Flavonoids and mitochondrial pharmacology:
a new paradigm for cardioprotection.
Lara Testai*
Department of Pharmacy, University of Pisa, via Bonanno, 6-56100, Pisa-Italy
Interdipartimental Center of Nutraceutical Research and Food for Healthy “Nutrafood”, Università di Pisa, via del Borghetto, 80-56124, Pisa-Italy
*mailing address: via Bonanno, 6-56100 Pisa-Italy e-mail: lara.testai@farm.unipi.it
phone:+390502219596 fax:+390502219609
ABSTRACT
Acute myocardial ischemia is one of the major causes of illness and of deaths in the western society, therefore the definition of the signaling pathways involved in the cardioprotection represent a challenging goal in order to discover novel pharmacological approaches. In this regard, a number of epidemiologic studies demonstrate a relationship between intake of flavonoid-rich foods and reduction of cardiovascular risk factors and mortality. Moreover, numerous experimental studies have examined flavonoid-induced cardioprotective effects on several animal models of myocardial ischemia/reperfusion. As concerns the mechanisms of action, although the antioxidant effect of flavonoids has been long thought to be a crucial factor accounting for cardioprotection, mitochondrial pathways (ion channels, protein kinases, etc.) are presently emerging as specific pharmacological targets more relevantly involved in the anti-ischemic effects of some flavonoids. Since these pharmacodynamic features seem to be poorly considered, this review examines the mitochondrial role in the cardioprotective mechanisms of some members of this phytochemical class, by describing the biological pathways and reporting an overview of the most important experimental evidence in this field.
1-INTRODUCTION
1.1- Myocardial ischemia and cardioprotective strategies
Acute myocardial ischemia is one of the major causes of illness in the western society and despite of the recent advances in therapy, it is still responsible for a number of deaths. Indeed, the reduced coronary blood supply leads to cell death and loss of cardiomyocyte population, resulting in serious and often irreversible consequences on myocardial functionality (Lloyd-Jones et al., 2010). The myocardial cell death during an ischemic episode is mainly caused by necrosis, due to the irreversible opening of the mitochondrial permeability transition pore (mPTP) (Vanlangenkker et al., 2008; 2012). The most effective strategy to reduce ischemic damage is an early reperfusion, but paradoxically the reperfusion in itself is responsible for additional damage, due to apoptotic cell death; indeed, the global myocardial damage is referred to as ischemia-reperfusion (I/R) injury (Yellon et al., 2007).
The beginning of the reperfusion is associated with a burst of reactive oxygen species (ROS) production, probably formed by complex I and complex III of the respiratory chain (Kevin et al., 2003). The mitochondrial proteins are particularly susceptible to ROS-induced damage: ROS have direct effects on the respiration and play a critical role in the opening of the mPTP (Halestrap et al., 1998, 2004; Solaini and Harris, 2005; Pacher et al., 2006).
mPTP is a high conductance mega-channel, anchored between the mitochondrial outer and inner membrane. When it is assembled, it allows the connection between the cytoplasm and mitochondrial matrix, during ischemia it is closed, because an acidic cytosolic pH (Hunter et al., 1976; Halestrap, 1991). Indeed the rapid energization of mitochondria at reperfusion leads to electrogenic uptake of Ca2+, previously accumulated into the cytosol during ischemia. This factor, together with the rise of ROS production and the recovery of neutral pH, promotes the opening of mPTP (Kim et al., 2006a; Kimura et al., 1992). As a consequence of mPTP opening, all small molecular weight solutes (< 1.4 kDa) equilibrate across the inner membrane; in contrast, the largest molecules (i.e. proteins) remain entrapped in the matrix, exerting an osmotic pressure that leads to
the uptake of water and matrix swelling. Although the unfolding of the cristae allows the matrix to expand without rupture of the inner membrane, the outer one breaks and leads to the release of pro-apoptotic proteins, confined in the inter-membrane space, such as cytochrome c (Bernardi, 1999; Doran and Halestrap, 2000; Martinou and Green, 2001). There is an increasing evidence that time of mPTP opening is closely correlated with the extent of I/R damage; indeed, inhibitors of the pore opening (such as cyclosporine A) protect the heart from I/R injury. On the other hand, many strategies, aimed at inhibiting mPTP opening, represent challenging potential approaches to contain extension of myocardial damage; these phenomena are called ischemic pre-conditioning (IPreC) and ischemic post-conditioning (IPostC) (Javadov et al., 2003; Griffiths and Halestrap, 1993). Then, IPreC consists of transient brief episodes of I/R (typically of 2-5 minutes) before a severe prolonged episode of I/R, it was first described in dog hearts (Murry et al., 1986), and thereafter confirmed in many mammalian species, including humans (Edwards et al., 2000; Yellon and Downey, 2003; Kloner and Rezkalla, 2006).
