European Review for Medical and Pharmacological Sciences 2018; 22: 1084-1093 Che-1 attenuates hypoxia/reoxygenation-induced cardiomyocyte apoptosis by upregulation of Nrf2 signaling D. WANG, T.-Y. CHEN, F.-J. LIU The Second Ward of Cardiovascular Medicine Department, Ankang City Central Hospital, Ankang, China Abstract. – OBJECTIVE: Hypoxia/reoxygen- Introduction ation (H/R)-induced cardiomyocyte apoptosis plays a critical role in the development of myo- Myocardial ischemia/reperfusion injury is one cardial infarction. Che-1 has been reported as of the leading causes of death and disability after an anti-apoptotic gene in response to various 1 cellular stresses. However, whether Che-1 reg- cardiac surgery and myocardial infarctions . Cell ulates cardiomyocyte apoptosis in myocardi- apoptosis induced by ischemia/reperfusion is one al infarction remains unclear. In this study, we of the major mechanisms of myocardial injury2. aimed to investigate the role of Che-1 in regulat- The sudden loss of oxygen and nutrient supply to ing H/R-induced cardiomyocyte apoptosis and the cardiomyocytes induces the overproduction the underlying molecular mechanism. MATERIALS AND METHODS: of reactive oxygen species (ROS) and activates The expression 3 of mRNA and protein was detected by Real-time the apoptotic pathway . The cardiomyocytes are quantitative polymerase chain reaction and West- terminally differentiated cells that lack a regen- ern blot. Cell viability was detected by cell count- eration ability4. Therefore, preventing the loss of ing kit-8 assay. Cell cytotoxicity was measured cardiomyocytes is essential for maintaining car- by lactate dehydrogenase assay. Cell apoptosis diac function during myocardial ischemia/reper- was assessed by caspase-3 activity assay. Intra- cellular ROS generation was determined using a fusion injury. Reactive Oxygen Species Assay Kit. The activity Che-1 (also known as apoptosis antagonizing of antioxidant response elements was detected transcription factor, AATF) is a human RNA by luciferase reporter assay. polymerase II binding protein, which regulates RESULTS: We found that Che-1 expression was the transcription of various genes5-7. The Che-1 significantly upregulated in cardiomyocytes in re- gene is located on chromosome 17, which is very sponse to H/R treatment. Functional experiments showed that silencing of Che-1 promoted H/R-in- rich in segmental duplications and protein-coding duced cell apoptosis and oxidative stress. By con- genes and is home to genes implicated in various trast, overexpression of Che-1 significantly alle- human genetic diseases8. Che-1 is highly con- viated H/R-induced cell apoptosis and oxidative served and ubiquitously expressed in eukaryotes stress. Interestingly, we found that Che-1 promot- playing important roles in regulating cell cycle, ed the expression of nuclear factor erythroid 2-re- cell proliferation, and cell apoptosis9,10. Moreover, lated factor 2 (Nrf2) and upregulated the activity of antioxidant response elements. Moreover, Che- Che-1 is suggested as a strong inhibitor of apop- 1 significantly upregulated the expression of Nrf2 tosis and is involved in regulating neurotoxicity, downstream target genes, including heme oxy- tumorigenesis and chemoresistance9,11-13. Previous genase-1 and NADPH-quinone oxidoreductase 1. reports have also shown that Che-1 is responsive CONCLUSIONS: Our results showed that Che- to various cellular stresses, such as hypoxia14,15 1 alleviates H/R-induced cardiomyocyte apopto- 16 sis by upregulation of Nrf2 signaling. Our study and endoplasmic reticulum stress . However, suggests that Che-1 may serve as a potential whether Che-1 regulates cardiomyocytes during and promising therapeutic target for the treat- myocardial ischemia/reperfusion injury remains ment of myocardial infarction. unclear. Nuclear factor-E2-related factor 2 (Nrf2) is a Key Words: highly conserved transcription factor that regu- Che-1, Hypoxia/reoxygenation, Myocardial infarc- 17-19 tion, Nrf2. lates the antioxidant process . Under oxidative stress, Nrf2 transfers into the nucleus and binds 1084 Corresponding Author: Fajun Liu, MD; e-mail: [email protected] Role of Che-1 in H/R-induced cardiomyocyte apoptosis to the antioxidant responsive elements (ARE), F12 and were pre-plated for 90 min to remove which activates antioxidant enzymes, such as non-cardiomyocytes and enrich the culture with heme oxygenase-1 (HO-1) or NADPH-quinone cardiomyocytes at 37°C for 1 h. Finally, the oxidoreductase 1 (NQO1)20. The activation of non-adherent cardiomyocytes were collected Nrf2/ARE prevents oxidative cardiac cell injury and plated on 1% gelatin-coated well plates and in vitro and in vivo21,22. Therefore, Nrf2 has been cultured at 37°C in a humidified incubator con- proposed as a promising target for the treatment taining 5% CO2/95% air. To establish the H/R of myocardial ischemia/reperfusion injury. model, cardiomyocytes were cultured in a tri- In this study, we aimed to investigate the gas incubator containing 94% N2/5% O2/1% CO2 potential role of Che-1 in regulating cardiomyo- for 6 h followed by reoxygenation