The Role Of PI3K/Akt Signaling Pathway In Myocardial Ischemic Preconditioning In The Evolution Of Diabetic Rats

The role of PI3K/Akt signaling pathway in myocardial ischemic preconditioning in the evolution of diabetic ratsObjectiveThe protection effects of ischemic preconditioning were observed in non-diabetic myocardium. In contrast, relatively little is known whether preconditioning can afford protection to diabetic hearts as well and whether mechanisms of adaptation to ischemica are operating in diabetic myocardium. To investigate whether development of diabetes mellitus affects ischemic preconditioning in the rat heart, we have chosen a model of streptozotocin-induced diabetes(1 week and 9 weeks), the myocardial ischemia/reperfusion and ischemic preconditioning were induced by occlusion of the left anterior descending(LAD) coronary artery. To test the effect of phophatidylinositol-3-kinase(PI3K)/Akt signaling on diabetic myocardial ischemic preconditioning. To study whether insulin protects myocardial reperfusion injury and evaluate the role of the PI3K/Akt pathway.Methods1. The evolution of diabetic response to ischemic preconditioning in rat heartsFollowing 1 week and 9 weeks of streptozotocin-induced(45 mg/kg, i.v.)diabetic rats. All hearts were divided randomly into nine groups: non-diabetic sham group(CS group), non-diabetic ischemic/reperfuaion(I/R) group(CIR group), non-diabetic ischemic preconditioning(IPC) group(CIP group), 1-week diabetic sham group(1wS group), 1-week diabetic I/R group(1wIR group), 1-week diabetic IPC group(1wIP group), 9-week diabetic sham group(9wS group), 9-week diabetic I/R group(9wIR group)and 9-week diabetic IPC group(9wIP group). In sham group, there were made no procedures after surgery. In I/R group, the injury was induced by occlusion of the left anterior descending(LAD) coronary artery for 30 min followed by reperfusion for 60 min In IPC group, preconditioning consisted of three cycles of 5 min ischemia and 5 min reperfusion, prior to ischemia. At the end of the experiment, measured the serum CK, CK-MB, quantified the severity of arrhythmias by a scoring system, observed the ultrastrucural change of the myocardium, checked infarct size by TTC staining.2. The role of PI3K/Akt signaling pathway on ischemic preconditioning in the evolution of diabetic rat heartsAt the end of the experiment, the expressions of phosphor-Akt and phosphor-Bad, Bax, Bcl-2 were assessed by immunoblotting.3. Myocardial protection of insulin administration in diabetic ratsFollowing 9 weeks of streptozotocin-induced(45 mg/kg, i.v.)diabetes. All hearts were subjected to 30 min of myocardial ischemia followed by 60 min reperfusion. The rat were divided randomly into six groups: non-diabetic control group, non-diabetic insulini group, non-diabetic wortmannin group, diabetic control group, diabetic insulini group and diabetic wortmannin group. The rats were randomly treated with intravenous infusion of saline, insulin(60 U/L), or insulin+wortaminin(1ug/kg/h).Results1. Blood glucose level and relative heart weightAfter one and nine weeks of diabetes duration, blood glucose levels significantly increased 21.75 mmol/L±4.82 mmol/L and 27.76 mmol/L±4.68 mmol/L, as compared to 4.89 mmol/L±0.49 mmol/L in non-diabetic control group(P＜0.05, respectively). Relative heart weight was increased at 9 weeks of diabetes as compared to controls (3.44 mg/g±0.30 mg/g vs 2.54 mg/g±0.18 mg/g, P＜0.05) which was moderately increased only in the 1 week of diabetes (2.64 mg/g±0.11 mg/g vs 2.54 mg/g±0.18 mg/g, P＞0.05) .2. The effects of ischemic preconditioning in non-diabetic heartsCompared with that of CIR group, the elevation of plasma CK, CK-MB corresponding to reperfusion injury were reduced significantly (2441.1 U/L±280.3U/L vs 5367.0 U/L±357.4 U/L, 1407.4 U/L±222.0 U/L vs 3441.4 U/L±324.1U/L, P＜0.05, respectively). The severity of arrhythmias in the CIP group was decreased(from the score 4.25±0.89 tol.75±0.71, P＜0.05). And the myocardial infarct size were reduced significantly(31.0%±7.5% vs58.5%±8.3%, P＜0.05). The most significant ultrastructura changes were mitochondrial damage, which were more severe in CIR group. Compared with that of CIR group, the expression of phosphor-Akt and phosphor-Bad and Bcl-2 in CIP group were increased, whereas Bax expression decreased.3. The effects of ischemic preconditioning in 1-week diabetic heartsCompared with that of 1wIR group, the elevation of plasma CK, CK-MB corresponding to reperfusion injury were reduced significantly(2519.1 U/L±273.9 U/L vs 3366.1 U/L±385.4 U/L, 1503.4 U/L±276.9 U/L vs 1929.4 U/L±276.0 U/L, P＜0.05, respectively). The severity of arrhythmias were no significantly different between 1 wIP group and 1wIR group(the score 2.25±0.71 with 2.88±1.23, P＞0.05). And the myocardial infarct size were reduced significantly(36.8%±8.3% vs 45.8%±8.7%, P＜0.05). The most significant ultrastructura changes were