The number of patients with diabetes and the metabolic syndrome reaches epidemic proportions [1
]. The incidence of myocardial infarction in diabetic patients is twice that of the general population [3
]. Infarct size for a given ischemic insult is larger in diabetic patients than in non-diabetic patients [3
]. This, on top of diabetic cardiomyopathy [8
], contributes to progressive heart failure. Therefore, new or more potent strategies to protect them against cardiovascular complications are needed. Our mice models of type II diabetes and the metabolic syndrome are, similar to the clinical reality, more vulnerable for IR injury. In other experimental diabetic models, disagreement exists about the sensitivity to IR injury [25
]. Depending on the reports, infarct size in animal models of diabetes was larger, smaller or similar to non-diabetic controls [26
]. There are numerous differences in experimental protocols and a single factor cannot entirely explain the discrepancies. Duration of the diabetic state and plasma level of insulin (type I versus type II) seems to influence the myocardial susceptibility to IR injury [26
]. Although type II diabetes represents 90% of the diabetic patients, the majority of the experimental studies are performed in type I diabetic rat models, induced by streptozotocin [5
]. Most of them report a similar or reduced myocardial infarct area after IR injury [25
]. Rodrigues et al. [25
] reported that 3
months after IR injury, also ventricular dysfunction was attenuated and the profile of calcium handling proteins was better. Nevertheless, the mortality was higher in the type I diabetic group, due to increased autonomic dysfunction [25
]. The sensitivity to IR injury in this model seems to be time-dependent. Some studies report a resistance to IR early after diabetes induction by streptozotocin, which disappears after 2
]. Others however demonstrated an increased infarct size after coronary occlusion as early as 8
days after streptozotocin injection [26
], indicating that duration of diabetes is not the only factor responsible for the heterogeneous results. Disagreement also exists about the effect of acute hyperglycemia to IR injury. Although some studies report a larger or similar susceptibility to IR injury in a hyperglycemic state, other reports demonstrate a significant protection, related to a better profile of calcium handling proteins [25
The difference in presence or absence of hyperinsulinemia may also be responsible for the heterogeneous data. With the exceptions of a few studies in type II diabetic Zucker and Goto-Kakizaki rats, all type II diabetic models with obesity and hyperinsulinemia showed increased myocardial susceptibility to IR injury [26
]. In the past decade, transgenic mouse models of type II diabetes and the metabolic syndrome have been developed. These models develop left ventricular diastolic and systolic dysfunction comparable with the diabetic cardiomyopathy seen in patients [8
]. We demonstrated in this study that these mice models of type II diabetes and the metabolic syndrome are more vulnerable for IR injury than their WT littermates. This correlates well with data from patient studies and previous studies in transgenic mice with type II diabetes [6
]. Nevertheless, the underlying mechanisms by which type II diabetic and metabolic syndrome myocardium is more vulnerable to IR injury remains incompletely understood. Several hypotheses were suggested.
First, the diabetic myocardium exhibits abnormal cardiac substrate utilization and impaired energetic efficiency. In non-ischemic conditions, the myocardium uses preferentially fatty acids as energy substrate. This is even more the case in type II diabetes and the metabolic syndrome. The use of fatty acids requires 12% more oxygen per ATP generated than glucose. During ischemia, the normal myocardium switches to glucose as preferred energy substrate. This possibility is lost in case of diabetes, because oxidation of fatty acids inhibits the catabolism of glucose [29
Second, endothelial dysfunction and reduced nitric oxide (NO) bioavailability is present in type II diabetes and the metabolic syndrome [11
]. The beneficial or detrimental effect of NO depends on its concentration and cellular origin [5
]. Although constitutive production of NO by endothelial cells (eNOS) is thought to be cytoprotective, NO generated by the inducible form (iNOS) is generally regarded as pro-inflammatory and cytotoxic. In diabetic myocardium, basal iNOS-levels are higher and the increase of iNOS-levels after IR injury is larger than in non-diabetic myocardium [30
is crucial in postischemic myocardium by significantly decreasing intracellular Ca2+
]. It was shown before that SERCA2a
-activity is depressed in ob/ob and DKO mice [8
]. In the streptozotocin type I diabetic model, SERCA2a
-expression after IR injury is increased versus WT animals. This is a possible explanation for the better preserved contractility after IR injury in this model [25
Fourth, diabetic and metabolic syndrome models exhibit increased production of reactive oxygen species (ROS) [32
]. ROS likely plays a central role in impairing mitochondrial energy metabolism, through mitochondrial uncoupling, but also through direct damage of mitochondrial components. This can lead to apoptosis [32
Fifth, the activity of aldose reductase, a NADPH-dependent enzyme, increases during IR injury [33