for 24 h in an cyte apoptosis during myocardial ischemia/reper- atmosphere containing 5% CO2/95% air at 37°C. fusion injury using an in vitro cellular model Cells cultured under normal conditions served induced by hypoxia/reoxygenation (H/R). We as control. This study was reviewed and ap- found that Che-1 expression was significantly proved by the Institutional Animal Care and Use upregulated in cardiomyocytes in response to Committee of Ankang City Central Hospital. H/R treatment. Functional experiments showed The experiments were performed in accordance that silencing of Che-1 promoted H/R-induced with the National Institute of Health Guide (Na- cell apoptosis and oxidative stress, while over- tional Institute of Health Publications No. 80-23, expression of Che-1 showed protective effects. Revised 1978) for the care and use of Laboratory Interestingly, we found that Che-1 promoted the Animals for experimental procedure. expression of Nrf2 and upregulated the activity of ARE. Moreover, Che-1 significantly upregulated Real-Time Quantitative Polymerase the expression of Nrf2 downstream target genes, Chain Reaction (RT-qPCR) including HO-1 and NQO1. Taken together, our Total RNA was extracted for cultured cardio- results showed that Che-1 alleviates H/R-induced myocytes using TRIzol (Invitrogen, Carlsbad, cardiomyocyte apoptosis by upregulation of Nrf2 CA, USA) and RNA was then reverse transcribed signaling, providing a novel and promising ther- into cDNA using M-MLV Reverse Transcrip- apeutic target for the treatment of myocardial tase (TaKaRa, Dalian, China) according to the ischemia/reperfusion injury. manufacturer’s instructions. The PCR reactions were performed with SYBR Premix Ex Taq II assay (TaKaRa) on an ABI 7500 Real-Time PCR Materials and Methods System (Applied Biosystems, Foster City, CA, USA) following the thermal cycle conditions: 30 Cell Culture s at 95°C, followed by 40 cycles of 95°C for 40 s, The primary neonatal cardiomyocytes were 60°C for 35 s and 72°C for 40 s. β-actin was used isolated from 1-day-old neonatal C57BL/6 as internal control for normalization of mRNA mouse using enzymatic disassociation method expression. Relative expression was calculated as previously described23. In brief, the mice using the 2-ΔΔCt method. were euthanized, and the ventricular myocardial tissues were dissected under aseptic conditions. Western Blot Analysis After washing with phosphate-buffered saline Equal amounts of proteins were separated us- (PBS), the tissues were cut into small pieces ing a 12% sodium dodecyl sulfate polyacrylamide and digested using 1.2 mg/ml pancreatin and gel (SDS-PAGE) and then transferred to polyvi- 0.625 mg/ml type II collagenase in a 37°C wa- nylidene difluoride (PVDF) membranes (Milli- ter bath for 40 min. The cardiomyocytes were pore, Billerica, MA, USA). Non-specific binding then re-suspended in Dulbecco’s Modified Eagle sites on the membranes were blocked by 5% fat- Medium (DMEM)/F12 (Gibco, Rockville, MD, free milk at 37°C for 1 h. The membranes were USA) containing 10% fetal bovine serum (FBS) incubated with primary antibodies including an- Gibco, Rockville, MD, USA) and 1% penicillin/ ti-Che-1, anti-Nrf2 and anti-β-actin (Santa Cruz streptomycin (Sigma-Aldrich, St. Louis, MO, Biotechnology, Santa Cruz, CA, USA) at 4°C USA). The isolated cells were then filtered with overnight. The membranes were then probed with a 70 μm cell strainer (Millipore, Billerica, MA, secondary antibodies for 1 h at room temperature. USA). After centrifugation at 500 × g for 5 The protein bands were visualized by enhanced min, the cells were re-suspended in DMEM/ chemiluminescence using a Western blot detec- 1085 D. Wang, T.-Y. Chen, F.-J. Liu tion kit (Millipore, Billerica, MA, USA). Band sity reflecting ROS levels was measured using a intensity was measured by Image-Pro Plus 6.0 fluorescence spectrophotometer (Bio-Tek, Win- software for relative protein quantitation. ooski, VT, USA). Cell Transfection Luciferase Reporter Assay The siRNAs for targeting Che-1 were purchased For detection of ARE activity, cells were trans- from Santa Cruz Biotechnology (Santa Cruz, CA, fected with Che-1 siRNA or a Che-1 expression USA) and transfected into cells according to the vector in the presence of an ARE reporter vector manufacturer’s instructions. The Che-1 expression and a phRL-TK Renilla luciferase vector (Prome- vector was generated by inserting the Che-1 open ga, Madison, WI, USA). Luciferase activity was reading frame into a pcDNA3.1 vector and it was determined using the Dual-Luciferase Reporter transfected into cells using Lipofectamine 2000 Assay system (Promega, Madison, WI, USA) ac- (Invitrogen, Carlsbad, CA, USA) according to the cording to the manufacturer’s instructions. manufacturer’s protocol. Statistical Analysis Cell Counting Kit-8 (CCK-8) Assay Data are expressed as mean ± standard devi- Cell viability was evaluated using a CCK-8 as- ation (SD). Statistical analyses were carried out say. Briefly, cells were seeded into 96-well plates using SPSS 18.0 statistics software (SPSS Inc., at 3 × 105 cells/well and cultured overnight.