CIRBP-OGFR axis safeguards against cardiomyocyte apoptosis and cardiotoxicity induced by chemotherapy

Figures

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  Figure 1. Reduction in cold-inducible RNA-binding protein (CIRBP) expression is involved in cardiomyocyte apoptosis and cardiotoxicity induced by chemotherapeutics. A, Representative immunoblots assessing apoptosis induced by three conventional chemotherapeutics (doxorubicin (DOX), cisplatin, and 5-fluorouracil (5-FU)) in immortalized human ventricular myocytes (AC16). B, Densitometric quantification of the relative protein levels of CIRBP and Cleaved-Caspase 3 in A after treatment with 500 nM DOX, 30 µM cisplatin, and 100 µM 5-FU. C, Representative immunoblots assessing apoptosis of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) induced by the aforementioned chemotherapeutics. D, Representative immunoblots assessing apoptosis of neonatal rat ventricular myocytes (NRVMs) induced by the aforementioned chemotherapeutics. E, Representative immunofluorescence images of heart tissue sections from mice receiving vehicle or DOX. Nuclei, DAPI (blue); cardiomyocytes, cardiac troponin T (cTnT) (green); CIRBP (red). Scale bar, 20 µm. F, Quantification of the relative CIRBP fluorescence intensity in 6 microscopic fields from 3 hearts per group in E. G, Immunoblot analysis of…

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  Figure 2. CIRBP ablation exacerbates DOX-induced cardiomyocyte apoptosis. A, Representative immunoblots assessing DOX-induced apoptosis of AC16 cells transfected with negative control siRNA (NC) or CIRBP siRNA (SiCIRBP). B, Densitometric quantification of the relative protein levels of CIRBP and Cleaved-Caspase 3 in A. Vehicle-treated cells transfected with NC siRNA served as controls for quantification. C, Representative immunoblots assessing DOX-induced apoptosis of NRVMs transfected with NC or SiCIRBP. D, Densitometric quantification of CIRBP and Cleaved-Caspase 3 protein levels in C. Vehicle-treated cells transfected with NC served as controls for quantification. E, Flow cytometry analysis of apoptosis of the cells described in A. F, Statistical analysis of the apoptosis rate in E. G, Quantification of the relative mRNA levels of BAX and BCL2 (BAX/BCL2) for assessment of DOX-induced apoptosis of AC16 cells transfected with NC or SiCIRBP. The data are presented as the mean ± SD of three independent experiments. And statistical significance was analysed by one-way ANOVA followed by Tukey-Kramer multiple comparisons **, p < 0.01; ***, p < 0.001.

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  Figure 3. CIRBP mitigates mouse myocardial apoptosis and cardiotoxicity to protect against DOX insult. A, Echocardiographic analysis of cardiac function in mice pre-injected with an adeno-associated virus 9 (AAV9) vector harbouring a control (AAV9-Ctrl) or CIRBP (AAV9-CIRBP)-expressing cassette in the presence or absence of DOX (vehicle or DOX). EF, ejection fraction; FS, fractional shortening. Vehicle + AAV9-Ctrl, n = 9; DOX + AAV9-Ctrl, n = 8; DOX + AAV9-CIRBP, n = 10. B, Statistical analysis of the heart weight/tibia length (HW/TL) ratio of the mice in A. C, Biochemical determination of the relative serum levels of creatine kinase-myocardial band (CK-MB), cTnT, lactate dehydrogenase (LDH), and N-terminal pro-B type natriuretic peptide (NT-proBNP) to assess cardiac injury in the mice in A. D, Representative TUNEL staining images of heart tissue sections from the mice in A. Nuclei, DAPI (blue); cardiomyocytes, cTnT (green); TUNEL (red). Scale bar, 50 µm. E, Quantification of TUNEL staining in 3 microscopic fields from the hearts of the mice in A. F, Immunoblot analysis of protein levels in heart tissues from the mice in A. G, Densitometric quantification of CIRBP and Cleaved-Caspase 3…

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  Figure 4. Exploration of the downstream effector of CIRBP that likely accounts for cardiomyocyte apoptosis. A, Volcano plot of differentially regulated transcripts in AC16 cells transfected with NC siRNA compared with those transfected with SiCIRBP. Opioid growth factor receptor (OGFR) is indicated by the arrow. B, Gene ontology (GO) enrichment analysis of the genes with altered expression between the NC siRNA- and SiCIRBP-transfected group, as determined by transcriptomics analysis. C, Heatmap showing differentially expressed proteins between the NC siRNA- and SiCIRBP-transfected groups identified by proteomics analysis. OGFR is indicated by the red dashed rectangle and indicated by the arrow. D, Relative levels of OGFR mRNA in human cardiomyocytes infected with an adenovirus vector expressing a control shRNA (Ad-Ctrl)- or CIRBP shRNA (Ad-shCIRBP)-expressing cassette or transfected with NC siRNA or SiCIRBP. Left, hiPSC-CMs; middle, AC16 cells; Right, T0519 cells. E, Immunoblot analysis of cellular proteins isolated from different cardiac myocytes described in D. The data are presented the mean ± SD of three independent experiments. Statistical significance was analysed using Student's…

