Nogo-C regulates post myocardial infarction fibrosis through the interaction with ER Ca2+ leakage channel Sec61α in mouse hearts

Figures

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  Fig. 1. Nogo-C is upregulated in post MI mouse hearts and rat cardiac fibroblasts treated with Ang II or TGF-β1. a, b Western blot showing Nogo-C and fibronectin (FN) protein levels in sham and myocardial infarction (MI) mouse hearts 1day (a) or 28 days (b) after operation. n = 3 mice in every group. **P < 0.01 versus sham mice. c Western blot and average data showing Nogo-C and FN protein levels in rat neonatal cardiac fibroblasts treated with 0.1 μM Ang II for indicated times. d Western blot and average data showing Nogo-C and FN protein levels in rat neonatal cardiac fibroblasts treated with 10 ng/ml TGF-β1 for indicated times. n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus untreated control cells

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  Fig. 2. Nogo-C increases expression of profibrogenic proteins in cardiac fibroblast. a Western blot and average date showing the Nogo-C protein level in rat cardiac fibroblasts transfected with Ad-Nogo-C at 25 and 50 m.o.i or Ad-LacZ for 48 h. n = 3 independent experiments. b Western blot and average data of protein levels of profibrogenic proteins in cardiac fibroblasts transfected with Ad-Nogo-C or Ad-Lacz for 48 h. n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus Ad-LacZ transfected cells. c Immunofluorescence staining showing FN in cardiac fibroblasts transfected with Ad-Nogo-C or Ad-LacZ for 48 h. Scale bar = 25 μm

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  Fig. 3. Knockdown of Nogo-C inhibits Ang II or TGF-β1-mediated expression of profibrogenic proteins in cardiac fibroblasts. a Nogo-C protein level in cardiac fibroblasts transfected with Ad-sh-Nogo-C or Ad-scramble for 72 h. n = 3 independent experiments. b Western blot and average date showing profibrogenic proteins CTGF, FN, collagen type I and TGF-β1 in cardiac fibroblasts transfected with Ad-sh-Nogo-C or Ad-scramble with/without Ang II (0.1 μM) stimulation. c Immunofluorescence staining of FN in cardiac fibroblasts transfected with Ad-sh-Nogo-C or Ad-scramble with/without Ang II stimulation. Scale bar = 25 μm. d Western blot and average date showing profibrogenic proteins CTGF, FN, collagen type I and α-SMA in cardiac fibroblasts transfected with Ad-sh-Nogo-C or Ad-scramble with/without TGF-β1 (10 ng/ml) stimulation. e, f Immunofluorescence staining of FN (e) or α-SMA (f) in cardiac fibroblasts transfected with Ad-sh-Nogo-C or Ad-scramble with/without TGF-β1 stimulation. n = 3 independent experiments. Scale bar = 25 μm. *P < 0.05, **P < 0.01, ***P < 0.001 vs Ad-scramble. #P < 0.05, ##P < 0.01, ###P < 0.001 vs Ang II or TGF-β1 groups

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  Fig. 4. Knockout of Nogo-C inhibits post-MI fibrosis and improves cardiac function. a Western blot and average date showing levels of profibrogenic proteins in sham and MI hearts from wild-type and Nogo-C−/− mice. n = 3 mice for each group. b Picric acid Sirius red and HE staining showing collagen deposition in MI cardiac border zone. n = 15 images from 3 mice for each group. Scale bar = 200 μm. c Representative M-mode images of echocardiography, and average data of EF (left) and FS (right) in 2-dimensional images. n = 8 mice for each group. *P < 0.05, **P < 0.01, ***P < 0.001 vs wild-type sham. #P < 0.05, ##P < 0.01, ###P < 0.001 vs wild-type MI

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  Fig. 5. Nogo-C regulates fibrotic responses throughincreasing intracellular Ca2+ concentration. a Immunofluorescence staining showing the co-localization of Nogo-C with ER in cardiac fibroblasts with/without TGF-β1 or AngII stimulation. Scale bar = 10 μm. b Representative images (upper) and average data (bottom) showing the ER Ca2+ release in response to caffeine (10 mM) stimulation in cardiac fibroblasts transfected with Ad-Nogo-C or Ad-LacZ, indicated by Fluo-4 AM fluorescent signal. n = 12 cells from 3 independent experiments for each group. c Representative images (left) and average data (right) showing cytosolic Ca2+ concentration of cardiac fibroblasts transfected with Ad-Nogo-C or Ad-LacZ with/without EGTA-AM (50 μM) treatment. n = 30 cells from 3 independent experiments for each group. d Profibrogenic proteins (CTGF, FN and collagen type I) in cardiac fibroblasts transfected with Ad-Nogo-C or Ad-LacZ with/without EGTA-AM (50 μM) treatment. n = 3 independent experiments. e Sec61α protein level in cardiac fibroblasts transfected with Ad-Nogo-C. f Sec61α protein level in cardiac fibroblasts in the presence of TGF-β1 (10 ng/ml) for 48 h. g Cytosolic Ca2+ concentration in cardiac…

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  Fig. 6. Knockdown of Sec61α inhibits Nogo-C- and TGF-β1-mediated fibrotic responses in cardiac fibroblasts. a Western blot and average date showing Sec61α and Nogo-C protein levels in cardiac fibroblasts transfected with Ad-sh-Sec61α or Ad-scramble for 72 h. n = 3 independent experiments. b Western blot and average date showing Nogo-C and Sec61α protein levels in cardiac fibroblasts transfected with Ad-sh-Nogo-C or Ad-scramble for 72 h. n = 3 independent experiments. c Immunofluorescence staining showing the co-localization of Nogo-C with Sec61α in cardiac fibroblasts with/without TGF-β1 or AngII stimulation. Scale bar = 10 μm. d CoIP of Nogo-C protein with Sec61α protein in cardiac fibroblasts. e Ubiquitination level of Sec61α protein in cardiac fibroblasts transfected with Ad-Nogo-C or Ad-LacZ in the presence of MG132 (10 μM). f Cytosolic Ca2+ concentration in cardiac fibroblasts transfected with Ad-Nogo-C with/without co-transfection of Ad-sh-Sec61α. g Western blot and average date showing levels of profibrogenic proteins TGF-β1, CTGF, FN, and collagen type I in cardiac fibroblasts transfected with Ad-Nogo-C with/without co-transfection of Ad-sh-Sec61α. h Profibrogenic proteins and…

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  Fig. 7. Schematic graph of Nogo-C in the regulation of post-MI fibrosis