In the present study we demonstrated that Kir6.2 knockout abolished the protection of resveratrol against myocardial ischemia/reperfusion injury in vivo and in vitro. Resveratrol enhanced the activity of AMPK and promoted the association of AMPK with Kir6.2. Suppression of AMPK attenuated the cardioprotection of resveratrol whereas activation of AMPK mimicked protective effects of resveratrol. In addition, Kir6.2 knockout reversed the cardioprotective effects of AMPK activation. Thus, Kir6.2/K-ATP channel is required for resveratrol-mediated protection against myocardial ischemia/reperfusion injury and AMPK-Kir6.2/K-ATP channel signal pathway mediates cardioprotection of resveratrol (Figure 5C).
K-ATP channel is a kind of molecular sensors that plays a critical role in cardiovascular adaptive response under the challenge of stress. The cardiac K-ATP channels are composed of Kir6.2 subunits in combination with SUR2A, while Kir6.1 subunit is a major molecular component of vascular smooth muscle K-ATP channels. Opening K-ATP channels blocks cardiomyocyte apoptosis in ischemia-reperfusion. However, the specific roles of Kir6.1 and Kir6.2 in myocardial protection remain unclear. As Kir6.1 knockout mice could not endure I/R injury (the mortality rate within 24 hours after I/R injury was 100%), Kir6.1+/- mice and Kir6.2 knockout mice were used to subject to I/R injury in the present study. We found that the infarct size was much larger in Kir6.1+/- mice compared to WT mice following myocardial I/R injury. But, no significant difference was detected in isolated cardiomyocyte apoptosis after OGD/R treatment between WT and Kir6.1+/- mice. Kir6.1/K-ATP channel has been demonstrated to be critical in the regulation of vascular tone, especially in the coronary arteries . As a result, more severe damage in Kir6.1+/- mice induced by I/R injury might be due to vasodilatation disorder, instead of cardiomyocyte dysfunction. Furthermore, there were no significant differences in infarct size between the WT and Kir6.2-/- mice after I/R injury, which was consistent with the report of Masashi Suzuki.
Recent studies suggest that resveratrol enhances activity of the K-ATP channels in rat hearts and activation of the K-ATP channel is necessary for resveratrol action. As opposed, the experiments on the mouse beta cell line revealed that resveratrol blocked K-ATP channels by directly binding to sulfonylurea receptor 1 (SUR1) of K-ATP channel. This discrepancy in the results obtained on the various experimental models strongly suggests tissue and species specificity of resveratrol. In addition, pancreatic β-cell K-ATP channels are thought to be composed of Kir6.2 and SUR1 subunits, and it is plausible that SUR1 may not be primarily expressed in ventricular myocytes. Resveratrol is specific to SUR1 subunits, and thus it causes opening of Kir6.2 SUR2A K-ATP channel in cardiac tissues but blocks Kir6.2 SUR1 K-ATP channel in pancreatic tissues. It is not immediately clear how to reconcile these different outcomes, and further work may be needed to determine whether there is a differential mechanism involved.
Emerging evidence indicates that resveratrol protects cardiomyocytes from I/R injury via a combination of suppression of superoxide levels and activation of potassium channels. However, the relation of K-ATP channel and resveratrol in I/R injury remains unknown. Thus, the aim of this study was designed to assess the roles of Kir6.2 or Kir6.1–containing K-ATP channel in cardioprotection of resveratrol under I/R injury. Our work found that resveratrol reduced the infarct size and decreased the activity of serum LDH and CK following I/R injury, and Kir6.2 knockout abolished the cardioprotective effects of resveratrol in vivo. In addition, Kir6.2 knockout also reversed the suppressive effects of resveratrol on cardiomyocyte apoptosis after OGD/R treatment in vitro. But, Kir6.1 knockdown failed to abolish the cardioprotection of resveratrol. These findings indicate that Kir6.2/K-ATP channel is required for resveratrol-mediated cardioprotection.
It is well known that the most prominent role of K-ATP channels in cardiovascular system is that opening of this channel can protect cardiac myocytes against ischemic injuries. K-ATP channels are present in multiple tissues and cell types within the cardiovascular system and these channels differ from each other in terms of their biophysical and pharmacological properties. The properties of K-ATP channels are different in various tissues due to the combinations of the subunits forming the channel. The “cardiac” K-ATP channel has conventionally been thought to consist of Kir6.2/SUR2A subunits. Although SUR1 subunits are not essential subunits of ventricular sarcolemmal K-ATP channels, the evidence is strong that SUR1 subunits are also expressed in the heart (particularly in atria) and that they may have a functional role. In contrast to Kir6.2-null and SUR2-null mice, SUR1-null mice are protected from ischemia/reperfusion. One interpretation is that SUR1 subunits might actually contribute to ischemic or post-ischemic damage. SUR1 levels are up-regulated after ischemic events; even in tissues where it is not normally expressed, such as the endothelium. Recent data suggest that SUR1 subunits associate with TRPM4 subunits, which may modulate the membrane potential and ionic gradients, thus contributing to post-ischemic injury. It was recently demonstrated that resveratrol is a natural SUR1 ligand that can induce apoptosis in a SUR1 isoform-specific manner. One might predict that resveratrol reduces infarct size after ischemia/reperfusion by blocking the cardiovascular SUR1-containing channels.
Resveratrol provides cardioprotection by triggering different endogenous signaling pathways including oxidative stress/antioxidant defense system, glucose/insulin metabolism, iNOS/nitrotyrosine, and preconditioning, which are associated with energy metabolism[34–36]. AMPK is an energy sensor protein that is activated in response to ATP depletion. AMPK activation plays a critical roles in ischemia/reperfusion injury in the heart[38, 39]. A recent report demonstrated that resveratrol protected ROS-induced cell death by activating AMPK in the H9c2 cardiac muscle cell line. In the present study, resveratrol enhanced AMPK activation in ischemic heart. Blockade of AMPK activity abolished the suppressive effects of resveratrol on cardiomyocyte apoptosis. Moreover, activation of AMPK mimicked protective effect of resveratrol on survival of cardiomyocytes. Taken together, these data indicate that resveratrol can exert beneficial actions on the cardiovascular systems partly through the AMPK-dependent mechanism. Furthermore, recent study has showed that AMPK mediates preconditioning in cardiomyocytes by regulating the activity and recruitment of sarcolemmal K-ATP channels, but the exact mechanism is still unclear. In our study, we found that AMPK interacted with Kir6.2 and resveratrol increased the association of Kir6.2 with AMPK. This interaction may trigger and promote Kir6.2/K-ATP channel opening, which mediates cardioprotection of resveratrol. Notably, Kir6.2 knockout abolished the cardioprotection of AMPK activation, indicating that Kir6.2/K-ATP channel might be downstream of AMPK. Thus, resveratrol enhances the AMPK phosphorylation, which in turn increases the association of AMPK with Kir6.2, and then promotes Kir6.2/K-ATP channel opening, subsequently protects against I/R injury.