]. Since aldose reductase activity is already increased in diabetes [34
], IR injury further reduces myocardial glycolysis and glucose oxidation and increases the glycosylation of advanced end-products. This leads to more extensive cross-linking of key structures within tissues and increased IR injury [34
Sixth, chronic hyperglycemia leads to cardiac hypertrophy and decreased microvasculature density in myocardium. This microcirculation dysfunction causes increased vulnerability to IR [28
Seventh, the exposure to abnormal substrates and cytokines makes the diabetic heart more prone to IR. It was recently shown that IL-33 is decreased in type I diabetic mice [28
]. It is known that IL-33 prevents apoptosis, attenuates myocardial infarction and improves cardiac function. In this study was also shown that chronic activation of protein kinase C-β in diabetes contributes to myocardial dysfunction and infarct [28
Finally, leptin deficiency leads to ventricular hypertrophy and heart failure [35
]. McGaffin et al. reported that leptin has a protective role after IR [36
]. Leptin deficient mice had worse survival, worse cardiac function and increased apoptosis, 4
weeks after IR injury. Leptin induces activation of signal transducer and activator of transcription-3, which induces transcription of several cardioprotective genes. These, in turn, inhibit reverse remodeling and apoptosis [35
]. It is thus possible that leptin deficiency leads to reduced contractility and larger infarct size versus WT, independently from the other risk factors. Nevertheless, the effect of leptin deficiency during the acute phase of IR, as in our experiments, is not known. It is unlikely that STAT-3 activation plays a role during the first hours after IR injury, since transcription of new cardioprotective genes is required.
In hypothesis 1 and 2, we postulated that food restriction respectively ACE-I could reduce the effects of IR injury in mouse models of type II diabetes and the metabolic syndrome. In our study, 12
weeks food restriction or ACE-I, was not capable to preserve in vivo cardiac contractility, nor limit infarct size after IR injury. Nevertheless, ACE-I significantly improved load-independent contractility, as measured by PRSW, in the non-ischemic group.
In food restricted mice, the daily intake was limited to the amount of ‘normal’ free fed wild type mice, measured to be 2.5
g/day. After 12
weeks, the weight loss of ob/ob and DKO mice was 35 à 40% of initial body weight. We did find a slight but non-significant restoration of contractility after this treatment. In a previous study by our group [37
], we found that food restriction with body weight reduction of 50 à 55%, partly restored contractility. So, it is possible that weight reduction of 35 to 40% is insufficient to significantly improve contractility in our mice models. Nevertheless, heart weight was reduced in ob/ob and even more in DKO, below the level of WT, demonstrating already the impact of the food restriction. A further explanation for the difference between the present and earlier studies is that the experimental conditions in the present study differed from the previous. In the study of Van den Bergh et al. [37
], hemodynamic measurements were performed in closed chest mice. In this study, an IR protocol was investigated. This implicates that also in the control groups, left ventricular contractility was measured in the same experimental conditions as the ischemia-reperfusion group, i.e. with an open thorax and time matched.
Food restriction has been shown to reduce IR injury in wild type rodent models [13
]. In renal IR injury, 30% food reduction leads to significant protection. The length of restriction, required for the onset of these renal benefits, is not known but also food restrictions, as little as 2
weeks, lead to significant protection. Interestingly, there is no increase in protection between 2 and 4
weeks of food restriction, suggesting that the maximal protection afforded by these short treatments was already reached after 2
]. Also in the heart, different short-term (2
weeks) food restriction protocols, ranging from 10% to 70%, improve cardiac function following IR injury in wild type rats [39
]. Yamagishi et al. [39
] reported that 70% food restriction during 11
days, resulting in a weight reduction of 38% compared to free-fed rats, has a different effect on pre-ischemic contractility and sensitivity to IR injury. In this study, pre-ischemic cardiac function was reduced but cardiac recovery after IR injury was improved [39
]. An improved ischemic tolerance via altered expression of functional proteins induced by low serum T3 levels, decreased coronary flow rate and change in metabolic flux, were suggested. Anaerobic glycolysis was more activated during ischemia in food restricted animals. This activation of glycolytic flux is fundamental to effective recovery of cardiac function during reperfusion after ischemia [39
]. Nevertheless, the boundary between food restriction and under nutrition is weak. More than 70% food restriction results in depressed cardiac function [39
]. The effect of food restriction on IR injury was never studied in the intended group, the obese type II diabetic patients and animal models. In our type II diabetic and metabolic syndrome mouse models, food restriction did not induce significant protection against IR injury.
