NPAS2 ameliorates myocardial ischaemia/reperfusion injury in rats via CX3CL1 pathways and regulating autophagy

Figures

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  Figure 1. NPAS2 was downregulated in myocardial ischaemia/reperfusion injury rats. (A, B) Typical images of H&E and Masson staining of myocardial tissue segments. (C, D) Typical images of TTC of myocardial tissue segments. The infarct size was measured and calculated as a percentage of the total area. (E, F) Typical images of Tunel of myocardial tissue segments. The relative percentages of apoptotic cells were calculated. (G, H) Typical echocardiographic images of M-mode and LVEF. (I) The mRNA level of NPAS2 in rat myocardial tissue was determined by qRT-PCR. (J) The protein level of NPAS2 (90kDa), LC3B (14 and 16kDa) and p62 (62kDa) in rat myocardial tissue was determined using Western Blot. Data are expressed as mean ± SEM (n = 6).

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  Figure 2. Overexpression of NPAS2 ameliorated rats ischaemia/reperfusion injury in vivo. (A, B) Typical images of H&E and Masson staining of myocardial tissue segments. (C, D) Typical images of TTC of myocardial tissue segments. The infarct size was measured and calculated as a percentage of the total area. (E, F) Typical images of Tunnel of myocardial tissue segments. The relative percentages of apoptotic cells were calculated. (G, H) Typical echocardiographic images of M-mode and LVEF. (I) The mRNA level of NPAS2 in rat myocardial tissue was determined by qRT-PCR. Data are expressed as mean ± SEM (n = 6).

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  Figure 3. Overexpression of NPAS2 inhibited autophagy in ischaemia/reperfusion injury rats. (A) The protein level of NPAS2 in rat myocardial tissue was determined using Western Blot. (B) The protein level of Cleaved-Caspase-3 (17kDa) and Caspase-3 17kDa) in rat myocardial tissue was determined by Western Blot. (C) The protein level of LC3B and p62 in rat myocardial tissue was determined using Western Blot. (D) Representative photomicrographs of LC3B (green) immunofluorescence in rat myocardial tissue. DAPI was used to counterstain nuclei.

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  Figure 4. Overexpression of NPAS2 ameliorated hypoxia/reoxygenation injury in vitro. (A, B) Flow cytometry detected the changes of apoptosis in H9c2 cells and quantified. (C) The protein level of Cleaved-Caspase-3 (17kDa) and Caspase-3 (17kDa)in H9c2 cells was determined by Western Blot. (D) The protein level of NPAS2 in H9c2 cells was determined by Western Blot. (E) The protein level of LC3B (14 and 16kDa) and p62 (62kDa) in H9c2 cells was determined by Western Blot. (F) Typical images of immunofluorescence staining of mRFP-GFP-LC3 in H9c2 cells. Typical profiles of autophagosomes (RFP+GFP+dots) and autolysosomes (RFP+GFP-dots). (G) Autophagic vacuoles (autophagosomes) determined by transmission electron microscopy (TEM). Representative TEM images are shown, and typical autophagosomes are marked with black arrows. Data are expressed as mean ± SEM (n = 3).

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  Figure 5. Cry2 interacted with NPAS2 in cardiomyocyte. (A, B) String database (https://string-db.org/) and BioGRID Database (biogrid.org) were used to predict Cry2 and NPAS2 interaction. (C) The protein level of Cry2 (67kDa) in rat myocardial tissue and H9c2 cells was determined using Western Blot. (D) Co-IP assay was performed with anti-NPAS2 or anti-Crry2 antibody was carried out using extracts prepared from rat myocardial tissue and H9c2 cells. The presence of Cry2 or NPAS2 in these IPs was determined using Western Blot. (E, F) Flow cytometry detected the changes of apoptosis in H9c2 cells and quantified. (G) The protein level of Cleaved-Caspase-3 (17kDa) and Caspase-3 (17kDa) in H9c2 cells was determined using Western Blot. (H) The protein level of NPAS2 (90kDa) in H9c2 cells was determined using Western Blot. (I) Representative photomicrographs of NPAS2 (green) and Cry2 (Red) immunofluorescence in H9c2 cells. DAPI was used to counterstain nuclei. Data are expressed as mean ± SEM (n = 3).

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  Figure 6. NPAS2 transcriptionally promoted CX3CL1 expression in vivo. (A) The protein level of CX3CL1 (100kDa) in rat myocardial tissue was determined by Western Blot. (B) The protein level of CX3CL1 (100kDa) in H9c2 cells was determined by Western Blot. (C) H9c2 cells were transfected with CX3CL1 promoter and the luciferase activity was determined after 24 hours. (D) Amplification of the CX3CL1 promoter sequence was performed BY ChIP assay in H9c2 cells. (E, F) Typical images of H&E and Masson staining of myocardial tissue segments. (G, H) Typical images of TTC of myocardial tissue segments. The infarct size was measured and calculated as a percentage of the total area. (I, J) Typical images of Tunnel of myocardial tissue segments. The relative percentages of apoptotic cells were calculated. (K, L) Typical echocardiographic images of M-mode and LVEF. (M) The protein level of CX3CL1 (100kDa), LC3B (14 and 16kDa), p62 (62kDa), Cleaved-Caspase-3 (17kDa), Caspase-3 (17kDa), p-AKT (60kDa), t-AKT (60kDa), p-mTOR (289kDa) and t-mTOR (289kDa) in rat myocardial tissue was determined by Western Blot. Data are expressed as mean ± SEM (n = 6).

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  Figure 7. NPAS2 transcriptionally promoted CX3CL1 expression in vitro. (A, B) Flow cytometry detected the changes of apoptosis in H9c2 cells and quantified. (C) The protein level of Cleaved-Caspase-3 (17kDa) and Caspase-3 (17kDa) in H9c2 cells was determined by Western Blot. (D) The protein level of CX3CL1 (100kDa) in H9c2 cells was determined by Western Blot. (E) The protein level of LC3B (14 and 16kDa) and p62 (62kDa) in H9c2 cells was determined by Western Blot. (F) The protein level of p-AKT (60kDa), t-AKT (60kDa), p-mTOR (289kDa) and t-mTOR (289kDa) in H9c2 cells was determined by Western Blot. (G) Typical images of immunofluorescence staining of mRFP-GFP-LC3 in H9c2 cells. Typical profiles of autophagosomes (RFP+GFP+dots) and autolysosomes (RFP+GFP-dots). (H) Autophagic vacuoles (autophagosomes) determined by transmission electron microscopy (TEM). Representative TEM images are shown, and typical autophagosomes are marked with black arrows. Data are expressed as mean ± SEM (n = 3).