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 3 – Melatonin protects F-actin organization in H9C2 cells against H/R via ERK1 in vitro. Representative confocal microscopy images show H9C2 cells stained with FITC-phalloidin. The results showed that simulated H/R induced more diffuse and irregular actin disposition compared with control group. Melatonin preserved more regular and well-defined actin organization and PD98059 (ERK1 inhibitor) reduced the protection of melatonin. bar = 20μm. (Control: control group; H/R:H/R group; H/R+mel: H/R+ melatonin group; H/R+mel+PD: H/R+ melatonin+PD98059 group) Control H/R H/R+Mel H/R+Mel+PD Figure 4 – Melatonin reduces Ca 2+ overload in H9C2 cells against H/R via ERK1 in vitro. Ca 2+ content was assessed using Fura-2/AM in 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 green fluorescence intensity by Fura-2 was obviously stronger in H/R group, and melatonin pretreatment reversed the change which was inhibited by ERK1 inhibitor.bar = 30 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) Control H/R H/R+Mel H/R+Mel+PD Control H/R H/R+Mel H/R+Mel+PD A B 6 5 4 3 2 1 0 Intensity of Ca 2+ fluorescence (Relative fold) ** S # ischemia-reperfusion injury. 9-14 Melatonin administration showed to contribute to the rehabilitation of contractile function on isolated heart during reperfusion and to reduce the sensitivity of mPTP opening to Ca 2+ . 36 Melatonin has also demonstrated to play a role in the mitochondrial adaptive changes. 37 Melatonin and its metabolites efficiently interact with various ROS and reactive nitrogen species, and additionally they up regulate antioxidant enzymes and downregulate pro-oxidant enzymes. 9,15,16 Previous studies confirmed that melatonin pretreatment attenuated IR injury by reducing oxidative damage and inhibiting mPTP opening. However, the evidence about melatonin’s effect and underlying mechanism on Ca 2+ overload under acute ischemia/reperfusion is rare. The present study demonstrated that melatonin performs cardioprotection through modulation of IP3R and SERCA2a to maintain calcium homeostasis via ERK1 pathway in cardiomyocytes. ERK1 pathway has been shown to have anti‑apoptotic effect during the process of reperfusion injury. 24,25 It is not clear if melatonin maintains calcium homeostasis through modulating IP3R and SERCA2a via ERK1. In the present study, the results showed that melatonin promote phosphorylation of ERK1 in cardiomyocytes against H/R, and pretreatment of PD98059 (ERK1 inhibitor) reduced phosphorylation of ERK1. In vitro results indicated melatonin prevents cardiomyocytes apoptosis against H/R. Meantime, melatonin can preserve structure of cardiomyocytes against reperfusion injury. Moreover, calcium overload induced by H/R is significantly reversed by melatonin. Moreover, the pretreatment of PD98059 inhibited the effect of melatonin 48