BNIP3L promotes cardiac fibrosis in cardiac fibroblasts through [Ca2+]i-TGF-β-Smad2/3 pathway

Figure 1. BNIP3L expression is consistent with myocardial hypertrophy and fibrosis in vivo. The rats were subjected to abdominal aortic constriction (AAC) surgery. (a) Western blot analysis showing the effects of pressure overload on BNIP3L expression. (b) Densitometric analysis of blots for determining BNIP3L normalized to GAPDH. (c) Representative histological images with hematoxylin and eosin staining of heart sections. (d) Quantification of muscle fiber thickness from hematoxylin and eosin staining of heart sections. (e) Western blot analysis showing the effects of pressure overload on fibronectin expression. (f) Densitometric analysis of blots for determining fibronectin normalized to GAPDH. (g) Masson’s trichrome staining showing the effects of pressure overload on fibrotic lesions. (h) Mean values of the ratio of collagen surface area to the myocardial surface area expressed as percentage of fibrosis. Data shown are mean ± SD, *P ≤ 0.05 vs. Control and sham, n = 8.

Figure 2. BNIP3L is up-regulated in CFs during pressure overload-induced cardiac remodeling. (a) Immunohistochemistry showing the cellular distribution of BNIP3L. (b) Immunofluorescence showing the co-localization of BNIP3L and DDR2.

Figure 3. The effects of NE on cell fibrosis and BNIP3L expression in CFs. Neonatal rat CFs were treated with different concentrations of NE for the indicated times. (a) Western blot analysis showing NE-induced fibronectin and collagen I expression. (b) Proliferation was measured using the MTT assay. (c) Cells were stained with PI and examined by FACS. The data were analyzed using the ModFit program. (d) Western blot analysis showing NE- induced BNIP3L expression. Each experiment repeated three times. Data shown are mean ± SD, *P < 0.05 vs. Control.

Figure 4. The roles of BNIP3L in CFs. Neonatal rat CFs were transfected with the BNIP3L and BNIP3L-siRNA adenoviruses. (a) Western blot analysis showing the effects of the adenoviruses on BNIP3L expression. (b) Western blot analysis showing the effects of BNIP3L on fibronectin and collagen I expression. (c) Proliferation was measured using the MTT assay. (d) Cell cycle stage was determined by FACS, and the data were analyzed using the ModFit program. Each experiment repeated three times. Data shown are mean ± SD, *P < 0.05 vs. Control.

Figure 5. The roles of BNIP3L in NE-treated CFs. Neonatal rat CFs were treated with NE after transfection with the BNIP3L and BNIP3L-siRNA adenoviruses. (a) Western blot analysis showing the effects of the transfection on NE-induced BNIP3L expression. (b) Proliferation was measured using the MTT assay. (c) Cell cycle stage was determined by FACS, and the data were analyzed using the ModFit program. (d,e) Western blot analysis showing the effects of BNIP3L on fibronectin and collagen I expression. Each experiment repeated three times. Data shown are mean ± SD, *P < 0.05 vs. Control, #P < 0.05 vs. NE.

Figure 6. BNIP3L plays an important role in fibrosis via the TGF-β/Smad signaling pathway. Neonatal rat CFs were transfected with the BNIP3L and BNIP3L-siRNA adenoviruses or were treated with NE after the transfection. (a,b) Western blot analysis showing the effects of the adenoviruses on the expression of TGF-β (*P < 0.05 vs. Control, #P < 0.05 vs. NE). (c) Western blot analysis showing the effects of TGF-β-siRNA on the expression of BNIP3L. (d,e) Immunofluorescence images demonstrating the nuclear accumulation of Smad2/3. (f,i) Western blot analysis showing the effects of NE, pcDNA3-BNIP3L and TGF-β-siRNA on the TGF-β and fibronectin protein levels (*P < 0.05 vs. NE, #P < 0.05 vs. BNIP3L). Each experiment repeated three times.

Figure 7. BNIP3L regulates Ca2+ in CFs. Neonatal rat CFs were loaded with Fura Red and analyzed by flow cytometry. Data are reported as fluorescence intensities. (a) The BNIP3L-siRNA transfected or NE-induced cells were loaded with Fura Red and analyzed as above (*P < 0.05 vs. Control, #P < 0.05 vs. NE.). (b) The adenoviruses of BNIP3L-transfected or NE-induced cells were loaded with Fura Red and and analyzed as above (*P < 0.05 vs. Control, #P < 0.05 vs. NE). (c) Co-immunoprecipitation showed the interaction of InsP3R with Bcl-XL and BNIP3L when the cells were transfected with pcDNA3-BNIP3L (*P < 0.05 vs. pcDNA3). (d) Co-immunoprecipitation showed the interaction of Bcl-XL with InsP3R and BNIP3L when the cells were transfected with pcDNA3-BNIP3L (*P < 0.05 vs. pcDNA3). Each experiment repeated three times. Data shown are mean ± SD.

Figure 8. BNIP3L expression was up-regulated by the NE-AR-PKC signaling pathway. (a) Western blot analysis showing the effects of doxazosin (DOX) and metoprolol (MET) on AAC-induced BNIP3L. (b) Western blot analysis showing the effects of propranolol (PRO), phenoxybenzamine (PHE) and bisindolylmaleimide I (BIM) on NE-induced BNIP3L expression. (c) Western blot analysis showing the effects of DOX and MET on AAC-induced fibronectin expression. (d) Masson’s trichrome staining showing the effects of DOX and MET on AAC-induced cardiac fibrosis (*P < 0.05 vs. sham, #P < 0.05 vs. AAC). (e) Echocardiographic assessment showing the effects of DOX and MET on AAC-induced cardiac dysfunction (n = 8, *P < 0.05 vs. sham, #P < 0.05 vs. AAC).