Deficit of glucocorticoid‐induced leucine zipper amplifies angiotensin‐induced cardiomyocyte hypertrophy and diastolic dysfunction

Figures

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  Figure 1. Higher GILZ mRNA expression in Ang II–infused WT mice. A, Experimental design. B, Real‐time quantitative PCR analysis of GILZ mRNA level was up‐regulated in the hearts of Ang II–treated WT mice compared with WT control mice. Data derived from 2 independent experiments and were presented relative to the expression of GAPDH mRNA. C, Representative immunofluorescent images showing higher expression of GILZ protein (green) in WT heart after Ang II administration. Nuclei were counterstained with DAPI (blue). Scale bars: 20 µm. D, Real‐time quantitative PCR analysis of L‐GILZ mRNA level was up‐regulated in the hearts of Ang II‐treated WT mice compared with WT control mice. Data derived from 2 independent experiments and were presented relative to the expression of β‐tubulin mRNA. Graphs were expressed as mean ± standard error of the mean

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  Figure 2. The lack of GILZ partly aggravated diastolic function after Ang II. A, Rise in mean blood pressure upon Ang II did not present any statistical difference between WT and KO mice. B, Unchanged ejection fraction and fractional shortening demonstrated unimpaired systolic function. C, Evidence of diastolic dysfunction upon infusion of Ang II; KO + Ang mice showed a higher increment of E‐wave deceleration time. *P < .05 vs strain‐matched control; °P < .05 vs WT + Ang

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  Figure 3. Increment of LV hypertrophy after Ang II treatment was more marked in KO animals. A, Posterior wall thickness (normalized for bodyweight) elevation was more striking in GILZ‐KO mice. B, Cardiomyocyte cross‐sectional area demonstrated by laminin immunostaining (green). C, Frequency distribution curve of myocyte cross‐sectional area was shifted rightwards in KO + Ang group in comparison with WT + Ang. D, FoxP3 transcript up‐regulation upon Ang II was less marked in GILZ‐KO hearts. E, Representative immunofluorescent images showing higher expression of FoxP3 (green) in the LV after exposure to Ang II. F, Up‐regulation of GATA4 only occurred in WT + Ang hearts. Real‐time quantitative PCR analysis of FoxP3 and GATA4 derived from 2 independent experiments and were presented relative to the expression of GAPDH mRNA. Graphs were expressed as mean ± standard error of the mean. G, Capillary rarefaction after Ang II in WT and KO hearts. H, Representative images showing lower capillary density in WT + Ang and KO + Ang mice compared with untreated animals. Endothelial cells were detected by isolectin B4 (green). Nuclei in (E, H) were counterstained with DAPI (blue). *P < .05 vs strain‐matched…

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  Figure 4. Pro‐inflammatory response in the heart enhanced by Ang II was not affected by GILZ. A, Real‐time quantitative PCR analysis of cytokine INF‐γ, IL‐1β and TNF‐α mRNA levels in the heart. B, Representative immunofluorescent images showing higher number of CD3+ (red, upper panels) and CD45+ (green, lower panels) cells in WT + Ang and KO + Ang hearts compared with WT. Nuclei were counterstained with DAPI (blue). C, Western blot analysis of total and phospho‐NF‐κBSer536 expression and densitometric analysis of phospho‐NF‐κBSer536/total NF‐κB. D, Western blot analysis of precursor pro–Caspase‐1 and active Caspase‐1 expression, and densitometric analysis of pro–Caspase‐1/lamin B1. Lamin B1 antibody served as loading control. Graphs were expressed as mean ± standard error of the mean. E, Real‐time quantitative PCR analysis of cytokine INF‐γ, IL‐1β and TNF‐α mRNA levels in the spleen. Data in (A, E) derived from 2 independent experiments and were presented relative to the expression of GAPDH mRNA. Graphs were expressed as mean ± standard error of the mean. *P < .05 vs strain‐matched control; °P < .05 vs WT + Ang; † P < .05 vs WT. Scale bars: 20 µm

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  Figure 5. Cell death, extracellular matrix deposit and fibroblast accumulation in the heart were more extended in Ang II–exposed mice. A, Higher apoptotic rate in WT + Ang and KO + Ang hearts compared with Ang II–untreated hearts. B, Apoptotic nuclei (green) in WT + Ang and KO + Ang hearts. Cardiomyocytes were immunostained with α‐sarcomeric actin (α‐SA, red). C, Real‐time quantitative PCR analysis of BAX mRNA levels in the heart. D, Representative immunofluorescent images showing excessive accumulation of fibrillar collagen I (green) and cardiac fibroblasts (vimentin, red) in WT + Ang and KO + Ang hearts compared with Ang II‐untreated hearts. E, Examples of perivascular fibrosis in WT + Ang and KO + Ang myocardium. Nuclei in (B, D, E) were counterstained with DAPI (blue). F, Real‐time quantitative PCR analysis of TGF‐β and CTGF mRNA levels in the heart. Data in (C, F) derived from 2 independent experiments and were presented relative to the expression of GAPDH mRNA. Graphs were expressed as mean ± standard error of the mean. *P < .05 vs strain‐matched control; °P < .05 vs WT + Ang. Scale bars: (B) 10 µm, (D, E) 50 µm