Notch3 Modulates Cardiac Fibroblast Proliferation, Apoptosis, and Fibroblast to Myofibroblast Transition via Negative Regulation of the RhoA/ROCK/Hif1α Axis

Figures

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  FIGURE 1. Notch3 was overexpressed and knocked down successfully in cardiac fibroblasts (CFs). (A,B) Rat CFs were transfected with ov-N3ICD plasmid or vector. Notch3 expression was detected by RT-qPCR and western blotting analysis (n = 3). (C) CFs were treated with small interfering RNA constructs (sc notch3, si notch3 1, si notch3 2, or si notch3 3), and mRNA expression of notch3 was quantified by RT-qPCR (n = 3). (D) Western blot analysis of notch3 expression after notch3 knockdown (n = 3). Values represent the mean ± SD. *P < 0.05, **P < 0.01.

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  FIGURE 2. Notch3 inhibits the proliferation of rat cardiac fibroblasts (CFs). (A,C) We used the EdU assay to measure CF proliferation after notch3 overexpression or knockdown (n = 6). Scale bars = 200 μm. (B,D) We next used the Cell Counting Kit-8 (CCK8) assay to determine CF proliferation (above, n = 3). Quantification of the CF proliferation determined by EdU assay (below, n = 6) and CCK8 assay (above, n = 3) in different groups, as indicated. Values represent the mean ± SD. *P < 0.05, **P < 0.01.

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  FIGURE 3. Notch3 promotes the apoptosis of rat cardiac fibroblasts (CFs). (A,B) Representative western blot and quantitative data of cleaved caspase3, total caspase3, and Bcl2 after notch3 overexpression or knockdown (n = 3). (C,D) The CF apoptotic rate after notch3 overexpression or knockdown, detected by flow cytometry. Q1-UR and Q1-LR were used to analyze the change of apoptotic rate in different experimental groups (n = 3). Values represent the mean ± SD. *P < 0.05, **P < 0.01. C-caspase3, cleaved caspase3; T-caspase3, total caspase3.

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  FIGURE 4. Notch3 inhibits cardiac fibroblast to myofibroblast transition. (A,B) Western blots measuring α-SMA, Col I, and Col III after notch3 overexpression or knockdown. The quantification of the protein bands is shown on the right (n = 3). Values represent the mean ± SD. *P < 0.05, **P < 0.01.

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  FIGURE 5. The effects of notch3 on Hif1α, RhoA, ROCK1, and ROCK2 in rat cardiac fibroblasts (CFs) with different treatments. (A) Representative immunofluorescence images and quantitative data of Hif1α (red) in neonatal rat cardiac fibroblasts after notch3 overexpression (n = 6). Nuclei were detected with DAPI (blue). Scale bars = 200 μm. (B) The protein expression of Hif1α, RhoA, ROCK1, and ROCK2 was determined by western blot after notch3 overexpression (n = 3). (C) Western blots of RhoA in the membrane and cytosol after notch3 overexpression (n = 3). (D) Hif1α (red) in neonatal rat cardiac fibroblasts after notch3 knockdown was detected by immunofluorescence and statistical analysis of mean intensity (n = 6). Nuclei were detected with DAPI (blue). Scale bars = 200 μm. (E) Hif1α, RhoA, ROCK1, and ROCK2 were detected by western blot after notch3 silencing (n = 3). (F) Western blots of RhoA and cadherins in the membrane and cytosol after notch3 silencing (n = 3). Values represent the mean ± SD. *P < 0.05, **P < 0.01. M-RhoA, membrane RhoA; C-RhoA, cytosolic RhoA; M-pan cadherin, membrane pan cadherin; C-pan cadherin, cytosolic pan cadherin; M-β-actin, membrane β-actin; C-β-actin, cytosolic…

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  FIGURE 6. Notch3 regulates cardiac fibroblast (CF) function via the RhoA/ROCK/Hif1α pathway. (A) CFs were pretreated with 2-ME for 2 h before notch3 knockdown. CF proliferation was quantified using the EdU assay (n = 6). Scale bars = 200 μm. (B) The Cell Counting Kit-8 (CCK8) assay was also used to determine CF proliferation (above, n = 3). Quantification of CF proliferation determined by EdU assay (below, n = 6). (C) Western blot analysis of cleaved caspase-3, total caspase3, and Bcl2 in different groups. The quantification of the protein bands is shown on the right (n = 3). (D) CFs were pretreated with DMOG for 2 h before notch3 overexpression. Flow cytometry quantified the percentage of apoptosis in each group (n = 3). (E) Western blot analysis of α-SMA, Col I, and Col III in CFs pretreated with 2-ME for 2 h before notch3 knockdown. The quantification of the protein bands is shown below (n = 3). (F) Representative Western blot and quantification of Hif1α in CFs pretreated with Y-27632 for 2 h before notch3 knockdown (n = 3). (G) Representative immunofluorescence images and quantification of Hif1α (red) in CFs pretreated with Y-27632 for 2 h before notch3 knockdown (n = 6). Nuclei were…

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  FIGURE 7. Notch3 alleviates cardiac fibrosis after myocardial infarction via the RhoA/ROCK/Hif1α pathway. (A,B) Notch3 overexpression in the myocardium after Ad-N3ICD injection was confirmed by RT-qPCR and western blot (n = 5). (C) Measurements of cardiac function indices of rats in each group 2 months after myocardial infarction (MI) injury (n = 5). (D) Masson staining of the rat heart tissues. The positively stained area (blue) represents fibrosis (n = 5). (E) Western blot analysis and quantitative results of α-SMA, RhoA, ROCK1, ROCK2, and Hif1α (n = 5). (F) Western blot analysis of membrane-bound RhoA and cadherins in rat hearts from different treatments. Values represent the mean ± SD. *P < 0.05, **P < 0.01. M-RhoA, membrane RhoA; C-RhoA, cytosolic RhoA; M-pan cadherin, membrane pan cadherin; C-pan cadherin, cytosolic pan cadherin; M-β-actin, membrane β-actin; C-β-actin, cytosolic β-actin.