Our study demonstrates that the long-term supplementation of a low fat diet with pasta enriched with BBG increases the survival rate of mice undergoing LV I/R injury. The magnitude of the LV infarct scar size and of the contraction necrotic bands was safely attenuated in mice fed with P-BBG for five weeks. The dietary intake of P-BBG also prevented an increase in body weight without altering the systemic glucose tolerance, cardiac function, and the well-being of mice. Compared to the Control group, the histological analysis revealed a higher density of coronary capillaries and arterioles in the explanted hearts of P-BBG mice, which support higher tissue viability downstream of the obstructed coronary tree. Regarding the cardioprotective pathways, our data revealed, for the first time, the relationship between myocardial VEGF, p53 and Parkin protein upregulation, vasculogenesis enhancement and reduction of caspase-3 cleavage at the early stages of post-ischemic reperfusion conferred by the sustained dietary intake of P-BBG.

It is largely recognised that cardiac ischemic preconditioning enhances the myocardial protein expression of VEGF33, a growth factor that induces myocardial angiogenesis and cell survival34. In line with the cardioprotective effects of VEGF, other researchers have perfused the heart with VEGF35 or have upregulated the myocardial VEGF expression using gene therapy36,37 in order to enhance the local adaptive response naturally occurring under oxidative microenvironment. However, none of these invasive approaches may be ethically permissible in healthy subjects. Hence, the urgent need to develop a noninvasive strategy to enhance collateral artery growth in the adult myocardium and to promote the survival of cardiac cells following I/R injury.

In a previous study, we demonstrated that chronic exposure of endothelial cells to water-soluble BBG enhances the pro-angiogenic and pro-survival expression of manganese superoxide dismutase (MnSOD)22, which is a mediator of ischemic preconditioning38.

In the current study, we thus hypothesized that the daily dietary intake of water-soluble BBG might promote the formation of new coronary vessels in healthy subjects and prevent myocardial I/R injury.

For this purpose, healthy mice were fed with a low fat diet supplemented with pasta made with a mixture of barley flour and wheat flour, which guarantees a high content of viscous polysaccharides and a regular BBG intake. Our hypothesis is supported by the evidence that pasta safely provides a BBG-enriched diet27,39. To the best of our knowledge, no previous study has assessed the effects of diet supplemented with BBG-enriched pasta to counteract the onset of myocardial I/R injury.

First, we have observed that P-BBG-enriched diet precludes gain weight in mice. Our data are consistent with recent clinical study showing that daily intake of barley beta-D-Glucan prevents visceral fat obesity and body weight gain in humans40. In fact, the ability of soluble BBG to form highly viscous solutions in the human gut slows gastric emptying, digestion and absorption of dietary fat41.

In our study, the survival rate at an early stage of post-ischemic reperfusion was significantly higher in mice fed with P-BBG than with the wheat pasta. Compared to the Control group, the IS/AAR ratio revealed that the left ventricles of P-BBG mice were less injured and the contraction band necrosis, a hallmark of immediate necrotic cell death during the first minutes of reperfusion42, was undetectable in treated hearts. Since myocardial injury is induced after an overnight fasting period, it is conceivable that cardioprotection depends on increased myocardial angiogenesis due to the sustained intake of P-BBG. Our hypothesis is well supported by previous clinical evidence showing that individual tolerance to cardiac post-ischemic reperfusion injury is due to a higher density of coronary collaterals4,5.

In line with our hypothesis, the myocardial density of coronary capillaries and arterioles was higher in mice fed with a diet supplemented with P-BBG than with regular pasta. Although the myocardial protein levels of dectin-1, a receptor that mediates endothelial β-D-Glucan effects43, were similar in both experimental groups, the marked increase in VEGF protein levels in the P-BBG hearts is noteworthy and is closely linked to the regular intake of BBG and to the weight loss. Indeed, we cannot exclude that BBG-based diet may induce VEGF expression in other tissues. It is known that an increase in VEGF expression in adipose tissue can result in increasing vascular density44, in reducing adipocyte size and in precluding gain weight in mice45,46.

In addition, the myocardial levels of anion superoxide decreased significantly in the reperfused hearts of mice fed with P-BBG rather than in the Control mice.

These new in vivo findings strongly support our previous study22. In fact, VEGF likely enhances the local expression of MnSOD23, a key anti-oxidant enzyme, which contributes to the angiogenesis22 and higher cardiac tolerance against ischemia/reperfusion injury47 in P-BBG mice.

Since natural compounds may promote VEGF/antioxidant signaling and inhibit the onset of apoptosis in hearts undergoing I/R48, we assessed the myocardial levels of cleaved caspase-3, an established pro-apoptotic factor. The levels of cleaved caspase-3 were significantly lower in P-BBG hearts, even though the myocardial protein expression of HIF-1α, a transcriptional factor involved in the induction of VEGF expression49 as well as in the inhibition of apoptosis50, was similar in both experimental groups. These results are in agreement with our previous in vitro study22 and suggest the activation of different signaling pathways by BBG.

