Epigenetic Repression of Chloride Channel Accessory 2 Transcription in Cardiac Fibroblast: Implication in Cardiac Fibrosis

FIGURE 1. Chloride channel accessory 2 (Clca2) expression is down-regulated in activated cardiac fibroblasts. (A,B) C57B/6 mice were subjected to the LAD procedure to induce myocardial infarction. The mice were sacrificed 7 day after the surgery and primary cardiac fibroblasts were isolated. Clca2 expression was examined by qPCR and Western. N = 6 mice for each group. (C,D) C57B/6 mice were subjected to the TAC procedure to induce myocardial hypertrophy. The mice were sacrificed 7 day after the surgery and primary cardiac fibroblasts were isolated. Clca2 expression was examined by qPCR and Western. N = 6 mice for each group. (E,F) Primary cardiac fibroblasts were isolated from C57B/6 mice and treated with TGF-β (2 ng/ml). Cells were harvested at indicated time points and Clca2 expression was examined by qPCR and Western. Error bars represent SD (*p < 0.05, two-way Student’s t-test). All experiments were repeated three times and one representative experiment is shown.

FIGURE 2. TWIST1 mediates Clca2 trans-repression in cardiac fibroblasts. (A) Different Clca2 promoter-luciferase constructs were transfected into mouse embryonic fibroblasts (MEFs) followed by treatment with TGF-β. Luciferase activities were normalized by protein concentration and GFP fluorescence. (B) Wild type and mutant Clca2 promoter-luciferase constructs were transfected into MEFs followed by treatment with TGF-β. Luciferase activities were normalized by protein concentration and GFP fluorescence. (C) Primary cardiac fibroblasts were treated with TGF-β (2 ng/ml) and were harvested at indicated time points. ChIP assays were performed with indicated antibodies. (D,E) Primary cardiac fibroblasts were transfected with indicated siRNAs followed by treatment with TGF-β (2 ng/ml). Gene expression was examined by qPCR and Western. Error bars represent SD (*p < 0.05, two-way Student’s t-test). All experiments were repeated three times and one representative experiment is shown.

FIGURE 3. TWIST1 represses Clca2 transcription by promoting histone deacetylation. (A) Primary murine cardiac fibroblasts were transfected with indicated siRNAs by treatment with TGF-β (2 ng/ml). ChIP assays were performed with indicated antibodies. (B,C) Primary murine cardiac fibroblasts were treated with TGF-β (2 ng/ml) in the presence or absence of different HDAC inhibitors. Clca2 expression was examined by qPCR and Western. (D,E) Primary murine cardiac fibroblasts were transfected with indicated siRNAs by treatment with TGF-β (2 ng/ml). Clca2 expression was examined by qPCR and Western. (F) Primary cardiac murine fibroblasts were transfected with indicated siRNAs by treatment with TGF-β (2 ng/ml). ChIP assays were performed with indicated antibodies. Error bars represent SD (*p < 0.05, two-way Student’s t-test). All experiments were repeated three times and one representative experiment is shown.

FIGURE 4. TWIST1 interacts with and recruits HDAC1 to repress Clca2 transcription. (A) Primary cardiac murine fibroblasts were transfected with indicated siRNAs by treatment with TGF-β (2 ng/ml). ChIP assays were performed with indicated antibodies. (B) Immunoprecipitation was performed with whole cell lysates from primary murine cardiac fibroblasts. (C) Primary murine cardiac fibroblasts were treated with or without TGF-β (2 ng/ml) for 48 h. Re-ChIP was performed with indicated antibodies. (D) Wild type and mutant Clca2 promoter-luciferase constructs were transfected into MEFs with increasing doses of HDAC1 followed by treatment with TGF-β. Luciferase activities were normalized by protein concentration and GFP fluorescence. Error bars represent SD (*p < 0.05, two-way Student’s t-test). All experiments were repeated three times and one representative experiment is shown.

FIGURE 5. Chloride channel accessory 2 (Clca2) regulates activation of cardiac fibroblasts. (A–E) Primary murine cardiac fibroblasts were infected with adenovirus carrying a Clca2 expression vector (Ad-FLAG-Clca2) or an empty vector (Ad-EV) followed by treatment with TGF-β. Expression levels of Clca2 were examined by qPCR (A) and Western (B). Pro-fibrogenic genes were examined by qPCR (C) and Western (D). Cell proliferation was examined by EdU incorporation (E). (F–J) Primary murine cardiac fibroblasts were transfected with indicated siRNAs followed by treatment with TGF-β. Expression levels of Clca2 were examined by qPCR (F) and Western (G). Pro-fibrogenic genes were examined by qPCR (H) and Western (I). Cell proliferation was examined by EdU incorporation (J). Error bars represent SD (*p < 0.05, two-way Student’s t-test). All experiments were repeated three times and one representative experiment is shown. (K) A schematic model.