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  Figure 5. CIRBP represses OGFR expression by destabilizing OGFR mRNA. A, Analysis of the half-life (t1/2) of OGFR (left) and GAPDH (right) mRNA in AC16 cells transfected with NC siRNA or SiCIRBP after exposure to actinomycin D by real-time qPCR. B, RNA immunoprecipitation (RIP) with IgG and CIRBP antibodies using lysates prepared from AC16 cells. GAPDH mRNA served as a control. C, Schematic depiction of truncated fragments of OGFR mRNA used for RNA pull-down, crosslinked RNA immunoprecipitation coupled with reverse transcription polymerase chain reaction (CLIP-PCR), and the generation of luciferase reporter constructs. D, RNA pull-down analysis of the interactions between CIRBP and the different truncated RNA fragments shown in C. The protein lysates used for the pull-down assay were prepared from AC16 cells. GAPDH served as a control. E, Mapping of the CIRBP-binding regions in OGFR mRNA in AC16 cells through CLIP-PCR. IgG and GAPDH mRNA served as controls at different levels. The amplified specific bands are indicated by the red arrowheads. F, Measurement of luciferase activity in AC16 cells expressing different reporter constructs and transfected with NC siRNA or SiCIRBP. n=8 per group,…

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  Figure 6. CIRBP-mediated OGFR repression plays an essential role in protecting cardiomyocytes from apoptosis induced by DOX. A, Representative immunoblots assessing DOX-induced apoptosis in AC16 cells transfected with NC siRNA, SiCIRBP, or siRNAs targeting both CIRBP and OGFR (SiCIRBP + SiOGFR). B, Densitometric quantification of the relative protein levels of CIRBP, OGFR, P53, P21, P16, and Cleaved-Caspase 3 in A. DOX-treated cells transfected with NC siRNA served as controls for quantification. C, Flow cytometry analysis of apoptosis of the cells described in A. D, Statistical analysis of the apoptosis rate in C. E, Flow cytometry analysis of DOX-induced apoptosis of AC16 cells treated with NC siRNA (DOX + NC), SiCIRBP (DOX + SiCIRBP), SiCIRBP plus naltrexone (NTX) (DOX + SiCIRBP + NTX), or SiCIRBP plus alvimopan (ALV) (DOX + SiCIRBP + ALV). F, Statistical analysis of the apoptosis rate in E. G, Representative immunoblots assessing DOX-induced apoptosis in AC16 cells co-transfected with pCDNA and pGL3 constructs (pCDNA + pGL3), pCIRBP and pGL3 constructs (pCIRBP + pGL3), pCIRBP and pGL3-WT constructs (pCIRBP + pGL3-WT), or pCIRBP and pGL3-ADdel constructs (pCIRBP + pGL3-ADdel). H,…

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  Figure 7. OGFR blockade ameliorates CIRBP deficiency-aggravated myocardial apoptosis and cardiotoxicity during DOX administration. A, Echocardiographic analysis of cardiac function in mice in the vehicle + AAV9-Ctrl groups (pre-injected with a control AAV9 vector and not treated with DOX), the DOX + AAV9-Ctrl group (pre-injected with control a AAV9 vector and treated with DOX), the DOX + AAV9-shCIRBP group (pre-injected with an AAV9 shCIRBP vector and treated with DOX), the DOX + AAV9-shCIRBP + NTX group (pre-injected with an AAV9 shCIRBP vector and NTX and treated with DOX), and the DOX + AAV9-shCIRBP + ALV group (pre-injected with an AAV9 shCIRBP vector and ALV and treated with DOX). Representative echocardiograms for each group. Vehicle + AAV9-Ctrl, n = 8; DOX + AAV9-Ctrl, n = 8; DOX + AAV9-shCIRBP, n = 7; DOX + AAV9-shCIRBP + NTX, n = 8; DOX + AAV9-shCIRBP + ALV, n = 7. Statistical analysis of EF, FS (B) and the HW/TL ratio (C) of mice in A. D, Biochemical determination of the relative serum levels of CK-MB, cTnT, LDH, and NT-proBNP for assessment of cardiac injury in the mice in A. E, Representative TUNEL staining images of heart tissue sections from the mice in A. Nuclei, DAPI…