In this study, using in vivo ischemia reperfusion model incorperating with the application of different durations of ischemia and reperfusion in the same disease model, we demonstrated that the diabetic hearts, even at the early stage of the disease, are more sensitive to IRI than hearts from non-diabetic subjects. Furthermore, we showed that the severity of the post-ischemic myocardial injury of the diabetic hearts depended more on the duration of ischemia than that of reperfusion. The severity of oxidative stress and reduction of endogenous antioxidant capacity and the impairment of protective signaling pathway related to the activation of STAT3 and Akt are likely the major mechanisms responsible for the increased post-ischemic myocardial injury in diabetes after prolonged ischemic insult. To our knowledge, this is the first systematic study to investigate the mutual impact of duration of ischemia and reperfusion on myocardial tolerance to IRI in diabetes, particularly, in the early phase of diabetes. Our findings may help to resolve the discrepancy regarding whether or not hearts from diabetes at the early stage of the disease are less resistant to ischemic insult.
Although clinical studies have convincingly demonstrated that the diabetic heart is more vulnerable to IRI, results from experimental studies are still inconsistent, especially at the early stage of the disease. Smaller infarct size was observed after 30 min ischemia in diabetic rat hearts at 1 week after STZ injection than in controls, but this infarct size limiting effect was disappeared 8 weeks later . Similarly, Ma et al.  reported that the diabetic hearts were more sensitive to IRI only at the late phase of the diabetes. However, enlargement of infarct size was observed as early as 8 days in STZ-induced diabetes . Thus, despite of the consensus that hearts from chronic diabetic subjects are more sensitive to IRI than hearts from controls, results obtained from studies conducted at the early stage of diabetes are mostly inconsistent. Of note, there are numerous differences in experimental preparations and protocols in the reported studies, and a single factor cannot entirely explain the discrepancy in the effects of diabetes on the sensitivity of the heart to ischemia reperfusion injury. Therefore, in the present study, we applied different duration of ischemia and reperfusion in the same diabetic model to examine whether or not the diabetic heart is more vulnerable to IRI at its early stage. Studies in non-diabetes subjects have shown that a sigmoidal relationship exists between myocardial infarct size and duration of ischemia, where, 20 min of ischemia only induced extremely small infarct size [23, 24], and significant impact of the duration of reperfusion only observed when ischemia time was shorter than 60 min in non-diabetes . However, our previous study showed that myocardial infarct size in rats at 4 weeks of STZ-induced diabetes did not significantly differ from that in the non-diabetic rats when the rats were subjected to 30 min ischemia followed by 2 hours of reperfusion . Therefore, in the current study we applied different durations of ischemia (30 min and 45 min) followed by either 2 hours or longer duration (3 hours) of reperfusion to see whether or not diabetic rats were more sensitive to IRI than non-diabetc rats. We found that enlargement of infarct size was only detectable when the diabetic rats were subjected to a longer enough (45 min) duration of ischemia with either 2 or 3 hours reperfusion, wherever the release of plasma CK-MB and plasma LDH were significantly increased in diabetic rats relative to controls after either 30 or 45 min of ischemia with 2 or 3 hours reperfusion. These indicate that myocardial cellular injury is more severe in diabetic than that in the control rats during reperfusion, despite that significant difference of infarct size was only observed when rats subjected to 45 min of ischemia with 2 or 3 hours reperfusion. Infarct size is considered as the gold standard in assessing the severity of heart damage after ischemia reperfusion, while CK-MB is regarded as the diagnostic hallmark for assessing post-ischemic myocardial infarction in patients after acute myocardial ischemia [25, 26]. Reports from different studies showed that these two reliable parameters, infarct size and CK-MB, not always reach their peak values at the same time. In patients with acute myocardial ischemia, plasma CK-MB reaches its peak level at about ten hours after ischemia and predicts well with myocardial infarct size which reaches its peak level at five to seven days after reperfusion with thrombolytic therapy . Similar findings have also been reported in a rat model, which showed that the increase in post-ischemic CK-MB peaked minutes after reperfusion while significant myocardial infarct size did not become apparent until after one hour of reperfusion . In the present study, significant higher post-reperfusion CK-MB levels and LDH levels (a marker of cell necrosis) in diabetic rats correlated with more severely impaired post-reperfusion left ventricular function and increased numbers of apoptotic cardiomyocytes as compared to controls. In the current study, significant difference in post-reperfusion infarct size between diabetic and control rats were only detected when the rats were subjected to 45 minutes of ischemia with 2 or 3 hours of reperfusion but not after 30 min of ischemia followed by 2 hours reperfusion despite of the significant increase in CK-MB level and LDH levels. This indicates that the reperfusion time of 180 minutes may not be long enough to catch significant increase in infarct size in the diabetic group when the duration of ischemia was set at 30 min. In other words, enlargement of infarct size depended more on the duration of ischemia but not (or to a less degree) on that of reperfusion. In line with our present results, studies in dogs  and patients  showed that myocardial necrosis is time dependent and ischemia time serves as a determinant of infarct size and subsequent left ventricular dysfunction in individual patients with myocardial infarction. Significantly higher levels of CK-MB and LDH as well as more severe impairment in post-ischemic left ventricular function and increased apoptotic cardiomyocytes in the diabetic rats as shown in the present study indicate that myocardial injury is more severe in diabetic than in control rats, even at the early stage of the disease (4 weeks of diabetes). More importantly, results from current study also suggest that inconsistence of experimental protocols may be responsible for the discrepancy of the previously reported results.
