Role Of MnSOD Nitration In Memory Effect Of Myocardial Injury Induced By High Glucose

ObjectiveWe use cultured cardiomyocytes of SD rat as a model, reserch the injury ofmyocardial cell in different concentrations of glucose, observe the memoryeffect of high glucose and relationship of memory effect and high glucoseduration, explore the role of MnSOD nitration in the "metabolic memory" effect.Methods1,Take the heart from newborn SD rats, isolate cells using trypsin andculture the cells. The cultured cardiomyocytes were divided into four groups:group A (glucose concentration of5.6mmol/L); group B (glucose concentrationof16.7mmol/L); group C (glucose concentration of25mmol/L); group D(glucose concentration of33.3mmol/L). The cell viability was measured by MTTand apoptosis was detected by flow cytometry, respectively, to observe the celldamage of different glucose concentrations,clear the best glucoseconcentrations to the myocardial cell damage.2,Know the optimal concentration of the cell injury, according to the cellculture days were divided into A, B, and C three groups, and then cultured indifferent high glucose days were divided into three subgroups: group A (A1: lowglucose5.6mmol/L x6d, A2: high glucose25mmol/L x2d+low glucose5.6mmol/L x4d, A3: The high glucose25mmol/L x6d), group B (B1: low glucose5.6mmol/L x8d, B2: high glucose25mmol/L x4d+low glucose5.6mmol/L x4d,B3: high glucose25mmol/L x8d), group C (C1: low glucose5.6mmol/L x10d,C2: high glucose25mmol/L x6d+low glucose5.6mmol/L x4d, C3: high glucose25mmol/L x10d). Detect ROS content of each group use Fluorescencespectrophotometer, observe the "metabolic memory",and clear the best durationof high glucose caused by the memory effect of myocardial cells.3,Clear the optimal glucose concentration and the best duration of highglucose,cultured myocardial cells is divided into four groups:continued lowglucose group (5.6mmol/L D-glucose×8d), B: sustained high glucose group(25mmol/L D-glucose×8d), C: high-sugar+sugar group (25mmol/L D-glucose×4d+5.6mmol/L D-glucose×4d), D:high glucose+low glucose+aminoguanidinegroup (25mmol/L D-glucose×4d+5.6mmol/L D-glucose×4d+0.5mmol/L aminoguanidine). Measured3-nitrotyrosine(3-NT) content byELISA and analyzed its expression in the cytoplasm by immunocytochemistry,detect the content of ROS by fluorescence spectrophotometer, xanthine oxidaseassay MnSOD activity, nitration of tyrosine residues in MnSOD was analyzed byimmunoprecipitation and Western blot.Results1,with group A (5.6mmol/L), myocardial cell’s viability and apoptosis rate ofgroup B (16.7mmol/L) were no significant changes, in group C (25mmol/L)and group D (33.3mmol/L), myocardial cellsactivity was reduced, the apoptosisrate increased (P <0.05); compared with group C, group D cell’s viability wasreduced, apoptosis rate increased (P <0.05), suggesting that the25mmol/Land33.3mmol/L glucose concentration cancells to produce injury, and33.3mmol/L glucose concentration on cell injury is more serious.2,with the group A1(low glucose5.6mmol/L x6d), ROS of group A2(highglucose25mmol/L x2d+low glucose5.6mmol/L×4d) had no significantchanges, group A3(high glucose25mmol/L x6d)(P <0.05) increased; with thegroup B1(low glucose5.6mmol/L x8d), ROS content in group B2(high glucose25mmol/L x4d+low glucose5.6mmol/L x4d) and group B3(high glucose25mmol/L x8d) were significantly increased (P <0.05); compare with group C1(low glucose5.6mmol/L x10d), ROS content in the C2group(high glucose25mmol/L x6d+low glucose5.6mmol/L×4d) and C3group (high glucose25mmol/L×10d) were significantly increased (P <0.05). The results showed thatgroup A did not produce the memory effect, group B and group C have asignificant memory effect, suggesting that the high sugar continued to4daysand6days of cell damage can produce memory effect.3,compare with group A (continuous low glucose),3-NT content in groupB(sustained high glucose group) and group C (high glucose+low glucose)increased (P <0.05), the level of expression in the cells increased,MnSODactivity decreased (P <0.05), ROS content increased (P <0.05). with the groupB(sustained high glucose group),3-NT contents in group C (high glucose+lowglucose) decreased (P <0.05), MnSOD activity increased and the nitrationlevels decreased (P <0.05), ROS contents have no significant changes.Compared with the group C (high glucose+low glucose),3-NT content in groupD(high glucose+low glucose+aminoguanidine) decreased, the level ofexpression in the cells significantly reduced, MnSOD activity increased andnitration levels decreased (P <0.05),ROS content decreased (P <0.05).Conclusion1,Myocardial cell injury could induced by high glucose in vitro, and thedegree related with the glucose concentration.2,“Metabolic memory” effect is existed in high glucose-induced myocardialinjury,and memory effect related with the duration of the first high glucose.3,Nitrification stress involved in “metabolic memory ", and the MnSODnitration may play an important role in the memory effect.