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

Cold-inducible RNA-binding protein (CIRBP) is documented to be required for maintaining cardiac function, however, its role in chemotherapy-induced cardiotoxicity remains obscured. Herein, we report that CIRBP decreases cardiomyocyte apoptosis and attenuates cardiotoxicity through disrupting OGF-OGFR signal. CIRBP deficiency is involved in diverse chemotherapeutic agents induced cardiomyocyte apoptosis. Delivery of exogenous CIRBP to the mouse myocardium significantly mitigated doxorubicin-induced cardiac apoptosis and dysfunction. Specifically, OGFR was identified as a downstream core effector responsible for chemotherapy-induced cardiomyocyte apoptosis. CIRBP was shown to interact with OGFR mRNA and to repress OGFR expression by reducing mRNA stability. CIRBP-mediated cytoprotection against doxorubicin-induced cardiac apoptosis was demonstrated to largely involve OGFR repression by CIRBP. NTX as a potent antagonist of OGFR successfully rescued CIRBP ablation-rendered susceptibility to cardiac dyshomeostasis upon exposure to doxorubicin, whereas another antagonist ALV acting only on opioid receptors did not. Taken together, our results demonstrate that CIRBP confers myocardium resistance to chemotherapy-induced cardiac apoptosis and dysfunction by dampening OGF/OGFR axis, shedding new light on the mechanisms of chemo-induced cardiotoxicity and providing insights into the development of an efficacious cardioprotective strategy for cancer patients.

Four weeks post-AAV9 injection, the mice were injected via the tail vein with DOX (5 mg/kg) once weekly for 4 weeks to generate a preclinical model of chemotheray-induced cardiotoxicity as described in previous studies. (3,4) For NTX or ALV treatment, the mice were given intraperitoneal injection daily with either 15 mg/kg NTX, 15 mg/kg ALV, or 0.2 ml sterile saline (vehicle) once DOX administration. The mice were housed 5 or 6 per cage, and all mice in one cage belonged to the same treatment group. The mice were observed daily and weighed per week, and after 8 days of DOX treatment, they were subjected to echocardiography before being sacrificed. Mice were sacrificed under the isoflurane inhalation (1.4%) and followed by cervical dislocation

RNA sequencing (RNA-seq)
Total RNA was extracted from tissue using TRIzol® reagent according to the manufacturer's instructions (Invitrogen), and genomic DNA was removed using DNase I (Takara). Then, RNA quality was determined with a 2100 Bioanalyser (Agilent) and quantified using an ND-2000 spectrophotometer (NanoDrop Technologies). Only high-quality RNA samples (OD260/280 = 1.8~2.2, OD260/230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0, > 1 μg) were used to construct a sequencing library. An RNA-seq transcriptome library was prepared using a TruSeqTM RNA Sample Preparation Kit from Illumina (San Diego, CA) and 1 μg of total RNA. After quantification with a TBS380 instrument, the paired-end RNA-seq sequencing library was sequenced with an Illumina HiSeq xten/NovaSeq 6000 sequencer (2 ×150 bp read length). Differential expression analysis was performed using DESeq2, DEGseq, and EdgeR with a Q value ≤ 0.05 (genes with a |log2FC| > 1 and Q value ≤ 0.05 according to DESeq2 and EdgeR and a Q value ≤ 0.001 according to DEGseq were considered significantly differentially expressed genes (DEGs)).

iTRAQ quantitative proteomics
The samples were suspended in protein lysis buffer (8 M urea, 1% SDS) containing an appropriate protease inhibitor to inhibit protease activity. Then, the mixture was ultrasonicated at 40 kHz and 40 W for 2 min and then incubated on ice for 30 min. After centrifugation at 12000 × g and 4 °C for 30 min, the concentration of the protein supernatant was determined by the BCA method with a BCA Protein Assay Kit (Thermo Scientific). Protein quantification was performed according to the protocol of the kit, and then analysis was performed using standard procedures. Briefly, the peptides were labelled with iTRAQ tags (Applied Biosystems) and analysed by on-line electrospray tandem mass spectrometry on a Nano Aquity UPLC system (Waters Corporation) connected to an LTQ Orbitrap XL mass spectrometer (Thermo Scientific) equipped with an on-line nano electrospray ion source (Michrom Bioresources). Proteins identified by two or more unique peptides were selected for further analysis. Differentially expressed proteins were selected according to the following criteria: (1) an adjusted p value < 0.05 and (2) a fold change < 0.83 or fold change > 1.2.