Larger amount of studies support the concept that increased oxidative stress, such as a burst of reactive oxygen species production and reduction of antioxidant capacity contribute to myocardial tissue injury secondary to ischemia and reperfusion , especially in diabetic heart, in which hyperglycemia enhances myocardial oxidative stress and subsequently aggravates diabetic heart damage to IRI . It has been widely accepted that prolongation of ischemia would dramatically increase myocardial pro-inflammatory cytokine TNFα production , which will subsequently increase the formation of nitrotyrosine and concomitantly reduce the activities of antioxidant enzymes, leading to a burst of ROS production which is detrimental to the myocardium . As shown in the present study, STZ-induced diabetic rats exhibited increased myocardial oxidative stress manifested as increased myocardial 15-F2t-Isoprostane production and reduced plasma SOD activity, which was accompanied with increased myocardial TNFα protein expression, reduced myocardial nitric oxide production and increased myocardial nitrotyrosine formation. It has been documented that during ischemia and reperfusion, especially in the early few minutes of reperfusion, increased myocardial oxidative stress, such as augment of 15-F2t-IsoP production and reduction of SOD activity contribute to myocardial injury . Similarly, in the present study, after ischemia reperfusion, diabetic rats displayed more severe oxidative stress, shown as significantly increased myocardial 15-F2t-IsoP production and reduction of plasma SOD activity relative to control. Of note, progressively significant increase of myocardial 15-F2t-IsoP production was observed when diabetic rats subjected to longer duration (from 30 min to 45 min) of ischemia but not that of reperfusion, which suggests that 15-F2t-IsoP production occurs mainly at the time of ischemia or in the early minutes of reperfusion and maintains at certain level when reperfusion sustained. This is consistent with our previous report showing that 15-F2t-IsoP increased at the time of reperfusion and progressively reduced during reperfusion and that 15-F2t-IsoP per se can exacerbate myocardial IRI in isolated rat hearts . We observed a remarkable compensatory increase of plasma SOD activity in diabetes when reperfusion last for a longer time (from 2 hours to 3 hours), which is similar to our previous study showing that in 9 weeks of STZ-induced diabetic rats, a compensatory increase in myocardial total antioxidant capacity occurred as a consequence of the increase of cytosolic Cu/Zn-SOD, but yet it was not sufficient to prevent hyperglycemia-induced oxidative stress . It should be noted that oxidative stress represents a major cause of reduced myocardial nitric oxide (NO) availability. NO, produced by enzymes called NO-synthases (NOS), has been demonstrated to play an important role in contributing to cardiovascular homeostasis and enhanced bioavailability of endogenous NO has been shown to protect the heart from myocardial IRI . Physiologically, NO is mostly produced from endothelial NO-synthases (eNOS), which exhibits beneficial effects to the heart. Under pathological condition, including diabetes and ischemia stimuli, inducible NOS (iNOS) is excessively expressed and produces large amount of NO, which in the concomitant presence of excessive superoxide formation results in the formation of peroxynitrite, leading to exacerbated myocardial injury at the setting of ischemia reperfusion . In the present study, reduced myocardial NO and increased nitrotyrosine were observed in diabetic rats compared to control both before and after ischemia reperfusion, which suggest that an reduction of NO bioavailability was existed in diabetic heart.
Two important signaling pathways namely, the reperfusion injury signaling kinase (RISK) pathway including PI3K/Akt signaling cascade and the protective survivor activating factor enhancement (SAFE) pathway including Jak2/STAT3 signaling cascade play key roles in enhancing myocardial NO bioavailability. Our previous reports [12, 21] showed that myocardial phosphorylation of Akt and STAT3 were decreased significantly in diabetic rats at the early stage of the disease followed by decreased eNOS activation, which subsequently reduced NO production, and rendered the heart less tolerant to ischemic insult. This hyperglycemia-induced increase of IRI in diabetes can be prevented by treatment with antioxidants, N-Acetylcysteine and allopurinol, by activating both the Akt and STAT3 involved signaling pathways . Activation of Akt may subsequently increase FoxO phosphorylation, resulting its inactivation via nuclear exclusion, and thus attenuate cardiac dysfunction caused by hyperglycemia . Similarly, in the present study, there are reduced Akt phosphorylation at site Ser 473 and reduced STAT3 phosphorylation at site Ser727 in diabetic heart relative to controls, accompanied by reduced phosphorylation of glycogen synthase kinase (GSK)-3β at site Ser 9. GSK-3β is a downstream target of RISK signaling pathway, which is phosphorylated by Akt, and plays important roles in necrosis and apoptosis of cardiomyocytes . Studies showed that activation (phosphorylation) of Akt can phosphorylate (inactivate) GSK-3β, which inactivates GSK-3β activity and confers cardioprotection . However, in diabetes, phosphorylation of Akt is reduced and the activity of GSK-3β is increased, which may lead the heart damage to ischemic insult . In the present study, both Akt and GSK-3β phosphorylation were reduced in diabetic heart both before and after IRI, accompanied with increased apoptotic cardiomyocytes. This indicates that impairment of activation of STAT3 and Akt may represent the fundamental mechanism responsible for the increased susceptibility of the diabetic heart to IRI.