Signaling via the Interleukin-10 Receptor Attenuates Cardiac Hypertrophy in Mice During Pressure Overload, but not Isoproterenol Infusion

Figures

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  FIGURE 1. Effects of IL-10 treatment and IL-10R1 gene silencing in TNF-α-induced cardiomyocyte hypertrophy. (A) Western blot analysis showing the expression of IL-10R1 in NRCM treated with adenovirus expressing either control shRNA or shRNA for IL-10R1 for 1–4 days. (B) Representative images of NRCM stained with α-actinin (red) and DAPI (blue) expressing either shRNA control or shRNA IL-10R1 after treatment with TNF-α (10 ng/ml) or with or without addition of IL-10 (20 ng/ml) for 48 h. Scale bar = 25 μM (C) Quantification of cell surface area (N = 3 independent experiments, with a minimum of n = 100 cells per experiment,*p < 0.05 vs. no TNF, #p < 0.05 vs. TNF without IL-10 (D) NRCM treated with 1 µM isoproterenol for 48 h ± IL-10. Scale bar = 25 µM and (E) quantification of myocyte cross-sectional area (N = 4 independent experiments, minimum 50 cells each, ***p < 0.001).

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  FIGURE 2. Pressure overload-induced hypertrophy model in IL-10R1-/- mice. (A) Representative Western blot of total heart extracts from IL-10R1-/- mice and WT littermates showing complete ablation of IL-10R1 expression in the knockout mice. (B) Analysis of heart weight/body weight ratio (HW/BW) of WT and IL-10R1-/- mice following transverse aortic constriction (TAC) for 2 weeks. IL-10R1-/- mice showed a significant increase in hypertrophic response compared to WT littermates (WT-sham, n = 13; WT-TAC, n = 19; KO-sham, n = 11; KO-TAC, n = 12, *p < 0.05, ***p < 0.001). (C) Histological sections of heart tissues stained with hematoxylin and eosin and (D) quantification of cardiomyocyte cross-sectional area showed enhanced cardiomyocyte size in IL-10R1-/- mice after TAC. (E) Masson’s trichrome staining and (F) quantification of fibrotic area indicated significantly higher fibrosis in IL-10R1-/- mice (WT-sham, n = 7; WT-TAC, n = 8; KO-sham, n = 8; KO-TAC, n = 7) Scale bars = 50 µM.

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  FIGURE 3. Echocardiography analysis of IL-10R1-/- mice following TAC. (A) Left ventricular end diastolic dimension (LVEDD) and (B) Left ventricular end systolic dimension (LVESD) were not different between IL-10R1-/- mice and WT controls basally and after TAC. (C) Ventricular septal wall thickness at diastole was bigger in IL-10R1-/- mice after TAC, while (D) posterior wall thickness did not differ between these genotypes. (E) There was no significant difference in ejection fraction between all experimental groups (WT-sham, n = 13; WT-TAC, n = 15; KO-sham, n = 11; KO-TAC, n = 12,*p < 0.05,***p < 0.001).

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  FIGURE 4. Invasive hemodynamics assessment using micro-catheter. (A) End systolic ventricular pressure was significantly increased in both genotypes following TAC, however there was no difference between IL-10R1-/- mice vs. WT. (B) There was no statistically significant difference in end systolic pressure (C) dP/dt max and (D) dP/dt min in all experimental groups(WT-sham, n = 13; WT-TAC, n = 15; KO-sham, n = 11; KO-TAC, n = 12, **p < 0.01, ***p < 0.001).

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  FIGURE 5. Effects of isoproterenol infusion in IL-10R1-/- mice and WT littermates. (A) Heart rate and (B) end systolic pressure were significantly increased in both genotypes following isoproterenol infusion (10 mg/kg BW/day) for 10 days. Consistently, (C) the rate of developed ventricular pressure (dP/dt max) and (D) the rate of ventricular relaxation (dP/dt max) were increased following isoproterenol treatment. However, there was no significant difference between IL-10R1-/- mice vs. WT controls (WT-vehicle, n = 3; WT-Iso, n = 6; KO-vehicle, n = 3; KO-Iso, n = 7, *p < 0.05,**p < 0.01,***p < 0.001).

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  FIGURE 6. Hypertrophic and fibrotic response in isoproterenol model. (A) Although there was significantly increased HW/BW ratio following isoproterenol infusion, there was no significant difference between IL-10R1-/- and WT mice (WT-vehicle,n = 3; WT-Iso, n = 6; KO-vehicle, n = 3; KO-Iso, n = 7,*p < 0.05,**p < 0.01). (B) Histological sections of heart tissues stained with hematoxylin and eosin and (C) quantification of cardiomyocyte cross-sectional area (D) Masson’s trichrome staining and (E) quantification of fibrotic area following isoproterenol infusion (WT-vehicle, n = 3; WT-Iso, n = 4; KO-vehicle, n = 3; KO-Iso, n = 3,***p < 0.001) Scale bars = 25 µM.

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  FIGURE 7. Echocardiography analysis of IL-10R1-/- mice in isoproterenol model. (A) Echocardiography assessment revealed that there was no difference in LVEDD and (B) LVESD among experimental groups. (C) Septal wall thickness was augmented in both groups after isoproterenol treatment but there was no difference between genotypes. (D) No difference in posterior wall thickness and (E) Ejection fraction among experimental groups at the end of isoproterenol treatment (WT-vehicle, n = 3; WT-Iso, n = 6; KO-vehicle, n = 3; KO-Iso, n = 7,*p < 0.05,**p < 0.01).

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  FIGURE 8. TNF-α levels in TAC and isoproterenol models. (A) Analysis of TNF-α levels in heart tissue extracts from WT and IL-10R1-/- mice following TAC as measured by ELISA (n = 5) (B) Cardiac TNF-α mRNA expression relative to β-actin as loading control did not differ between isoproterenol treated WT and IL-10R1-/- mice (WT-vehicle, n = 3; WT-Iso, n = 4; KO-vehicle, n = 3; KO-Iso, n = 3,*p < 0.05).

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  FIGURE 9. IL-10 induced activation of STAT3. (A) Representative western blot showing STAT3 phosphorylation in shRNA control or shRNA IL-10R1 NRCM basally, and following the addition of 20 ng/ml IL-10 for 10 min or 4 h (B) Ratio of phosphorylated:total STAT3 (N = 5,*p < 0.05) (C) Representative western blot showing STAT3 phosphorylation in shRNA control or shRNA IL-10R1 NRCM following the addition of 1 µM isoproterenol with or without 20 ng/ml IL-10, along with loading control GAPDH (D) Ratio of phosphorylated:total STAT3 (N = 3).