We aimed to evaluate the cardioprotective effect and mechanism of propofol in anthracycline-induced cardiomyocyte apoptosis. We selected the rat myocardial cell line, H9c2, and primary cardiomyocytes for
The use of anthracycline (ANT) for the chemotherapy of various malignancies has been hampered by their cardiac toxicity. The most severe is the chronic forms of cardiotoxicity, characterized by irreversible cardiac damage and congestive heart failure, which remain a major problem 50 years after the discovery of daunorubicin and doxorubicin (DOX), also known as Adriamycin (ADM), and their introduction in clinics [
Recent studies have demonstrated that microRNAs, as endogenous regulators of gene expression, were involved in the regulation of cardiovascular disease in several biological processes, including endothelial cell dysfunction, cell adhesion, and cardiomyocyte proliferation and apoptosis [
Propofol (2,6-diisopropylphenol), known as “milk of anesthesia,” is one of the most popular intravenous anesthetic agents in modern medicine. It is commonly used for the induction and maintenance of anesthesia and procedural and critical care sedation in children [
Doxorubicin (Adriamycin) was provided by Haimen Pharmaceutical (Zhejiang, China). Propofol was provided as a clinical formulation by the Pharmacy of Tianjin Medical University Cancer Institute and Hospital. Doxorubicin was diluted with sterile water to a final concentration of 100 mM and stored at 4°C. Propofol was diluted with DMSO to a final concentration of 10 mg/ml and stored at 4°C.
The rat myocardial cell line H9c2 was used from our laboratory, cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin double-antibiotic at 37°C in a 5% CO2 incubator.
Sprague–Dawley and F344 rats, between 6 and 9 weeks old, were purchased from Laboratory Animal Science Department of the Peking University Health Science Center (license number SCXK (Jing) 2006–2008). The animals were maintained in a naturally ventilated room, with a temperature of 25–28°C, relative humidity of 70%–85%, and a 12 h light/dark cycle. The animals were provided with standardized laboratory rat pellet food and water ad libitum and weighed weekly.
The flow cytometer (BD FACS Aria I), ELISA reader, incubator, centrifuges, and other experimental instruments were provided by the Core Laboratory of the Tianjin Medical University Cancer Institute and Hospital.
Twenty Sprague–Dawley (SD) rats were used for the isolation and culture of primary cardiomyocytes. After whole hearts were extracted aseptically, the pericardium and atrium were removed and washed several times with ice-cold PBS to remove residual blood and debris. The residual ventricular parts of the hearts were minced into 1-2 mm3 tissue blocks under sterile conditions. The tissues were then repeatedly digested with freshly prepared compound digestive enzyme [collagenase II (Gibco) 1 mg/ml, hyaluronidase (Gibco), 0.2 mg/ml in Hank’s solution (Gibco), sterile filtered, and pH 7.2] for 20 min in a 37°C incubator until the tissue blocks were completely digested and then filtered through a 200
The MTT (3-(4-5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide dye) reduction assay was performed to compare the effects of propofol, ADM, and the combination treatment. Each condition was replicated in five wells. The inhibitory concentration of propofol in the combination treatment was maintained at 1
Twenty-four hours after treatment, 20
H9c2 cells and primary cardiomyocytes were placed in 6-well plates. The cells were divided into four groups: (1) control group: blank control; (2) prop group: 1
After the treatments were applied to the cells, total RNA was extracted from H9c2 cells and primary cardiomyocytes by using the Trizol Reagent (Invitrogen). The RT primers sequence of miR-181a was 5
The H9c2 or primary cardiomyocytes were grown in 6-well plates, divided into the four groups as described above, and treated for 24 h. Subsequently, the cells were collected for protein extraction and lysed in RIPA buffer (1% NP-40, 1 mmol/l Na3VO4, 1 mmol/l NaF, and 0.5 mmol/l PMSF) on ice for 30 min. The lysate was clarified by centrifugation, and the supernatant was removed. The protein concentrations were assessed by using the BCA Protein Assay Kit (Pierce), and the absorbance was read at 490 nm by means of ELISA reader. Cell lysate containing 30
Rno-miR-181a inhibitor and rno-miR negative control were synthesized by GenePharma (Shanghai). Twenty-four hours prior to transfection, H9c2 cells were plated in 6-well plates (2.5 × 105 cells/well) and then transfected with miR-181a inhibitor (50 nM) or miR negative control (50 nM) by using Lipo2000 (Invitrogen). A Bcl-2-3
The body weights of F344 rats at the start of the experiment were 210 ± 15 g. The forty rats were randomly assigned into four groups of ten animals: (1) control group: injection of the same volume of saline at the same time; (2) prop group: 50 mg/kg propofol; (3) ADM group: 0.8 mg/kg ADM; and (4) ADM + prop group: 50 mg/kg propofol plus 0.8 mg/kg ADM. The administration was conducted by intraperitoneal injection every 3 weeks, for 6 weeks. The rats in each group were observed for psychological status, appetite, and activity, and the number of the surviving animals was recorded, and the survival rate was calculated. The body weights of the animals were weighed weekly. At the 3rd day after the last dose, the animals were sacrificed.