Analysis of mRNA stability
To measure the half-life of endogenous OGFR mRNA, actinomycin D (2 μg/ml) was added to the cell culture medium, and total RNA was isolated at the indicated times and subjected to RT-qPCR analysis using OGFR-specific primers.
GAPDH mRNA was used as a negative control (NC).

RNA immunoprecipitation (RIP)
For crosslinking of ribonucleoprotein (RNP) IP complexes, cells were exposed to UVC (254 nm, 400 mJ/cm 2 ), and whole-cell lysates were prepared for immunoprecipitation using an anti-CIRBP antibody. Briefly, the lysates were

Dual-luciferase reporter assay
Cultured AC16 cells were transfected with reporters using Lipofectamine 2000 (Invitrogen). The cells were cotransfected with the PGL3 promoter vector as an internal control. Firefly and Renilla luciferase activities were measured with a double luciferase assay system (Promega) following the manufacturer's instructions. Firefly luciferase activity was normalized to Renilla luciferase activity in the same sample.

Immunofluorescence staining
Paraffin-embedded sections (3 μm) were deparaffinized in xylene, rehydrated by washing in ethanol and rinsed in PBS.
Antigen retrieval was performed by pressure cooking the sections for 6 min in citrate buffer (pH 6.0, target retrieval solution, Dako, Santa Clara, CA, USA), and then the sections were blocked with 2.5% BSA. The sections were stained overnight at 4 °C with the following primary antibodies: rabbit anti-CIRBP, rabbit anti-cleaved caspase 3, and mouse anti-cTnT. After three washing steps, the sections were incubated with secondary antibodies conjugated to DyLight Fluor 488 and DyLight Fluor 550 for 1 h at room temperature, the nuclei were stained with 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI, Sigma Aldrich), and the slides were mounted with Permafluor mounting medium (Thermo Scientific). Images were taken on a Zeiss Axio observer Z1 microscope using Tissue FAXS software (version 6.06.245.103, Tissue Gnostics, Vienna, Austria). All histologic examinations were performed by an independent observer blinded to the treatment groups and time points.

Evaluation of apoptosis in cardiomyocytes and tissue sections
For flow cytometry, AC16 cells were grown overnight in 10 cm plates and treated with DOX for 24 h. Following treatment, the cells were trypsinized, pelleted, and resuspended in 100 μl of 1X Annexin V Binding Buffer. Then, 5 μl of FITC-conjugated Annexin V antibody and 5 μl of 7-amino-actinomycin D (7-AAD) were added. The cells were incubated in the dark at room temperature for 15 min, and then 400 μl of 1 × Annexin V Binding Buffer was added. The samples were analysed by an LSRFortessa flow cytometer (BD Biosciences) in one hour.
For TUNEL staining, tissues sections were dewaxed, hydrated and subjected to antigen retrieval. TUNEL staining was performed using an in situ cell death detection kit (no. 11684795910, Roche) according to the manufacturer's protocol.
The apoptotic index was measured by counting TUNEL-positive puncta in 100 randomly selected actinin-positive cells in multiple randomly chosen fields at 400× magnification. Images were captured with a laser-scanning confocal microscope (SP8, Leica).

Echocardiographic assessment
Echocardiograms were recorded from conscious, gently restrained animals using a Vevo 3100 system with an MS400C scanhead. M-mode recordings were obtained at the level of the papillary muscles to measure systolic and diastolic left ventricular internal dimensions. Ejection fraction (EF) and fractional shortening (FS) were calculated as (LVIDd-LVIDs)/LVIDd and are expressed as percentages.