Effects Of All-trans Retinoic Acid On Myocardial Ischemia-reperfusion Injury

[Backgroud]Coronary heart disease is the leading cause of death and disability worldwide. Althoughreperfusion therapy has been widely used in clinical practice and greatly increasedoutcomes after myocardial infarction, it also brings so called ischemia-reperfusion injury(IR injury) which causes progressive myocardium apoptosis and enormously reduces theprotective effects of reperfusion therapy. It has been reported that infarcted area causedby IR injury could be counted up to50%of total and30day adverse cardiac outcomes(occurrence rate5%-6%) are related with IR injury. Since the mechanism of cardiac IRinjury is still unclear, and by far no prevention or interference is available, it has attractedbroad attention and is turning into a troublesome issue demanding prompt solution inthe basic research field of cardiology.All-trans retinoic acid (ATRA) is an active form of Vitamin A in human body. Multiplestudies have shown its antioxidant and anti-apoptosis effects. But these studies focus onhaematology and neurology, and few is available in the field of cardiology.[Objective]There are two objectives in the current study:1) To verify the effects of all-trans retinoic acid on myocardial ischemia-reperfusioninjury by isolated experiments.2) To investigate the mechanisms of ATRA in preventing myocardial ischemia-reperfusioninjury. [Methods]Part1. The effects of all-trans retinoic acid on myocardial ischemia-reperfusion injury:H9c2cells were pretreated with ATRA for24h and then subjected tohypoxia/re-oxygenation injury (6-hour hypoxia and1-hour re-oxygenation). The effect ofATRA on cell viability was measured using a WST-1assay, and cell apoptosis wasquantified by TUNEL assay and Annexin V-FITC based flow-cytometric analysis.Apoptosis-related bcl-2, activated caspase-3and mitogen-activated protein kinase(MAPKs) signaling was measured by Western blot.Part2. The mechanisms of ATRA in preventing myocardial ischemia-reperfusion injury:H9c2cells were pretreated with ATRA for24h and then subjected tohypoxia/re-oxygenation injury (6-hour hypoxia and1-hour re-oxygenation).Mitogen-activated protein kinase (MAPKs) signaling was measured by Western blot.Reactive oxygen species (ROS) production was measured by fluorescent probe.[Results]Part1: WST-1assay revealed greater cell viability with ATRA treatment (p <0.05). TUNELassay and flow-cytometric analysis demonstrated ATRA’s anti-apoptotic effect after IR inH9c2cells (p <0.05). Western blot revealed that ATRA pre-treatment increased Bcl-2expression, reduced the level of activated caspase-3compared to control cells (all p <0.05).Part2: Western blot revealed that ATRA pre-treatment reduced the phosphorylation ofp38, JNK, and ERK, compared to control cells (all p <0.05). ATRA pre-treatment decreasedproduction of ROS in H9c2cells after IR.[Conclusions]ATRA pre-treatment has protective effect on myocardial ischemia reperfusion injury.ATRA effectively reduced cardiomyocyte apoptosis after I/R in vitro. This anti-apoptoticeffect might be attributed to its anti-oxidant effects and its ability to down-regulate MAPKs signaling.