Hypoxic preconditioning was capable to induce a strong protection in WT mice, but not in the DKO model of MS. Furthermore, the relationship between infarct limitation and improved contractility after preconditioning was different in DKO than in WT mice. In DKO, preconditioning induced a proportionally smaller improved PRSW per reduced infarct area versus WT mice. In ob/ob, preconditioning induced only a partial preservation of left ventricular contractility. The degree of protection was inferior to that in WT.
Since we have not directly investigated the mechanisms underlying the reduced or abolished delayed preconditioning potential, we can only hypothesize on them. This should further be investigated in depth to allow specific, additional therapeutic interventions. Potential underlying mechanisms are numerous.
First, our models are known with raised concentrations of non-esterified fatty acids, insulin resistance and hyperglycemia [14, 20, 21]. These metabolic anomalies induce atypical isoforms of protein-kinase C , which is crucial in the pathway of delayed preconditioning .
Second, insulin induces a preconditioning effect . It was shown that at 24 weeks, both ob/ob and DKO mice have increased insulin levels [14, 20]. It is therefore possible that mouse models with hyperinsulinemia are in a permanent preconditioned state or require a higher threshold to preconditioning [5, 25].
Third, previous studies demonstrated the importance of mitochondrial reactive oxygen species (ROS), generated in response to a preconditioning stimulus [1, 2]. Increased oxidative stress is demonstrated in our mouse models [14, 20]. It is therefore possible that increased ROS-levels induce a permanent preconditioned state or induce a higher threshold to preconditioning.
Fourth, the role of NO in IR injury and preconditioning is complex and depends on its concentration and cellular origin [1, 26]. eNOS is a trigger in the delayed preconditioning pathway. Endothelial dysfunction and reduced nitric oxide (NO) bioavailability is present in T2D and the MS . During IR injury, iNOS has a beneficial role in normal myocardium since iNOS knock-out non-diabetic mice show a larger infarct size versus WT-controls . The small magnitude of increase in iNOS levels in this situation is important, because the up regulation of iNOS has a pronounced dose-dependent effect: beneficial at low levels but toxic at high levels [1, 26]. In diabetic myocardium, basal iNOS-levels are 3 times higher than in non-diabetic myocardium . Furthermore, the increase of iNOS-levels in ischemic areas after IR injury is 2.6 times larger than in the ischemic areas of non-diabetic myocardium . The detrimental role of these high iNOS-levels during IR injury in diabetic myocardium was confirmed in another study , in which iNOS knock-out diabetic mice showed a smaller infarct size and reduced caspase-3 activity versus control diabetic mice. This might explain the increased sensitivity of our diabetic mice to IR injury. In the delayed preconditioning pathway, iNOS has a critical beneficial role . Targeted deletion of the iNOS-gene abrogates delayed preconditioning, suggesting that iNOS is a common effector of cardioprotection . Since basal iNOS levels are higher, it is conceivable that diabetic mice have a disturbed threshold or are even already in a permanent maximally protected or preconditioned state.
Fifth, delayed preconditioning is regulated through an up regulation of several other cardioprotective proteins . Previous studies focused mainly on the gene expression profile after delayed preconditioning in healthy subjects  and the effect of diabetes or the metabolic syndrome on this has never been investigated.
Finally, although a plethora of mechanisms are involved in preconditioning, it is widely accepted that all these mechanisms converge at the inhibition of the mitochondrial permeability transition pore (mPTP) opening at early reperfusion . Inhibiting mPTP opening will prevent the cardiomyocytes to undergo cellular necrosis and apoptosis. No data are available about mPTP-inhibition in our mouse models of T2D and the MS.
In an attempt to restore delayed preconditioning in ob/ob and DKO mice, therapeutic strategies were investigated. After food restriction or ACE-I, hypoxic preconditioning reduced infarct size and preserved PRSW up to the level without ischemia in both ob/ob and DKO. Furthermore, ACE-I restored the proportional effect of preconditioning on infarct limitation and contractility improvement to the WT-level. Comparable with our previous study , we found that diet or ACE-I without preconditioning did not reduce the impact of IR injury in these mice models, in contrast with the regained protection by delayed preconditioning potential in this study. The regained preconditioning potential is thus independent from the direct effects of ACE-I on IR injury. A possible explanation is that food restriction and ACE-I are not capable to influence insulin levels, glycemia, cholesterol, ROS, eNOS and iNOS sufficiently to see an effect on IR injury without previous preconditioning. Nevertheless, these parameters might have been restored sufficiently to induce again a threshold for delayed preconditioning.
It is already shown that food restriction reduces insulin levels and ACE-I improves insulin sensitivity [14, 19]. Furthermore, ACE-I reduces glycemia in ob/ob and DKO mice and food restriction lowers cholesterol levels in ob/ob mice . This can have lowered the threshold to delayed preconditioning since insulin has a preconditioning effect [5, 25], and hyperglycemia and hypercholesterolemia are key player in the induction of atypical isoforms of protein-kinase C , which is crucial in the delayed preconditioning pathway . In the aging heart, an impaired protein-kinase C translocation is one of the reasons responsible for the impaired preconditioning potential .
Food restriction partially restores classical preconditioning in senescent animals, but in combination with exercise, this restoration becomes complete. A restored norepinephrine release was suggested as the underlying mechanism . Furthermore, food restriction induces up-regulation of eNOS in DKO mice . ACE-I is capable to block the degradation of bradykinin into inactive peptides and to increase eNOS-levels [15, 16, 19, 33]. eNOS is a trigger in the delayed preconditioning pathway , which might explain the regained preconditioning potential.
Food restriction prevents ROS production  and also ACE-I has well known anti-oxidative activity [15, 16, 19]. Since an increase in ROS formation is an important trigger of delayed preconditioning , it is possible that these treatments reduce the increased baseline ROS-level in diabetic myocardium and restore the threshold for delayed preconditioning .
Another possible explanation why the treatments induced cardioprotection after preconditioning but did not have a direct effect during IR injury, is the different role and levels of iNOS in both pathways. As described earlier, basal iNOS-levels are higher and the increase of iNOS-levels after IR injury is larger than in non-diabetic myocardium  with a deleterious role in diabetic myocardium during IR injury . In contrast with this, a small increase in iNOS-levels is critical to induce its effects as common end-effector in the delayed preconditioning pathway . No studies were conducted to study the effect of food restriction on iNOS-levels in myocardium, but it was shown in a normotensive rat model that ACE-I reduces iNOS-levels with 24% . It is tempting to speculate that ACE-I did not reduce iNOS-levels sufficiently to reduce its toxic high-dose level during IR injury but enough to restore the threshold to induce delayed precondition (by a small iNOS increase). Further investigations need to be performed to elucidate the role of these underlying mechanisms.