Role of mito-KATP channels in sevoflurane postconditioning-induced inhibition of oxygen-glucose deprivation and restoration-induced pyroptosis in primary rat cardiomyocytes

Abstract

Objective To evaluate the role of mitochondrial ATP-sensitive potassium (mito-KATP) channels in sevoflurane postconditioning-induced inhibition of oxygen-glucose deprivation and restoration (OGD/R)-induced pyroptosis in primary rat cardiomyocytes. Methods Cardiomyocytes of newborn Sprague-Dawley rats (<48 h after birth) were cultured in vitro and seeded in 6-well dishes (2 cm in diameter) or in 96-well plates.The cells were divided into 6 groups (n=15 each) using a random number table: control group (group C), OGD/R group (group O), sevoflurane postconditioning group (group Sev), sevoflurane postconditioning plus 5-hydroxydecanoate (5-HD) group (group SH), 5-HD group (group H) and OGD/R plus 5-HD group (group HO). The cardiomyocytes were subjected to oxygen-glucose deprivation for 4 h followed by restoration of oxygen-glucose supply for 24 h. After oxygen-glucose restoration, the cardiomyocytes in the culture media were exposed to 2% sevoflurane for 1 h to perform sevoflurane postconditioning.At 1 h before oxygen-glucose deprivation, a specific mito-KATP channel blocker 5-HD 100 μmol/L was added to the culture media.Cardiomyocytes were cultured in normal culture atmosphere in group C. Cardiomyocytes were collected at 24 h of oxygen-glucose restoration.Cell pyroptosis was detected by double flow cytometry AlexaFour488 (caspase-1 FLICA staining) and TMR red (DNA staining) staining.The pyroptosis rate was calculated.The cell survival rate was measured by methyl thiazolyl tetrazolium assay.The content of reactive oxygen species (ROS) in mitochondria was determined by 2′, 7′-dichlorofluorescin diacetate assay.The mitochondrial membrane potential (MMP) was measured by using JC-1 fluorescent probe.The expression of interleukin-1beta (IL-1β) was determined by Western blot. Results Compared with group C, the pyroptosis rate and ROS content were significantly increased, the cell survival rate and MMP were decreased, and the expression of IL-1β was up-regulated in group O (P<0.05). Compared with group O, the pyroptosis rate and ROS content were significantly decreased, the cell survival rate and MMP were increased, and the expression of IL-1β was down-regulated in group Sev (P<0.05). Compared with group Sev, the pyroptosis rate and ROS content were significantly increased, the cell survival rate and MMP were decreased, and the expression of IL-1β was up-regulated in group SH (P<0.05). Compared with group SH, the pyroptosis rate and ROS content were significantly increased, the cell survival rate and MMP were decreased, and the expression of IL-1β was up-regulated in group HO (P<0.05). Conclusion The mechanism by which sevoflurane postconditioning inhibits OGD/R-induced pyroptosis in primary rat cardiomyocytes is probably associated with increasing mito-KATP channel opening. Key words: Anesthetics, inhalation; Anoxia; Myocytes, cardiac; Cell death; Postconditioning