Effects Of Acute Hyperglycemia On Myocardial Ischemia/reperfusion Injury And Cardioprotection Of GIK And Its Underlying Mechanism

BackgroundAn association between hyperglycemia and an increased risk of mortality and poor prognosis after acute myocardial infarction (AMI)was well noted not only in patients with diabetes but also in patients without diabetes. Recent clinical observations have demonstrated that acute hyperglycemia is highly harmful to multiple organs, including heart, in critically ill patients. Mechanisms for the association between stress hyperglycemia and adverse outcomes after AMI in non-diabetic patients are not well understood. Direct evidence to support a causative role of acute hyperglycemia in exacerbating myocardial ischemia/reperfusion (I/R) injury is not currently available and the underlying mechanisms responsible for the effect remain unidentified.Glucose-insulin-potassium (GIK) has been applied for AMI as an assistant therapy for more than 40 years. We have previously reported that administration of GIK exerts significant cardioprotective effects in myocardial ischemia/reperfusion and that insulin is largely responsible for the observed beneficial effects. But the results of some GIK clinical trials are very different and controversial. Some studies such as ECLA and DIGAMI suggest a beneficial effect of GIK in AMI patients with and without diabetes. However, some clinical trials of GIK (DIGAMI 2, CREATE-ECLA) showed no treatment benefits in the presence of hyperglycemia. It is possible that hyperglycemia obscure the protection effect afforded by GIK and the underlying mechanisms responsible for the effect remain unclear.Recently, it was demonstrated that hyperglycemia can induce oxidative stress, increase the generation of free radical and proinflammatory cytokine, further impair activation of Akt and increase apoptosis in cultured cadiocytes. Studies have shown that activation of the PI3K/Akt pathway was impaired in diabetic animals. But it is not clear if acute hyperglycemia impairs the activation of Akt induced by insulin in ischemia/reperfusion myocardium in vivo. The study was designed to investigate whether acute hyperglycemia may aggravate myocardial ischemia/reperfusion injury and whether acute hyperglycemia during the ischemic phase may impair GIK-mediated cardioprotection against ischemia/reperfusion injury.Objective1. To determine whether acute hyperglycemia during ischemia exacerbates myocardial ischemia/reperfusion injury in vivo.2. To evaluate the hypothesis that acute hyperglycemia blocks GIK-induced myocardial protection in ischemia/reperfusion heart.3. To investigate the mechanisms involved in the adverse effect of acute hyperglycemia on ischemic heart and on GIK.Methods1. Rats were anesthetized and myocardial ischemia was produced by exteriorizing the heart through a left thoracic incision and placing a 6-0 silk and making a slipknot around the left anterior descending coronary artery. After 30 minutes of ischemia, the slipknot was released and the myocardium was reperfused for 4 hours or 6 hours.2. A micro-catheter was inserted into left ventricular through right carotid artery to measure the left ventricular pressure. Hemodynamic data were continuously monitored on a polygraph (RM-6200C) and simultaneously digitized by using a computer interfaced with an analog-to-digital converter.3. Rats were randomized to receive one of the following treatments: (1) Sham; (2)Vehicle; (3) HG1; (4) HG2; (5) GIK; (6) GIK plus HG; (7) GIK plus wortmannin; (8) GIK plus HG and wortmannin. 4. Blood samples were drawn from caudal vein before ischemia, 30 minutes after ischemia, 2 hours and 4 hours after reperfusion respectively to measure blood glucose and plasma insulin levels.5. Plasma creatine kinase (CK) and lactate dehydrogenase (LDH) activities were measured spectrophotometrically (Beckman DU 640, USA) at 4 hours after reperfusion.6. Myocardial apoptotic index was analyzed by TUNEL assay. A double-staining technique was used, i.e., TUNEL staining for apoptotic cell nuclei and DAPI staining for all myocardial cell nuclei.7. The myocardial infarct size was determined by means of a double-staining technique and was analyzed by a digital imaging systems. Evan's blue stained area, TTC stained area and TTC stained negative area were measured digitally using Image Pro Plus software.8. Akt expression and activation (total Akt,total GSK-3βand pAkt,pGSK-3β) were determined by western blotting.Results1. Blood glucose and plasma insulin concentrations did not change in the vehicle group. Blood glucose level increased during administration of supplemental intravenous HG. Plasma insulin concentrations increased during administration of HG and GIK.2.±LVdP/dtmax increased in GIK group and decreased in HG group compared with vehicle group after 2 h and 4 h of reperfusion. At 4 hours after reperfusion,±LVdP/dtmax decreased by 11.4 % and 10.1 % respectively in GIK plus HG group compared with GIK group (P < 0.05).3. There were no significant differences in AAR/LV among all groups. GIK significantly decreased IS while HG significantly increased IS (P < 0.05). Moreover, administration of high glucose (HG) or wortmannin together with GIK completely abolished the protective effect of GIK (P<0.01). Interestingly, there was no significant difference in IS between HG plus GIK and HG plus wortmannin groups. More importantly, HG could not further exacerbate the inhibiting effects of wortmannin on the GIK-induced infarct size reduction.4. At 4 hours after reperfusion, CK and LDH activities were reduced in rats treated with GIK (P <0.01 vs. vehicle group), and increased in rats treated with HG (P <0.05 vs. vehicle group). Interestingly, GIK plus HG treatment increased the reperfusion associated CK and LDH elevations by 27.3% and 25.1% compared with those in rats treated with GIK alone (both P < 0.05).5. GIK during reperfusion exerted a significant antiapoptotic effect by reduced TUNEL-positive staining. HG exacerbateed myocardial apoptotic death as evidenced by increased TUNEL-positive staining. TUNEL-positive staining myocytes increased in GIK plus HG or wortmannin and the antiapoptotic effect of GIK were abolished. More importantly, HG could not further exacerbate the inhibiting effects of wortmannin on the GIK-induced apoptosis reduction.6. GIK exerts cardioprotective effect via activation of Akt in a PI3Kinase-dependent fashion in the I/R myocardium in vivo. Treatment with GIK resulted in a 1.7-fold increase in Akt phosphorylation compared with vehicle (P < 0.01), while Akt phosphorylation was decreased in HG group (0.5-fold of vehicle, P < 0.05 vs. vehicle). Akt phosphorylation induced by insulin were remarkably suppressed when infusion of HG during ischemia or pretreatment with wortmannin before reperfusion. More importantly, HG could not further aggravate the inhibiting effects of wortmannin on the GIK-induced Akt activation.7. Consistent with the levels of pAkt, GIK and HG resulted in 2.3-fold increase and 0.6-fold decrease in GSK-3βphosphorylation in myocardium in I/R rats. When I/R rats were infused with HG during ischemia or wortmannin before reperfusion, insulin-induced GSK-3βphosphorylation were remarkably suppressed (P < 0.05 vs. GIK group). There was no significant difference in IS between HG plus GIK and HG plus wortmannin groups. More importantly, HG could not further exacerbate the inhibiting effects of wortmannin on the GIK-induced GSK-3βphosphorylation.There was no difference in total GSK-3βin all groups.Conclusions1. Hyperglycemia during ischemia significantly exacerbates myocardial ischemia/reperfusion injury and almost completely blocks the cardioprotective effect afforded by GIK, as evidenced by significantly enlarged infarct size, increased myocardial apoptosis, and worse cardiac function following myocardial I/R.2. Hyperglycemia decreased basal and GIK-induced Akt phosphorylations. At least in part, hyperglycemia-induced decrease of myocardial Akt activation induced by GIK is a likely mechanism that accounts for the findings that hyperglycemia blunts cardioprotection effect afforded by GIK.