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 Figure 1 – Melatonin promoted activation of ERK1 in H9C2 cells against H/R. H9C2 cells incubated in normal condition or in simulated H/R condition, in simulated H/R condition plus pretreatment with melatonin, or in simulated H/R condition plus pretreatment with melatonin and PD98059 (ERK1 inhibitor).The expression levels of phosphorylated ERK1 (p-ERK1) were evaluated by Western blotting (A) and were quantitatively analyzed (B).All values are presented as the mean±SD. n = 3. SSp < 0.01 vs. H/R group; #p < 0.05 vs. H/R+Mel group.(Control: control group; H/R:H/R group; H/R+mel: H/R+ melatonin group; H/R+mel+PD: H/R+ melatonin+PD98059 group) A B Expression levels (Relative density) Control H/R H/R+Mel H/R+Mel+PD Control H/R H/R+Mel H/R+Mel+PD 2.0 1.5 1.0 0.5 0 SS # p-ERK1 t-ERK1 (50 mg/kg). The animals were then incubated and ventilated by a volume- regulated respirator during surgery. After a left lateral thoracotomy and pericardectomy, the left coronary artery was identified and gently ligated with a 6.0 prolene suture. Successful AMI was confirmed by the typical ST segment elevation in electrocardiography. Myocardial ischemia lasted for 30mins and reperfusion for 2 hours. Freshly preparedmelatonin (Sigma-Aldrich, St. Louis, MO, USA) was administered intraperitoneally at a dose of 20 mg/kg 12 hours prior to MI. PD98059 (ERK1 inhibitor, Sigma,USA) was administered with intraperitoneal injection at a dose of 5 mg/kg 4 hours prior to melatonin treatment. At the end of the reperfusion period, the hearts were excised for the preparation of sections (4 μm thickness) to detect the expression of SERCA2a and IP3R by immunofluorescence staining. Detection of myocardial SERCA2a and IP3R expression by immunofluorescence staining After being treated as above, the heart sections were fixedwith 4% paraformaldehyde in PBS for 15 min at room temperature, permeabilized with 0.1% Triton X-100 for 10 min, and then blocked with 5% BSA for 1 h. Then, samples were incubated overnight at 4°C with monoclonal mouse anti-rat SERCA2a antibody (1:100;Abcam, Cambridge, USA). After being washed with PBS for three times, samples were incubated with goat anti- mouse polyclonal IgG (1:400; Abcam,Cambridge, USA) at room temperature in the dark for 2 h. For nuclear counterstaining, samples were incubated with 4’, 6-diamidino-2-phenylidone (DAPI; Sigma, USA) for 5 min. Finally, the immunofluorescence images were obtained by inverted fluorescence microscope (Olympus, Tokyo, Japan). Statistical analysis Data were described as the mean ± SD of at least three independent analyzed experiments. The differences among more than 2 groups were evaluated through 1-way ANOVA (all data met the variances homogeneity and normal distribution),and LSD method was used to compare the statistical difference in the post-hoc analysis. A value of P<0.05 was considered statistically significant. All of the statistical analyses were performed with SPSS for Windows version 16.0 (SPSS Inc., Chicago, IL). Results Melatonin promoted activation of ERK1 in cardiomyocytes against H/R At 4h after reoxygenation, we investigated the effect of melatonin on phosphorylation of ERK1 (p-ERK1) using Western blot. The expression level of p-ERK1 did not show significant difference between control and H/R group. Melatonin significantly promoted the expression of p-ERK1 in cardiomyocytes which was reversed by PD98059 (ERK1 inhibitor) (Figure 1). Melatonin prevents H/R-induced apoptosis of cardiomyocytes via ERK1 pathyway in vitro The apoptosis of H9C2 cells was detected at 4h after reoxygenation by TUNEL staining. The results demonstrated H/R induce apoptosis of H9C2 cells in vitro. Pretreament with melatonin decreased H/R-induced apoptosis of H9C2. The results showed percentage of apoptotic cells was obviously higher in H/R group compared to control group, however, which was significantly lower in melatonin group than H/R group. PD98059 (ERK1 inhibitor) reduced the effect of melatonin on preventing cardiomyocytes apoptosis against H/R (Figure 2). Melatonin protects F-actin organization in H9C2 cells against H/R via ERK1 pathway We investigated F-actin organization in H9C2 cells at 4h after reoxygenation by fluorescent FITC-phalloidin staining . Control cardiomyocytes showed regular and well-defined actin organization, while cardiomyocytes in H/R group showed a more diffuse and irregular F-actin disposition. 46