Cardioprotective effects of melatonin against myocardial ischaemia/reperfusion injury: Activation of AMPK/Nrf2 pathway

Abstract Although reperfusion is the most effective therapy for patients with acute myocardial infarction, reperfusion injury limits the therapeutic effects of early reperfusion. Oxidative stress plays a crucial role in myocardial ischaemia/reperfusion (I/R) injury. Melatonin, a circulating hormone, is well‐known as an antioxidant in cardiovascular diseases. In this short communication, we show that melatonin significantly improves post‐ischaemic cardiac function, reduces infarct size and decreases oxidative stress. Furthermore, melatonin markedly increases AMPK activation and Nrf2 nuclear translocation. Nevertheless, these melatonin‐induced changes are abrogated by compound C. In addition, ML‐385, an Nrf2 inhibitor, also withdraws the antioxidative effects of melatonin but has little effect on AMPK activation. In conclusion, our results demonstrate that melatonin alleviates myocardial I/R injury by inhibiting oxidative stress via the AMPK/Nrf2 signalling pathway.


| INTRODUC TI ON
Ischaemic heart disease is the leading cause of morbidity and mortality in the United States and other parts of the world. 1 Reperfusion is the major treatment of choice for preserving left ventricular function and limiting myocardial infarct size. 2,3 However, the process of restoring coronary blood flow to the ischaemic cardiac tissue can, in itself, induce myocardial injury and cardiomyocyte death, a phenomenon termed 'ischaemia/reperfusion (I/R) injury. 4,5 Oxidative stress is considered to be the key pathogenic mechanism of myocardial I/R injury. 6,7 Therefore, illustrating the molecular mechanisms that could inhibit oxidative stress may be useful to prevent myocardial I/R injury.
Adenosine monophosphate-activated protein kinase (AMPK) has been identified as a pivotal energy sensor and regulator of cellular metabolism that has an important role in the regulation of energy homoeostasis under normal and ischaemic conditions. 8,9 Nuclear factor erythroid 2-related factor (Nrf2) is an important transcription factor that translocates into the nucleus and controls the expression of many target genes. 10 Melatonin (Mel) is well-known for its antioxidant capacity by scavenging free radicals and activating the antioxidant enzymes in a variety of cardiovascular diseases. The present study was aimed to determine the protective actions of Mel against myocardial I/R injury and elaborate whether AMPK/Nrf2 signalling is associated with Mel-induced cardioprotective effects.

| Myocardial I/R protocol
Male C57BL/6 mice (8 weeks old, 20-25 g body weight), were provided by the Laboratory Animal Center at AIR Force Medical University. The myocardial I/R injury model was established as described previously. 11 Briefly, mice were anaesthetized and ventilated via tracheal intubation with a Harvard rodent respirator. The left anterior descending coronary artery was ligated by placing a 6-0 silk suture and making a slip knot. Myocardial I/R injury was inflicted by ischaemia for 30 minutes, following by reperfusion for 2 hours or 24 hours. Mel (20 mg/kg) was intraperitoneally injected 10 minutes prior to reperfusion. Compound C (0.25 mg/kg) and ML-385 (30 mg/ kg) were intraperitoneally injected 15 minutes prior to reperfusion.

| Echocardiographic measurements
As described in a previous study, 12

| Measurements of myocardial infarct sizes
As described previously, 11 at the end of reperfusion, the left anterior descending coronary artery was reoccluded, and 2% Evans Blue dye was injected into the left ventricular cavity. Then, the hearts were quickly frozen at −80°C for 1 hours and sectioned horizontally into six slices. The slices were incubated in 1% TTC at 37°C for 15 minutes in the dark and subsequently fixed in 4% paraformaldehyde overnight. Myocardial infarct size was determined using Image-Pro Plus software (Media Cybernetics).

| Determination of SOD activity and MDA content
The activity of SOD and the content of MDA in cardiac tissues were determined using ELISA kits from the Institute of Nanjing Jiancheng Bioengineering Institute.

| Western blot analysis
Left ventricular tissue lysates were prepared using RIPA lysis buffer (Thermo Fisher Scientific) containing 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail. Denatured samples were subjected to SDS-polyacrylamide gels and transferred onto PVDF membranes. The membranes were incubated with primary antibodies against phospho-AMPK (Abcam), AMPK (Abcam), Nrf2 (Abcam), histone H3 (Cell Signaling Technology) and GAPDH (Cell Signaling Technology), followed by horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature, as described previously.

| Statistical analysis
All data were expressed as mean ± SEM. The statistical significance of differences was determined by the Student t test between two groups or one-way ANOVA, followed by Bonferroni's multiplecomparison post hoc t test. A value of P < .05 was considered to be statistically significant.

| Compound C abolished Mel-induced alleviation of infarct size, cardiac function and AMPK/ Nrf2 pathway during myocardial I/R
As revealed in Figure 1, I/R treatment dramatically increased infarct size and decreased LVEF and LVFS. Moreover, I/R treatment could significantly reduce the protein level of intranuclear Nrf2 but had little effect on the protein levels of p-AMPK and cytoplasmic Nrf2.
In particular, compound C could significantly obstruct the cardioprotective effects and activation of the AMPK/Nrf2 pathway of Mel in myocardial I/R.

| ML-385 attenuated antioxidative effects and Nrf2 nuclear translocation but had little effect on AMPK activation in Mel-treated myocardial I/R
As shown in Figure 2

| D ISCUSS I ON
In the present study, we further demonstrated that the cardiopro-

CO N FLI C T S O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used in this study are available from the corresponding author upon request.