Figure 5. Cardiac fibroblast proliferation and collagen synthesis are mediated via α7 nAChR–EGFR transactivation.

(A) Representative fluorescence image of containing p-EGFR (Y¹⁰⁶⁸) (red) and nuclei (blue by Hoechst) in ARCF 30 minutes after vehicle, ACh (10 nM), or nicotine (600 nM) treatment in the presence or absence of Mec (20 μM). Scale bar: 20 μm. (B) Summary data from A (n = 9). (C) EGFR transactivation in ARCFs treated with vehicle, 600 nM nicotine, or 10 nM ACh for 30 minutes. EGFR activation was estimated by p-EGFR(Y¹⁰⁶⁸)/EGFR (n = 3). EGFR siRNA and scrambled siRNA-transfected cells were used to confirm the antibody specificities. (D) EGFR transactivation in HEK293T-overexpressing EGFR, α7 nAChR, and Myc-tagged NACHO, by nicotine (600 nM, 30 min) in the presence or absence of α-BTX. Cells stably overexpressing EGFP were used as a control. EGFR phosphorylation, overexpressed EGFR, α7 nAChR, and NACHO were detected by the antibodies against p-EGFR (Y¹⁰⁶⁸), GFP, α7 nAChR, and Myc, respectively. (E) Summary data from D (n = 6). p-EGFR/EGFR was normalized to that in cells without nicotine and α-BTX treatment. (F and G) Cell counts (n = 4) (F) and collagen content (n = 4) (G) of ARCF in response to ACh (10 nM) or nicotine (600 nM) in cells transfected with either EGFR specific or scrambled siRNA. (H and I) Cell counts (n = 5) (H) and collagen content (n = 5) (I) of ARCF in response to nicotine (600 nM) in presence or absence of EGFR inhibitor AG1478 (50 μM). Data are shown as mean ± SEM. ANOVA followed by Bonferroni comparison. *P < 0.05. ACh, acetylcholine; α-BTX, α-bungarotoxin; AG1478, EGFR inhibitor; Mec, mecamylamine; nAChR, nicotinic acetylcholine receptor.