Calcium and integrin binding protein 1 (CIB1) induces myocardial fibrosis in myocardial infarction via regulating the PI3K/Akt pathway

Figures

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  Fig. 1.. The diagram of treatments for CIB1 function analysis in the myocardial infarction (MI). MI model was established on 8-week-old male C57BL/6 mice with permanent ligation of the left anterior descending coronary artery (LAD). Multipoint intramyocardial injection of Cib1 RNA-interfering lentivirus (LV-shCib1) was performed before LAD ligation for CIB1 knockdown. PBS: phosphate buffer saline. LV-shNC: negative control lentivirus.

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  Fig. 2.. CIB1 expression is upregulated in the heart of MI mice. A. The expression and location of CIB1 in the penumbra of ischemic heart at 1 and 4 weeks post-MI detected with immunohistochemistry. Magnification: 400×. Scale bar=50 µm. B. Quantification of CIB1 expression in immunohistochemistry. C. The mRNA level of Cib1 in the penumbra of ischemic heart at 1 and 4 weeks post-MI. Data are expressed as the mean ± SD (n=6/group). **P<0.01 vs.Sham group.

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  Fig. 3.. CIB1 silencing relieves MI-induced myocardial damage and improves cardiac dysfunction. A–B. The images (A) and quantification (B) of CIB1 expression in the penumbra of the ischemic heart of the mice infected with Cib1 RNA-interfering lentivirus detected with western blot. C–D. Representative images (C) and quantification (D) of heart infarct size detected with tetrazolium chloride (TTC) staining at 4 weeks post-MI. Normal tissue area (red) and infarcted tissue area (white) were measured, and the ratio of infarcted tissue area to the entire area was calculated. N.D.: not detected. E. The left ventricle-to-body weight ratio (LV/BW) of mice at 4 weeks post-MI. F. Cardiomyocyte short axis diameter measured in hematoxylin and eosin stained heart tissue sections at 4 weeks post-MI. Data are expressed as the mean ± SD (n=6/group). **P<0.01.

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  Fig. 4.. CIB1 knockdown alleviates myocardial fibrosis caused by MI in mice. A. Masson’s trichrome staining of heart slides at 4 weeks post-MI. The collagen fibers stained blue and the cell cytoplasm stained red. Magnification: 200×. Scale bar=100 µm. B. Percentage of the fibrotic area in Masson’s trichrome staining. C. The representative images of FSP1 and α-SMA expression in the penumbra of ischemic heart in immunofluorescence staining. Magnification: 400×. Scale bar=50 µm. D. Quantification of α-SMA expression in the penumbra of ischemic heart at 4 weeks after MI modeling. E–G. The mRNA level of Vim (E), Col1a1 (F), and Col3a1 (G) in the penumbra of the ischemic heart measured with real-time PCR. Data are expressed as the mean ± SD (n=6/group). **P<0.01.

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  Fig. 5.. Identification of cardiac fibroblasts (CFs) and CIB1 expression in the CFs. A. Immunofluorescence staining of FSP1 for the identification of cardiac fibroblasts (CFs). Magnification: 400×. Scale bar=50 µm. B–C. Representative images (B) and quantification (C) of CIB1 protein expression in the CFs infected with Cib1 RNA-interfering lentivirus. Data are expressed as the mean ± SD (n=3/group). **P<0.01.

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  Fig. 6.. The diagram of the investigation of CIB1 function in angiotensin II (Ang-II)-induced cardiac fibroblasts fibrosis in vitro. To analyze the effects of CIB1 knockdown on CFs fibrosis, the CFs were infected with Cib1 RNA-interfering lentivirus (LV-shCib1) or its negative control lentivirus (LV-shNC) followed by Ang-II treatment at 48 h post-injection. For the signaling pathway analysis, the CFs were subjected to an inhibitor of the PI3K/Akt pathway, LY294002, before Ang-II treatment.

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  Fig. 7.. Down-regulation of CIB1 restrains angiotensin II (Ang II)-induced cardiac fibroblasts (CFs) transformation and collagen production in vitro. A–B. The images (A) and quantification (B) of immunofluorescent staining for α-SMA expression in Ang II-treated CFs. Magnification: 400×. Scale bar=50 µm. C–E. The mRNA level of Vim (C), Col1a1 (D), and Col3a1 (E) in the Ang II-treated CFs measured with real-time PCR. Data are expressed as the mean ± SD (n=3/group). **P<0.01.

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  Fig. 8.. CIB1 depletion represses the PI3K/Akt signaling pathway activated by Ang II to prevent cardiac fibroblasts (CFs) fibrosis. A–B. Representative images (A) and quantification (B) of phosphorylated PI3K-p85 (p-PI3K-p85) and total PI3K-p85 protein expression in Ang II-treated CFs, analyzed by western blot. C–D. Representative images (C) and quantification (D) of phosphorylated Akt (p-Akt) and total Akt protein expression in Ang II-treated CFs. E–F. Representative images (E) and quantification (F) of α-SMA and Collagen I levels in Ang II-treated CFs when CIB1 was knocked down or LY294002 (an inhibitor of the PI3K/Akt pathway) was added. Data are expressed as the mean ± SD (n=3/group). **P<0.01 vs. Ang II.

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  Fig. 9.. The diagram of the potential mechanism of CIB1 in myocardial infarction (MI).