mitochondrial damage, which were more severe in 1wIR group. Compared with that of 1wIR group, the expression of phosphor-Akt and phosphor-Bad and Bcl-2 in 1wIP group were increased, whereas Bax expression decreased.4. The effects of ischemic preconditioning in 9-week diabetic heartsCompared with that of 9wIR group, the elevation of plasma CK, CK-MB corresponding to reperfusion injury were significantly different(7682.8 U/L±361.1U/L vs 6772.2 U/I±129.2 U/L, 4710.2 U/L±327.4 U/L vs 3564.0 U/L±170.6 U/L, P＜0.05 respectively). The severity of arrhythmias were significantly different between 9wIP group and 9wIR group(the score 4.74±1.04 with 4.63±0.92, P＞0.05). And the myocardial infarct size were not reduced significantly(65.6%±7.4% vs 62.1%±8.8%, P＞0.05). The more severe significant ultrastructura changes were in 9wIP group. Compared with 9wIR group, the expression of phosphor-Akt and phosphor-Bad did increased in 9wIP group, while there were no significantly different in Bcl-2 and Bax.5. Compared ischemic preconditioning between 1-week diabetic heart and 9-week diabetic heartThe elevation of plasma CK, CK-MB corresponding to reperfusion injury in 9wIP were significantly higher than those of 1wIP group(7682.8 U/L±361.1 U/L vs 2519.1 U/L±273.9 U/L, 4710.2 U/L±327.4 U/L vs 1503.4 U/L±276.9 U/L, P＜0.05, respectively). The severity of arrhythmias in the 9wIP group was significantly higher than that of 1wIP group(from the score 4.74±1.04 to 2.25±0.71, P＜0.05). And the myocardial infarct size in 9wIP were higher significantly than that of 1wIP group (65.6%±7.4% vs 36.8%±8.3%, P＜0.05). The myocardial ultrastructura changes were more severe in 9wIP group. Compared with that of 1wIP group, the expression of phosphor-Akt and phosphor-Bad and Bcl-2 in 9wIP group were less, whereas Bax expression higher.6. Myocardial protection of insulin administration in non-diabetic ratsIn non-diabetic rats, compared with the I/R group, insulin administration significantly reduced the elevation of plasma CK, CK-MB((2652.3 U/L±243.2 U/L vs 5380.1 U/L±292.4 U/L, 1399.9 U/L±83.8 U/L vs 2720.3 U/L±160.8 U/L, P＜0.05, respectively). The severity of arrhythmias decreased in the insulin group compared with the I/R group(1.87±0.64 vs 3.88±1.13, P＜0.05). And reduced infarct size(32.7%±5.6% vs 44.8%±7.2%, P＜0.05). The myocardium ultrastructura changes were less in insulin group, phosphor-Akt and phosphor-Bad and Bcl-2 expression increased after insulin, whereas Bax expression decreased. These effects of insulin were inhibited by wortmannin.7. Myocardial protection of insulin administration in diabetic ratsIn diabetic group, compared with the I/R group, insulin administration significantly reduced the elevation of plasma CK, CK-MB(3363.0 U/L±192.7 U/L vs 6571.3 U/L±430.6 U/L, 1893.6 U/L±192.7 U/L vs 3463.5 U/L±249.1 U/L, P＜0.05, respectively). The severity of arrhythmias decreased in the insulin group compared with the I/R group(2.13±0.87 vs 4.25±1.28, P＜0.05). And reduced infarct size (41.8%±6.5%vs 55.3%±9.3%, P＜0.05). The myocardium ultrastructura changes were less in insulin group, phosphor-Akt and phosphor-Bad and Bcl-2 expression increased after insulin, whereas Bax expression decreased. These effects of insulin were inhibited by wortmannin.8. Compared myocardial protection of insulin administration between non-diabetic and diabetic ratsThe elevation of plasma CK, CK-MB in diabetics were significantly higher than non-diabetic groups(3363.0 U/L±192.7 U/L vs 2652.3 U/L±243.2 U/L, 1893.6 U/L±L192.7 U/L vs 1399.9 U/L±83.8 U/L, P＜0.05, respectively). The severity of arrhythmias was no significantly different between them(1.87±0.64 vs 2.13±0.87, P＞0.05). But infarct size of diabetic group was higher than non-diabetic group (41.8%±6.5% vs 32.7%±5.6%,＜<0.05).Conclusions(1)IPC could reduce the severity of reperfusion induced arrhythmias and myocardial infarct size, protect myocardial ultrastructure in non-diabetic rats. IPC in 1-week diabetcs afford the same myocardium protection as in non-diabetic rats. The protection effects of IPC were diminished in 9-week diabetic rat hearts.(2)PI3K/Akt pathway play a key role in mediating IPC in non-diabetics. Akt is activated by IPC as a result of PI3K activation, lead to modulate pro-protein(Bad) and antiapoptotic protein(Bcl-2).(3)With the evolution of the diabetics, there is possible impairment in Akt activation in diabetic rat myocardium These results suggest that different degree of Akt activation is involved in the mechanisms of IPC in different course of diabetics.(4)In non-diabetic rats, insulin administration during I/R can reduce myocardial infarction, is mediated via PI3K/Akt signaling pathway, which depend on activation of Akt and modulation of downstream prosurvival targets of Akt incluing pro-and antiapoptotic proteins.(5)In diabetic rats, insulin administration still protect reperfusion myocardium, but it is less than in non-diabetic rat.