ABC | Volume 110, Nº1, January 2018

Original Article Hu et al Melatonin’s modulation on calcium in cardiomyocytes Arq Bras Cardiol. 2018; 110(1):44-51 death in cardiac cells. 20-23 So in the present studywe hypothesized melatonin has protective effects on cardiomyocyte against reperfusion injury through modulating IP3R and SERCA2a to maintain intracellular calciumhomeostasis. Ischemia-reperfusion has been shown to activate the anti‑apoptotic pro-survival kinase signalling cascades including p42/p44 extra-cellular signal-regulated kinases (ERK 1/2) which have been implicated in cellular survival. 24,25 It is not clear if ERK1 plays important role during modulation of melatonin on calcium homeostasis in cardiomyocytes. The present study aimed to elucidate whether melatonin protects cardiomyocytes against reperfusion injury through modulating IP3R and SERCA2a to reduce calcium overload via ERK1 pathway. Methods Ethics statement The present study was performed in accordance with the guidelines of the Ethic Committee of Chinese PLA (People's Liberation Army) General Hospital, Beijing, China. H9C2 Cells culture H9C2 cells (derived from the rat embryonic cardiomyoblast) were obtained from Chinese Academy of Medical Sciences (Shanghai, China) were cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12, Thermo Fisher Biochemical Products, Beijing, China) supplemented with 10% fetal bovine serum (FBS, Invitrogen Life Technologies, Carlsbad, CA, USA) and 100 mg/mL penicillin/streptomycin (Beyotime Institute of Biotechnology, China). H/R injury induction in vitro and melatonin or plus with PD98059 treatment Hypoxic conditions were produced using fresh Hanks solution with 95% N 2 and 5 % CO 2 . The pH was adjusted to 6.8 with lactate to mimic ischemic conditions. The dishes were put into a hypoxic incubator (Invivo2-400, Ruskinn) that was equilibrated with 95% N 2 and 5% CO 2 and the actual oxygen concentration was zero. Ambient O 2 levels in the hypoxia incubator were monitored by an O 2 analyzer (series-2000, Alpha Omega Instruments). After hypoxic treatment, the culture medium was rapidly replaced with fresh DMEM with 1% FBS to initiate reoxygenation. Hypoxia/reoxygenation procedure was achieved by 4 h of hypoxia treatment (anoxia) and 4 h of reoxygenation treatment. For melatonin treatment, cultured cells were pre-incubated with melatonin (5 uM) 12 h before hypoxia, or plus with PD98059 with concentration of 10 uM prior to melatonin treatment. The dose of melatonin was chosen according to previous studies. 18,26 In vitro TUNEL apoptosis assay of cardiomyocytes by confocal microscopy The apoptosis of H9C2 cells was examined by TUNEL assay. Briefly, cultured cardiomyocytes were fixed with 4% paraformaldehyde (PFA) (Millipore, USA) and permeabilized with 1% Triton X-100 (Sigma Aldrich, USA) in phosphate‑buffered saline (PBS) (Invitrogen, USA) for 30 minutes, followed by 3 times (3×10 mins) wash with fresh PBS. Then, an Apo‑BrdU in Situ DNA Fragmentation Assay Kit (BioVision, USA) was applied for 1 hour, followed by incubating the treated plates with 5 µl anti‑BrdUFITC antibody. Fifteen minutes of DAPI immunostaining were performed to identify the nuclei of cardiomyocytes. Then, the images were taken with an inverted Leica TCS-SP2 AOBS confocal laser-scanning microscope (Leica, Germany). Apoptosis was quantified as the percentage of cultured cardiomyocytes. F-actin study with fluorescent phalloidin and confocal microscopy F-actin detection with phalloidin was done according to manufacturer’s instructions. Briefly, H9C2 were fixed on polylysine-treated glass with 3.7% paraformaldehyde and later washed with 0.1% Triton X-100-PBS. Thereafter they were stained with 0.8unit/ml fluorescent FITC-phalloidin conjugate solution (KeyGen Bio TECH Corp,China) for 10 min at room temperature. Finally, they were washed three times with PBS. Mounted samples were analyzed using confocal microscopy. Detection of intracellular Ca 2 + concentration Intracellular Ca 2+ wasmeasured using the calcium-dependent fluorescent dye Fura-2 according to the manufacturer’s instructions. Briefly, H9C2 cultures were transferred to 1 mL fresh DMEM containing 5 μL Fura-2-acetoxy-methylester (AM; 10 μM; Life Technologies, Carlsbad, CA, USA) and incubated in a CO 2 incubator at 37°C for 1 h. Fura-2-loaded cells were then placed on the stage of a confocal microscopy (Olympus) and viewed using a 60× oil immersion objective. Western blots Following the appropriate treatments, cultured cells were lysed with RIPA lysis buffer (Beyotime,China) for 30 min and centrifuged at 14,000xg for 30 min. Equal amounts of protein were separated by 10% sodium dodecyl sulfate‑polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore). After being blocked with 5% milk in Tris buffered saline containing 0.05% Tween20 (TBST) at room temperature for 1h, the membrane was incubated at 4°C overnight with the following primary antibodies: t-ERK1(1:2000, Abcam), p-ERK1(1:1000, Abcam), IP3R (1:1000,Abcam), and SERCA2a (1:1000,Ab-cam). After being washed with TBST and further incubated with the appropriate secondary antibody at 37°C for 60 min, the blots were visualized with an enhanced chemiluminescence (ECL) reagent. Myocardial ischemia/reperfusion (I/R) model and melatonin treatment Male Sprague–Dawley (SD) rats (250 ± 10 g) were purchased from the Experimental Animal Center, Chinese PLA General Hospital. All procedures were approved by the Institutional Animal Care and Use Committee of the Chinese PLA General Hospital. Rats were divided into the following groups (n=5 in each group): (1) Control group, (2) I/R group, (3) I/R+Melatonin group, (4) I/R+Melatonin+PD98059. Rats were intraperitoneally anaesthetized with sodium pentobarbital 45