I‐κB Kinase‐ε Deficiency Attenuates the Development of Angiotensin II‐Induced Myocardial Hypertrophy in Mice

Figure 1. The expression of IKKε was increased in WT mouse hearts after infusion of Ang II for 8 weeks. (a) Representative western blot showing the expression of IKKε in the heart tissue at the 8th week of Ang II infusion (n = 4 mice per experimental group). (b) Representative images of IHC staining showing IKKε in WT mouse hearts after 8 weeks of Ang II stimulation. (n = 4 mice per experimental group, 400x; ∗P < 0.05 vs. saline). (c, d) Quantitative western blot and IHC analyses of IKKε (∗P < 0.05 vs. saline).

Figure 2. IKKε deficiency attenuated the cardiac dysfunction after Ang II stimulation. (a) Representative images and quantitative analyses of western blot showing the expression of IKKε in the heart tissue of WT and IKKε-KO mice (∗P < 0.05 vs. WT). (b) Changes in blood pressure after Ang II infusion. (c) Representative images showing the echocardiography of WT and IKKε-KO mice that were infused with saline or Ang II for 8 weeks. (d) Analysis of echocardiography parameters (LVPWd, EF, FS, and E/A) of WT and IKKε-KO mice that were infused with saline or Ang II for 8 weeks (n = 6 mice per experimental group; ∗P < 0.05 vs. saline or WT Ang II).

Figure 3. IKKε deficiency alleviated the development of Ang II-induced myocardial hypertrophy. (a) Representative images showing panoramagrams, HE staining and WGA staining of WT and IKKε-KO mice (n = 4 mice per experimental group). (b, c) Analyses of heart weight/tibial length (HW/TL) and cross-sectional area of WT and IKKε-KO mice under Ang II stimulation (n = 10 mice per experimental group in HW/TL; n = 4 mice per experimental group in a cross-sectional area, 400x in HE and WGA; ∗P < 0.05 vs. saline or WT Ang II).

Figure 4. IKKε knockout decreased the inflammatory reaction and fibrosis in Ang II-induced myocardial hypertrophy. Relative mRNA levels of heart failure markers (a) (atriopeptin, brain natriuretic peptide, Acta-1, and β-MHC), inflammatory factors (b) (IL-6, TNF-α, IL-1β, and IL-10), and fibrosis-related factors (c) (TGF-β1, CTGF, Collagen 1a1, and Collagen 3a1) in the heart tissues of WT or IKKε-KO mice after Ang II infusion (n = 4 mice per experimental group; ∗P < 0.05 vs. saline or WT Ang II).

Figure 5. IKKε knockout reduced collagen deposition in Ang II-induced myocardial hypertrophy. Representative images showing picrosirius red staining in perivascular and interstitial areas of the heart tissue from WT and IKKε-KO mice after Ang II stimulation and the related quantitative analyses of left ventricular collagen volume (n = 4 mice per experimental group, 400x; ∗P < 0.05 vs. saline or WT Ang II).

Figure 6. The lack of IKKε reduced apoptosis in myocardial tissue after Ang II infusion. (a) Representative images and TUNEL assay of WT and IKKε-KO mice after 8 weeks of Ang II infusion (n = 4 mice per experimental group, 400x; ∗P < 0.05 vs. WT). (b, c) Representative western blots and quantitative analyses of apoptosis-related proteins in the heart tissue of WT and IKKε-KO mice that were infused with Ang II for 8 weeks (n = 4 mice per experimental group, ∗P < 0.05 vs. saline or WT Ang II).

Figure 7. The lack of IKKε reduced pyroptosis in myocardial tissue after Ang II infusion. (a, b) Representative IHC images and analyses of IL-1β and IL-18 in WT and IKKε-KO mouse heart tissue after 8 weeks of Ang II infusion (n = 4 mice per experimental group, 400x, ∗P < 0.05 vs. saline or WT Ang II). (c) Representative western blots and quantitative analyses of pyroptosis-related proteins in the heart tissue of WT and IKKε-KO mice that were infused with Ang II for 8 weeks (n = 4 mice per experimental group, ∗P < 0.05 vs. saline or WT Ang II).

Figure 8. IKKε deficiency inhibits the Ang II-mediated MEK1/2-ERK1/2 and P38 signaling pathways. (a, b) Representative western blots showing the phosphorylation and total protein levels of the MAPK pathway proteins MEK1/2, ERK1/2, JNK, and p38 and the NF-κB pathway proteins IκBα, p65, RelB, and p100 in the heart tissue of WT and IKKε-KO mice that were infused with Ang II for 8 weeks (n = 6 independent experiments). (c) Relative quantitative analyses of western blot analysis of the MAPK pathway (n = 6 mice per experimental group; ∗P < 0.05 vs. saline or WT Ang II). (d) Relative quantitative analyses of western blot analysis of the NF-κB pathway (n = 6 mice per experimental group; #P < 0.05 vs. saline).

Figure 9. IKKε combined with ERK and p38 in the heart tissue under Ang II stimulation. Coimmunoprecipitation analysis of IKKε, MEK, ERK, and p38 in the heart tissues of WT mice that were stimulated by saline or Ang II for 8 weeks.