Kir6.2/K-ATP Channel Is Required For Resveratrol-mediated Protection Against Myocardium Ischemia/reperfusion Injury

Actue myocardial infarction (AMI) is the leading cause of death of coronary artery disease. The cause of formation is the interruption of blood supply to a part of the heart, causing heart cells to die. AMI is a medical emergency which requires immediate attention. Treatment attemps to salvage as much myocardium as possible and to prevent further complications, thus the phrase"time is muscle". However, the restoration of blood flow to ischemia tissues causes additional damage, referred to as ischemia/reperfusion (I/R) injury. Over the past two decades, a great body of studies have shown that ROS, intracellular Ca+ overload and endothelial injury play a crucial role in I/R injury. Thus, the key of the treatment is not only how to ensure the early resumption of blood flow in ischemic tissue, but also reduce or even lift-reperfusion injury.Resveratrol (3, 4′, 5-trihydroxy-trans-stilbene, Res) is a polyphenol phytoalexin found in a variety of fruits, vegetables, and grape skins. Resveratrol has many pharmacological effects, such as anti-atherosclerosis, anti-inflammation, anti-oxidant, and anti-cancer properties. Numerous studies showed that resveratrol pretreatment could reduced myocardial I/R injury, such as significantly reduced myocardial infarct size and improved the recovery of ventricular function after I/R injury.ATP sensitive potassium (K-ATP) channels, which link cell metabolic state to excitability, consist of discrete pore-forming and regulatory subunits and are activated by a decrease in ATP/ADP radio. K-ATP channels are expressed at a high level in cardiomyocytes and play the role of metabolic sensor. Both sarcolemmal (sarcK-ATP) and mitochondrial membrane (mitoK-ATP) have been suggested to play an important role in cardioprotection. Recent studys showed ischemic- and diazoxide-induced preconditioning are both abolished in hearts from Kir6.2 knockout mice, and resveratrol-mediated cardioprotection is achieved through the preconditioning effect, rather than direct protection. However, the role of Kir6.1/K-ATP or Kir6.2/K-ATP channels in resveratrol-mediated cardioprotection remains elusive.Aims: To investigate the role of Kir6.1/K-ATP and Kir6.2/K-ATP channel in resveratrol mediated protective effect against myocardium I/R injury.Methods : (1) Wide type (WT), Kir6.2 knockout (Kir6.2-/-) and Kir6.1 heterozygote (Kir6.1+/-) mice were subjected to left anterior descending artery ligation followed by reperfusion. Evans blue/TTC double staining were performed to evaluate the infract size; the activity of actate dehydrogenase (LDH) and creatine kinase (CK) in serum were also measured to determine the impairment of each mouse. (2) The myocardiocyte cultures were prepared from left ventricle tissues of embryonic day 19 WT, Kir6.2-/- and Kir6.1+/- mice, and cultures were used after 4 days in vitro. Staining with Hochest 33324 was used to determine the apopototic of myocytes, and assary of LDH released in the media was used to determine the injury of myocytes. (3) Western blot was taken for analyses of the protein levels of Kir6.1, Kir6.2, conserved dopamine neurotrophic factor (CDNF), AMP-activated protein kinase (AMPK) and p-AMPK; (4) Real-time qPCR was performed to analyses of the mRNA of silent information regulator 1 (SIRT1).Results: (1) Western blot analyses showed pretreatment with resveratrol increased the level of Kir6.2 protein, but had no effect on Kir6.1 protein;(2) Administration of resveratrol diminished infarct size, and inhibited the activity of CK and LDH in WT and Kir6.1+/- mice, but not in Kir6.2-/- mice; (3) In cultured cardiac cells, resveratrol also inhibited oxygen-glucose deprivation (OGD) induced apoptosis and LDH leakage. However, Kir6.2 knockout abolished the protective effects of resveratrol; (4) Kir6.2 knockout increased the phosphorylated AMPK; pretreatment with resveratrol significantly increased the levels of AMPK phosphorylation in WT hearts; Kir6.2 deficiency abolished the protective effect of AMPK agonist 5-aminoimidazde-4-carboxamide (AICAR) against OGD injury, and AMPK inhibitor Compound C (CC) suppressed the protection of resveratrol; (5) Pretreatment with resveratrol significantly increased the mRNA levels of SIRT1, and SIRT1 inhibitor Nicotinamide (NAM) suppressed the protection of resveratrol.Conclusion:(1) Kir6.2/K-ATP channel not Kir6.1/K-ATP channel is required for resveratrol-mediated protection against myocardium ischemia/reperfusion injury in mice; (2) Kir6.2 is required for resveratrol-mediated protective effect against myocardium I/R injury via AMPK/SIRT1 signaling pathway; (3) Kir6.1/K-ATP channel is involved in protective effect against I/R injury in mice.The major contributions of the present study lie in:Kir6.2-/- and Kir6.1+/- mice have been used to investigate the effects of Kir6.2/K-ATP channel on resveratrol induced cardiopretection, which helps to obtain direct evidence that Kir6.2 is required for resveratrol-mediated protective effect against myocardium I/R injury via AMPK/SIRT1 signaling pathway.