Although several studies have found a key role for phosphorylated Akt to mediate the cardioprotective phosphatidylinositide 3-kinase (PI3K) signaling pathway51 during preconditioning, the myocardial levels of phospho-Akt/total-Akt ratio were the same in treated and untreated mice.

We then examined the role of the P-BBG diet on myocardial STAT3 signaling pathways since STAT3 activation has been implicated in the inhibition of caspase-3 cleavage52 and also in the upregulation of MnSOD53 and VEGF54 genes. Surprisingly, cardiac STAT3 was also similarly expressed and phosphorylated in both experimental groups.

Lastly, although active NF-kB promotes the VEGF-dependent modulation of MnSOD expression55, the myocardial levels of phospho-NF-kB/total-NF-kB ratio were similar in both the P-BBG and Control hearts.

Although the activation of the pathways so far investigated plays a role in regulating VEGF levels during preconditioning, it seems that they are not activated by BBG in our model. Since p53 has been recently considered an important regulator of cardiac gene transcription56, we have hypothesized that it may play a key role in mediating the BBG-induced cardioprotection.

Farhang Ghahremani et al.57 demonstrated that p53 induces the expression of VEGF. In our study, the expression of p53 in P-BBG hearts was higher than in the Control hearts. It is conceivable that p53 leads to VEGF promoter activation by acting synergistically with HIF-1α, as previously demonstrated by others57.

We have demonstrated, for the first time, that dietary intake of P-BBG induces myocardial p53 upregulation without promoting systemic toxicity or cardiac cell death. Our findings are not in agreement with a previous study that found that dietary anti-oxidants increase p53 levels to the point where they induce apoptosis58. This can be explained by the fact that P-BBG diet also increased the myocardial protein expression of Parkin, an E3 ubiquitin ligase, which plays a key role in promoting mitophagy59 and in delaying caspase-3 activation60. Since healthy mice were safely fed with the P-BBG-enriched diet for five weeks, our results would seem to suggest that the physiological interplay between higher levels of p53 and Parkin may contribute to the non-invasive regulation of the VEGF expression and myocardial viability status.

As β-Glucan receptors are expressed on the surface of endothelial cells61, the upregulation of Parkin protein was localized in the coronary endothelial cells of P-BBG hearts. The binding affinity of dectin-1 to β-D-Glucan and its biological activity are similar in humans and mice22,62, it would thus appear that we have revealed BBG-dependent mechanisms in cultured HUVECs at rest and under acute oxidative stress. On day 7 after the exposure of HUVECs to 3% BBG, the levels of acetylated H4 histone, VEGF and MnSOD increased and were not affected by acute oxidative stress. Our in vitro results suggest that the acute oxidative burst induced by I/R injury in vivo did not counteract the BBG-induced regulatory effects on the abovementioned proteins.

Finally, we have demonstrated that the 7-day treatment of HUVECs with BBG similarly increased the protein expression of p53 and Parkin in the presence of increased histone acetylation. Our data agree with previous studies showing that class I HDAC inhibitors, such as BBG, upregulate the protein expression of p5363 and Parkin64. As a function of expression levels, p53 can either prevent or promote apoptosis65. In vitro, however, BBG-induced p53 upregulation did not affect the viability of endothelial cells.

Although the inhibition of p53 activity is essential to enhancing the mitophagic activity of Parkin66, it is still unknown whether the VEGF and Parkin protein expression induced by BBG depends on p53 activity. Consequently we performed additional experiments by treating HUVECs with BBG in the presence of PFT-α, an established inhibitor of p53 activity66. Although PFT–α did not alter the expression of Parkin, the BBG-induced upregulation of Parkin was not affected by inhibition of p53 activity.

At least, BBG mitigates the H 2 O 2 -induced reduction of Parkin protein expression independently of p53 activity. Hence, our results suggest that the Parkin expression induced by BBG is independent of p53 activity. Conversely, the BBG-induced increase in VEGF protein levels was hampered by PFT–α, which confirms that BBG increased the VEGF expression in a p53-dependent manner. Similarly, PFT–α hindered the BBG-induced decrease in anion superoxide levels in stressed cells, which confirms that BBG decreased the oxidative stress in a p53-dependent manner. Our findings reveal for the first time the impact of BBG on the simultaneous endothelial expression of VEGF and Parkin interacting differently with p53 activity. Moreover, we have demonstrated that the anti-oxidant effects of BBG depends on the p53 activation.

Therefore, by enhancing class I HDAC inhibition, the long-term dietary intake of P-BBG may simultaneously promote the neoangiogenesis and higher tolerance to oxidative stress due to enhanced endothelial expression of VEGF/MnSOD signaling and Parkin (the proposed mechanism is summarized in Fig. 10).