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 2 – Melatonin prevents H9C2 cells apoptosis against H/R via ERK1 in vitro. Apoptosis was assessed by fluorescence TUNEL. Representative TUNEL staining images (A) and quantitative analysis in H9C2 cells(B). bar = 50 μm.All values are presented as the mean±SD. n = 3.**p < 0.01 vs. control group; Sp < 0.05 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) 70 60 50 40 30 20 10 0 Apoptotic cells (%) Control H/R H/R+Mel H/R+Mel+PD Control H/R H/R+Mel H/R+Mel+PD A B TUNEL DAPI Merge ** S # The differences can be visualized in the representative cardiomyocytes. Pretreatment of melatonin improved F-actin organization in cardiomyocytes compared with H/R group, but PD98059 damaged F-actin organization by inhibiting melatonin’s effect (Figure 3). Melatonin reduces Ca 2 + overload in cardiomyocytes against H/R via ERK1 At 4h after reoxygenation, we investigated effect of melatonin on H/R-induced Ca 2+ overload in cardiomyocytes using the calcium-dependent fluorescent dye Fura-2. The results showed the fluorescence was stronger in H/R group than in control group, meanwhile the fluorescence was decreased inmelatonin group compared with H/R group, which indicated that H/R caused a marked increase of cytosolic Ca 2+ concentration and that melatonin pretreatment significantly inhibited H/R-induced increase of cytosolic Ca 2+ concentration which was reduced by PD98059 (Figure 4). Melatonin modulated expression of IP3R and SERCA2a in cardiomyocytes against H/R via ERK1 At 4h after reoxygenation, we investigated the effect of melatonin on expression of IP3R and SERCA2a in H9C2 by Western blot. The results indicated H/R increase expression of IP3R and reduce expression of SERCA, respectively. Pretreatment of melatonin inhibited expression of IP3R and induced expression of SERCA, which were reversed by PD98059. (Figure 5). Melatonin modulated expression of IP3R and SERCA2a via ERK1 pathway in reperfused rat hearts In vivo, we investigated the effect of melatonin on expression of IP3R and SERCA2a in reperfused rat hearts. IP3R expression was higher in I/R group compared with control group, and melatonin reversed the change. The results demonstrated expression of SERCA2a was lower in I/R group compared with control group, but expression of SERCA2a was higher in melatonin group than I/R group. The pretreament of PD98059 reduced the effect of melatonin on expression of IP3R and SERCA2a in rat hearts against I/R (Figure 6). Discussion Reperfusion-induced death of cardiomyocytes that were viable at the end of the ischemic event is defined as lethal myocardial reperfusion injury (reperfusion infarction). 1,3,27 The existence of lethal myocardial reperfusion injury has been inferred in both experimental MI models and in patients with STEMI(1). The major contributory factors for reperfusion- induced death of cardiomyocytes include oxidative stress, calcium overload, mitochondrial permeability transition pore (mPTP) opening, and hypercontracture. 28,29 Ca 2+ overload is one of the main actors of this lethal reperfusion injury, 30 which results in part from excessive sarco/endoplasmic reticulum (SR/ER) Ca 2+ release and Ca 2+ influx through the plasma membrane. 31 Although ryanodine receptors (RyRs) are the major cardiac SR/ER Ca 2+ -release channels involved in excitation–contraction coupling and ischemia–reperfusion injury, 32,33 some studies reported an increasing role for inositol 1,4,5-trisphosphate receptors (IP3R) Ca 2+ -release channels in the modulation of excitation–contraction coupling and cell death. 22,23 Gomez et al 34 indicated that inhibition of IP3R Ca 2+ channeling complex limited SR/ER Ca 2+ release and reduced both cytosolic and mitochondrial Ca 2+ overload and inhibited subsequent PTP opening. Meantime, the cardiac SERCA2a is a key pump responsible for intracellular calcium handling and contractility in cardiac cells. Impaired calcium reuptake resulting from decreased expression and activity of SERCA2a is a hallmark of HF. 20 IP3R and SERCA2a have been confirmed to play important roles in maintaining intracellular calcium homeostasis in cardiomyocytes. 20,22,23,35 Melatonin as one type of neuroendocrine hormone, is synthesizedprimarilybythepinealgland. 7,8 Previousstudiesshowed melatonin confers important protective effects against myocardial 47