Fibroblast-specific IKK-β deficiency ameliorates angiotensin II–induced adverse cardiac remodeling in mice

Cardiac inflammation and fibrosis contribute significantly to hypertension-related adverse cardiac remodeling. IκB kinase β (IKK-β), a central coordinator of inflammation through activation of NF-κB, has been demonstrated as a key molecular link between inflammation and cardiovascular disease. However, the cell-specific contribution of IKK-β signaling toward adverse cardiac remodeling remains elusive. Cardiac fibroblasts are one of the most populous nonmyocyte cell types in the heart that play a key role in mediating cardiac fibrosis and remodeling. To investigate the function of fibroblast IKK-β, we generated inducible fibroblast-specific IKK-β–deficient mice. Here, we report an important role of IKK-β in the regulation of fibroblast functions and cardiac remodeling. Fibroblast-specific IKK-β–deficient male mice were protected from angiotensin II–induced cardiac hypertrophy, fibrosis, and macrophage infiltration. Ablation of fibroblast IKK-β inhibited angiotensin II–stimulated fibroblast proinflammatory and profibrogenic responses, leading to ameliorated cardiac remodeling and improved cardiac function in IKK-β–deficient mice. Findings from this study establish fibroblast IKK-β as a key factor regulating cardiac fibrosis and function in hypertension-related cardiac remodeling.


Supplemental Figure 2. Deficiency of IKKβ in cardiac fibroblasts attenuates lipopolysaccharide-induced NF-κB activation and inflammation.
(A) Representative immunofluorescent staining of NF-κB p65 subunit in cardiac fibroblasts isolated from IKKβ F/F and IKKβ ΔFib mice that treated with 100 ng/ml of lipopolysaccharide (LPS) or vehicle control for 1 hr (Scale bar, 100μm). (B) QPCR analysis of the mRNA levels of proinflammatory cytokines and adhesion molecules (n=3. Two-way ANOVA **P < 0.01 and ***P < 0.001).

Supplemental Figure 3. Angiotensin II-induced mouse model of cardiac remodeling.
(A) Experimental scheme of mice subjected to 1000 ng/kg/min of angiotensin II (Ang II) or control (saline) infusion for 1 and 4 weeks. Eight-week-old male mice were intraperitoneally injected with tamoxifen (2 mg/per day) for 5 consecutive days to induce CreER T -mediated recombination. At the age of 10 weeks, the mice were implanted with mini-osmotic pumps. (B and C) Body weight and mortality rate of male IKKβ F/F and IKKβ ΔFib mice after 4-week Ang II or control infusion. Eight-week-old male IKKβ F/F and IKKβ ΔFib mice were intraperitoneally injected with 2 mg tamoxifen per day for 5 days. At the age of ten weeks, those mice were infused with 1000 ng/kg/min of angiotensin II (Ang II) or vehicle control for 1 week. Representative images of immunofluorescence staining of Ki-67 and fibroblast cell marker (A) or cardiomyocyte marker (B) in the hearts of IKKβ F/F and IKKβ ΔFib mice (Scale bar, A, 50 μm; B, 100 μm).

Supplemental Figure 5. Deficiency of fibroblast IKKβ does not increase cell apoptosis in the heart of angiotensin II-infused mice. (A-B)
Eight-week-old male IKKβ F/F and IKKβ ΔFib mice were intraperitoneally injected with 2 mg tamoxifen per day for 5 days. At the age of ten weeks, the mice were infused with 1000 ng/kg/min of angiotensin II (Ang II) or vehicle control for 1 week (A) or 4 weeks (B). Representative TUNEL staining of hearts from IKKβ ΔFib and IKKβ F/F mice. Apoptotic nuclei fluoresce red. The nuclei were visualized with DAPI (blue), and the TUNEL-positive cells were indicated by arrows (Scale bar, 100 μm).

Supplemental Figure 6. Deficiency of IKKβ abolishes the impact of angiotensin II on proliferation or apoptosis-related gene expression in cardiac fibroblasts.
Cardiac fibroblasts were isolated from male IKKβ F/F and IKKβ ΔFib mice, and then stimulated with 10 -6 M of angiotensin II (Ang II) or vehicle control for 24 hr. The expression levels of genes related to proliferation and apoptosis were analyzed by QPCR (n=3, Two-way ANOVA, *P < 0.05, **P < 0.01, and ***P < 0.001).

Supplemental Figure 7. Fibroblast IKKβ deficiency modestly affects cardiac inflammation after 4 weeks of angiotensin II infusion.
Eight-week-old male IKKβ F/F and IKKβ ΔFib mice were intraperitoneally injected with 2 mg tamoxifen per day for 5 days. At the age of ten weeks, those mice were infused with angiotensin II (Ang II) at the dose of 1000 ng/kg/min for 4 weeks. (A) QPCR analysis of the mRNA levels of inflammatory cytokines and adhesion molecules in the heart of male IKKβ F/F and IKKβ ΔFib mice (n=5-6, Two-way ANOVA, *P < 0.05, **P < 0.01 and ***P < 0.001). (B) Representative images of immunofluorescence staining of CD68 in the heart of IKKβ F/F and IKKβ ΔFib mice (n=3, Scale bar, 100 μm).