The epidemic explosion in the incidence of chronic heart failure (CHF) in Western society resulted in great human suffering and has produced tremendous burden on health care and social services. Since the majority of the CHF develops consequently to MI, the most effective CHF prevention is the prevention of MI from occurrence or minimization of myocardial damage after MI occurrence. The former is targeted through changing life style and diet, leading to reduction of cholesterol and other risk factors, strategies whose arsenal was recently expanded by using pharmacological tools such as statins 
. The latter is achieved by rapid and vigorous revascularization and early pharmacological therapy. However, a large fraction of heart attacks is not associated with increased level of cholesterol and its etiology, and thus means of prevention, remain unclear. Therefore, dietary or behavioral manipulations, which could increase the myocardial resistance to ischemic damage, are extremely valuable. Thus far, only exercise has been shown experimentally to increase the resistance of the myocardium to ischemic damage 
. We have recently reported, in experiments on rats, that dietary regimen using intermittent, every-other-day fasting significantly increased the myocardial tolerance to ischemic damage 
. In the present study we have shown for the first time that a regular dietary supplement of blueberry extract attenuates coronary ligation-induced myocardial damage.
Three months of blueberry-enriched diet did not affect food intake or body weight of rats. It also did not have any effect on morphometric or functional echocardiographic indices of the heart. However, experiments with single cardiomyocytes isolated from the LV of BD and CD rats showed significantly increased threshold of ROS-susceptibility for the induction of mitochondrial permeability transition with the blueberry diet. In other words, at the mitochondrial level, the tolerance of cardiomyocytes to oxidative stress was increased with this dietary supplement.
The results of our in vivo
experiments showed that in the myocardium of rats maintained on a blueberry-enriched diet, 24 hrs after coronary ligation the number of cardiomyocytes in the area at risk stained positively for apoptosis was 4.5 fold reduced compared to that in rats on the control diet; the number of inflammatory cells was reduced almost in half; and MI size resulting from a coronary ligation was 22% smaller. A subset of rats, in which LV remodeling was followed after induction of MI via serial echocardiography, also showed a reduction in original myocardial damage (two weeks after surgery) in BD rats which was naturally translated into significant attenuation of post MI LV remodeling: 10 weeks after coronary ligation rats maintained on blueberry-enriched diet had 14% smaller EDV, 26% smaller ESV, and 82% higher EF than rats fed a control diet. Moreover, 10 weeks after coronary ligation the resulting MI in rats maintained on blueberry-enriched diet prior to and post MI was 31% smaller than in rats maintained on control diet. The positive effect of a blueberry enriched diet post-MI was also confirmed by the assessment of cardiomyocyte density in the myocardium. The reduction of cardiomyocyte density observed 10 weeks after induction of MI in rats maintained on control diet (CC) and reflecting usual loss of cell associated with chronic heart failure 
was significantly attenuated among BB and CB groups, but was not found in BC group.
This study was designed as a proof of concept and was not intended to analyze the signaling pathways responsible for reduction of necro-apoptosis and inflammation in the myocardium after MI in animals on blueberry-enriched diet. The one thing that could be stated with certainty on the basis of our finding is that this signaling was associated with increased mitochondrial permeability transition threshold. Most probably, based on our previous extensive research, the effect was mediated through a number of possible kinases (31), however the ROS scavenging mechanism also cannot be excluded (32). Some inferences can also be made on the basis of previous studies. Fruits and vegetable rich in polyphenolics are known to delay or reverse the deleterious effects of aging on neurocommunication and behavior [see 40 for review]
. Blueberry extract was shown to significantly inhibit the lipopolysacharide-induced inflammatory response in brain microglia. This effect was due to down-regulation of iNOS mRNA and suppression of iNOS proteins, i.e., blueberry extract may inhibit one of the primary steps in the inflammatory stress pathway 
. In the same model (BV-2 mouse microglial cells), blueberry extract treatment also inhibited COX-2 mRNA and protein expression 
, which is known to be associated with proinflammatory stimuli 
, and the proinflammatory cytokines IL-1β and TNF-α 
. Extrapolating from these findings, it is reasonable to assume that in the present study similar oxidative/inflammatory stress signals may be operational. Much more difficult is to explain the reduced necro-apoptosis in our model. Blueberry-enriched diet is known to have some anti-cancer properties 
and one of the anti-cancer mechanisms is the activation of apoptosis 
An additional promising direction in understanding the tissue-protective properties of blueberry might be in exploring the possible effects of BB treatment on glucose metabolism and, specifically, its insulin-like properties 
. Tissue-protective properties of insulin are expanded well beyond its control of hyperglycemia. It suppresses the production of TNF-α, IL-6, and other pro-inflammatory cytokines and enhances the synthesis of anti-inflammatory cytokines, IL-4 and IL-10 [for review see 49]
. Specifically, cardioprotective effects of insulin were demonstrated clinically 
and in experimental models 
. Multiple mechanisms of cardioprotection were proposed: coronary dilatation, anti-inflammatory and anti-apoptotic; however, the most fundamental to cardioprotection remained insulin
s ability to stabilize the mitochondrial permeability transition (52). Thus, it is conceivable that increased myocardial tolerance to hypoxia after prolonged feeding with a blueberry-enriched diet, as well as elevation of mitochondrial permeability transition threshold shown in our study, are due to up-regulation of insulin sensitivity.
Therefore, the Apreventive/at/ part of our experiment clearly demonstrated that blueberry-enriched diet increased tolerance to ischemic damage of myocardium in a rat model of a permanent coronary ligation. The Atreatment/at/ part of the study, however, while less obvious, is also very promising. The switching of the diet immediately after coronary ligation to its opposite, i.e., blueberry-enriched to control and control to blueberry-enriched, revealed a tendency to affect the post MI progression of LV remodeling by accelerating or attenuating it respectively. This outcome indicates the possibility of adding blueberry supplementation to the therapeutic arsenal which should be evaluated further in experimental models of CHF.
In summary, in experiments examining rats maintained on a blueberry enriched diet, we found increased myocardial tolerance to ischemic damage. At a cellular level, the blueberry diet increased cardiomyocyte survival by elevating the mitochondrial permeability transition ROS threshold. In in vivo experiments this diet reduced the size of myocardial infarction induced by a permanent coronary ligation by attenuating necro-apoptosis and inflammation in the area at risk. The beneficial effects of the blueberry diet were extended after coronary ligation by continuing the attenuation of the post-MI LV remodeling and MI expansion. The only non-pharmacological intervention capable to produce similar cardioprotective effect so far was the intermittent fasting (27). To the best of our knowledge, this is the first demonstration of the effectiveness of a readily available natural product in acceptable quantity to significantly limit myocardial damage resulted from induced ischemia.