Cardiomyocyte apoptosis was detected by the TUNEL assay: the paraffin blocks were serially sectioned, routinely dewaxed, antigen repaired at 95°C for 10 min, soaked in 3% hydrogen peroxide solution for 20 min, and digested in 20
The measurement data were presented as the mean ± S.D. and analyzed with statistical methods, including Student’s
The
Antiproliferative effect ADM alone or combination treatment with propofol in cardiomyocytes. Morphological changes of H9c2 cells (a) and primary cardiomyocytes (b) in different treatment (control, prop 1
To determine whether propofol preserved myocardial cell viability through intervening in the process of apoptosis, cardiomyocytes of all four groups were assessed by flow cytometry (Figure
Effect of the combination of propofol and ADM induction of early apoptosis in cardiomyocytes by flow cytometry. After exposure to the different treatments for 24 h, the proportion of early apoptotic cells was analyzed using Annexin V-FITC and PI in H9c2 cells (a) and primary cardiomyocytes (b). Compared with the Adriamycin-alone group, combination treatment induced a decrease in the population of cells undergoing early apoptosis in H9c2 cells (
To ascertain the role of miR-181a in ADM-induced cardiomyocyte apoptosis, miR-181a expression was tested by RT-PCR. A significant upregulation of miR-181a expression was found in both ADM-alone group and the combination group (Figures
Propofol reduced the upregulation of miR181a induced by ADM and inhibited ADM-induced STAT3 activating. The expression of miR-181a in different treatment on H9c2 (a) or primary cardiomyocytes (b) tested by RT-PCR. The combination treatment upregulation of miR-181a was reduced compared to the ADM-alone group. (c-d) Phospho-STAT3, Bcl-2, Bax, Caspase3, and Caspase9 protein levels were measured in different treatment in cardiomyocytes by Western blotting. (e-f) Semiquantitative data from densitometric analysis of phospho-STAT3, Bcl-2, Bax, Caspase3, and Caspase9 are presented as relative ratio of each protein to
To explore the effect of propofol on the STAT3 pathway and apoptosis-related proteins in cardiomyocytes, the change in protein expression of phospho-STAT3, Bcl-2, Bax, Caspase3, and Caspase9 after the different treatments was detected by Western blot (Figures
As shown previously, propofol played a protective role in ADM-induced apoptosis, whereas the expression of miR-181a was significantly reduced in the ADM + prop group compared with that in the ADM group. To confirm the critical role of miR-181a in ADM-induced cardiomyocyte apoptosis in H9c2 cells, we transfected rno-miR-181a inhibitor and rno-miR-181a control into H9c2 cells and detected changes in apoptosis. The qRT-PCR assays showed that the expression of miRNA-181a in H9c2 cells transfected with the miRNA-181a inhibitor plasmid was significantly lower than that of H9c2 cells transfected with a miRNA-181a NC plasmid (
miR-181a contributes to reducing ADM-induced cardiomyocyte apoptosis by repressing Bcl-2. (a) RNAs isolated from H9c2 cells transfected with miR-181a inhibitor or control were analyzed by RT-qPCR to access the levels of miR-181a. The expression of miR-181a was reduced by miR-181a inhibitor transfection. (b) The cell viability of H9c2 cells transfected with miR-181a inhibitor or control in different treatment was tested by MTT assay. (c-e) After 24 h exposure of ADM treatment, the proportion of apoptotic cells was decreased in miR-181a inhibitor cells compared with control cells (
To delineate the underlying mechanism of miR-181a in the protective effect of ADM-induced cardiomyocyte apoptosis, we searched for potential target genes of miR-181a by using several bioinformatics tools, such as TargetScan, miRanda, and PicTar. The computational analysis revealed that there was a binding site on the 3
F344 rats were randomly divided into the following groups: (1) control group; (2) prop group; (3) ADM group; (4) ADM + prop group. The apoptosis index in rat heart tissue after different treatments was determined by TUNEL staining, and the expression of miR-181a and phospho-STAT3 was determined by RT-qPCR or Western blotting (Figure
Propofol could reduce the apoptosis cells induced by ADM in cardiomyocytes
The potential significance of microRNAs in myocardial injury has been highlighted by recent mechanistic research [
In previous studies, ANT-induced cardiotoxicity was shown to involve cardiomyocyte apoptosis [
Propofol, an anesthetic with pluripotent cytoprotective properties against various toxic insults, was shown to ameliorate ischemia-reperfusion injury in multiple organs such as the heart [
miR-181a, widely present in human organs, has been reported to modulate cell proliferation, migration, apoptosis, and tumorigenesis [
In addition, we demonstrated that miR-181a suppressed Bcl-2 expression by directly targeting of the Bcl-2 transcript in H9c2 cells (Figure
In summary, we found that propofol reduced apoptosis induced by ANT in cardiomyocytes
The authors declare that they have no competing interests.
Hongwei Zhao and Xiaobei Zhang contributed to the writing up of the first draft of the manuscript. Hongwei Zhao and Kaiyuan Wang conceived and designed the study. Xiaobei Zhang, Ying Zheng, Xiaokun Wang, and Yuan Li performed the experiments. Nan Hu and Peng Zhou reviewed and edited the manuscript for important intellectual content and gave final approval of the version to be published. Hongwei Zhao and Xiaobei Zhang contributed equally to this study.
The authors acknowledge the financial support from Science Foundation of Tianjin Medical University no. 2015KYZQ18 (Xiaobei Zhang) and Ph.D. Development Foundation of Tianjin Medical University Cancer Institute and Hospital no. B1503-1 (Xiaobei Zhang).