The effects of food restriction and ACE-I on the previously described mechanisms of increased vulnerability of type II diabetic myocardium to IR injury are incompletely known. Data concerning the effects of these treatments on cardiac substrate utilization, energetics or aldose reductase activity are missing. Dyslipidemia seems to be a less important factor during IR injury, since infarct size and left ventricular contractility were not significantly different in DKO mice versus ob/ob. Although cholesterol-levels were normal after food restriction in ob/ob mice, this could not correct the increased sensitivity to IR injury.
ACE-I prevents downregulation of SERCA2a
and phospholamban protein expression in heart failure [40
]. Furthermore, ACE-I prevents the decrease in capillary density in type I diabetic rats [41
]. This might explain the improved contractility in ob/ob and DKO mice with ACE-I.
It is widely accepted that food restriction and ACE-I reduce oxidative stress [13
]. Nevertheless, nor food restriction nor ACE-I were capable to reduce IR injury in our mice models. This casts doubt on the role of oxidative stress in the increased vulnerability of diabetic myocardium to IR injury. No data are available concerning the effects of food restriction on capillary density, abnormal substrate utilisation or cytokinelevels in diabetic models.
Captopril has been reported to reduce infarct size in animal models, such as canine, porcine or rat models [16
]. In the study of Itoh et al. [21
], captopril did not protect WT mice against post-ischemic dysfunction and myocardial enzyme release. In contrast to this, a significant protection by captopril was found in mice with overexpression of p90 ribosomal S6 kinase. Since p90 ribosomal S6 kinase expression and activity are increased in diabetic mice, a protective effect of captopril was anticipated by Itoh et al. [21
]. Nevertheless, we did not find infarct size reduction in our models of type II diabetes and the metabolic syndrome.
In this study, we investigated the effects of food restriction and ACE-I, because these treatments are standard therapies in type II diabetic and metabolic syndrome patients. Several other drugs with possible cardioprotective effects during IR injury have been developed. Peroxisome proliferator-activated receptor-γ agonists, such as rosiglitazone, have been shown to possess insulin-sensitizing and lipid-lowering and anti-inflammatory properties [42
]. It has been demonstrated that peroxisome proliferator-activated receptor-γ agonists reduce IR injury in wild type and hypercholesterolemic models [42
]. Nevertheless, it was shown by Ren et al. that the vasculoprotective effects of rosiglitazone are at least partially mediated by a reduction in local angiotensin II concentration [42
]. Since ACE-inhibitors have similar effects on angiotensin II concentration and recent meta-analysis suggest that there is an increase in the risk of myocardial infarction, rosiglitazone was not investigated in this study [42
Sulfonureas, such as metformin, are insulin-sensitizing agents, which reduce infarct size in non-diabetic animals by activation of cAMP-activated protein kinase and Akt. However, clinical and experimental studies in diabetic patients and models report a significantly higher cardiovascular risk and controversial effects on the effect on IR injury [26
Recent evidence has indicated a possible importance of the endothelin system in the pathogenesis of diabetic complications [27
]. Endothelin receptor blockade limits myocardial contractile depression in type I diabetic models [27
]. The effect on IR injury was not studied. Since angiotensin II stimulates and bradykinin decreases endothelin-1 formation, it is conceivable that the majority of endothelin-1 formation is blocked by ACE-I. Furthermore, endothelin-receptor antagonism additional to ACE-I has no added benefit against cardiac dysfunction in type I diabetic rats [27
Recently, Ichinomiya et al. demonstrated that high-dose fasudil, a Rho-kinase inhibitor, preserves postconditioning under acute hyperglycemic state in rats [43
]. Rho-kinase activity is involved in diabetic endothelial dysfunction and regulation of Rho-kinase signaling is important for cellular function, such as contraction, mortality, proliferation and apoptosis. Correspondingly, Rho-kinase is known to be activated in ischemic myocardium. Inhibition of Rho-kinase activity is a matter of interest on myocardial protection against IR injury since it activates the ATP-sensitive potassium channels [43
]. The effect of fasudil on postconditioning and IR injury in chronic hyperglycemic states, such as diabetes, has to be determined.
We used Captopril as ACE-I. Other ACE-inhibitors, with different lipid solubility and/or tissue specificity, might have affected the impact of IR injury. In this study, we investigated the acute effects of IR injury after an ischemia/reperfusion time of respectively 30 and 60 minutes. It is possible that the beneficial effects of these treatments appear in a later phase, for example during the remodeling phase.
The combination of food restriction and ACE-I was not investigated. In ob/ob mice, food restriction reduces weight and cholesterol levels and ACE-I reduces glycemia. It is possible that the combination of these effects improves myocardial damage. In DKO mice the additional effect is unlikely, since there was no reduction of cholesterol after diet and only moderate reduction of glycemia after ACE-I. Further studies have to be conducted to study these effects.