aggravated cardiac hypertrophy in mice. (a) Schematic illustration of the experimental design. (b) Systolic blood pressure (SBP) collected before and at days 3, 10, 17, and 21 after AngII infusion, n = 13 per group. (c) Representative M-mode echocardiography at the end of the experiment and calculation of EF and FS, n = 13 per group. (d) Representative H&E staining of the heart sections and ratios of heart weight/body weight and heart weight/tibial length, n = 13 per group. (e) Representative WGA staining of the heart sections and quantitation of myocyte cross-sectional area, n = 5 − 7 per group. (f) Relative mRNA levels of ANF and BNP in the hearts, n = 5 per group. ∗∗∗p < 0.001; ns: not significant.

Figure 4. FA supplement attenuated HHcy-aggravated cardiac inflammation and fibrosis in mice. (a) Representative H&E and CD68 immunohistochemical staining of the heart sections and quantitation of CD68-positive fraction, n = 5 − 7 per group. (b) Relative mRNA levels of IL-6, IL-1β, and TNF-α, n = 5 per group. (c) Representative α-SMA immunohistochemical staining of the heart sections and quantitation of α-SMA-positive fraction, n = 5 − 8 per group. (d) Relative mRNA levels of Col 1, Col 3, and α-SMA, n = 5 per group. (e) Representative Masson staining of the heart sections and quantitation of fibrotic fraction, n = 5 − 8 per group. (f) Representative Western blots and quantitation of NF-κB p-p65/p65 and TGF-β, n = 4 per group. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; ns: not significant.

Figure 5. Calcineurin-NFAT signaling might be involved in the pro-hypertrophic effect of Hcy. (a) Representative Western blot and quantitation of cardiac Calcineurin and NFAT in hyperhomocysteinemic mice. n = 4 per group. (b) Representative Western blot and quantitation of cardiac Calcineurin and NFAT in hyperhomocysteinemic mice treated with FA. n = 4 per group. (c) Representative Western blots and quantitation of Calcineurin and NFAT in Hcy-treated NRCMs. n = 4 per group. (d) Representative Western blots and quantitation of Calcineurin and NFAT in FA-treated NRCMs. n = 4 per group. (e) Representative Western blot and quantitation of Calcineurin and NFAT in CsA-treated NRCMs. n = 4 per group. (f) A working model of Hcy in AngII-induced hypertensive cardiac hypertrophy. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; ns: not significant.