More recently, Zhao and colleagues defined the IPostC, demonstrating that brief intermittent cycles of coronary re-occlusion (typically 30 seconds) and reperfusion (30 seconds) during the first minute of reperfusion after a severe ischemic event, reduce the infarct size by about 40% in canine hearts (Zhao et al., 2003). As observed for the IPreC, also the IPostC has been confirmed in many mammalian species (Skyschally et al., 2009; Staat et al., 2005).
The definition of the signaling pathways of IPreC and IPostC, paved the way to develop pharmacologic strategies designed to produce cardioprotection with drugs able to trigger the same mitochondrial pathways.
The IPreC, as well as IPostC which recruits analogous signaling pathways, is mediated by numerous endogenous factors, including adenosine (Liu et al., 1991), acetylcholine (Yao and Gross, 1993), bradykinin (Schulz et al., 1998) and opioids (Zhang et al., 1996; Weil et al., 1998; Schulz et al., 1995; Schwarz et al., 1997; Fryer et al., 1999; Wang et al., 2001) and gaseous molecules, such as NO (Lochner et al., 2000; Jones and Bolli, 2006; Bolli, 2001) and H2S (Zhang et al., 2007).
Moreover, both IPreC and IPostC involve the activation of protein kinase C (PKC), and possibly other kinases. The translocation of PKCε can be considered as an additional mechanism for protection: the phosphorylation of the mPTP components directly inhibits the pore opening (Ping et al., 1997; Baines et al., 2002; Bianes et al., 2003). Furthermore, a plethora of other kinases are thought to be involved in the protective signaling pathways, such as: phosphoinositide 3-kinase/Akt (PI3K/Akt), mitogen-activated protein kinase (MAPK), caspase, Bcl2/Bax, Janus kinase/Signal Transducer and Activator of Transcription (JAK/STAT) and Protein kinase G (PKG), cyclic AMP-dependent protein kinase A (PKA) (Simkhovich et al., 2013; Madonna et al., 2013; Jeyaraman et al., 2012). In addition to kinases, several types of potassium channels present in the inner mitochondrial membranes have been suggested to be end-effectors in cardioprotection. Presently, both ATP-sensitive (Inoue et al., 1991) and calcium-activated (Siemen et al., 1999; De Marchi et al., 2009) potassium channels have been recognized (Figure 1).
2- EVIDENCE OF CARDIOPROTECTION BY FLAVONOIDS
2.1- Epidemiological studies
A number of epidemiological studies demonstrate a relationship between intake of foods containing flavonoids and reduction of cardiovascular disease mortality and its risk factors. One of the first prospective studies demonstrating such a correlation was the Zutphen Elderly Study, published from Hertog and colleagues in 1993 (Hertog et al., 1993a). This study was carried out on a small cohort of 805 men in the Netherlands assuming flavonols and flavones; but from 1976 to today over 20 prospective cohorts, in western countries and in the United States of America, have been performed (Basu et al., 2010; Erdman et al., 2007; Robbins et al., 2006; Rudkowska and Jones, 2007; Rudkowska, 2008; Scheid et al., 2010; Schroeter et al., 2010; Sies, 2010; Steinberg et al., 2003; Mursu et al., 2008; Cassidy et al., 2013; Mink et al., 2007; Knekt et al., 1996; Higher et al., 2013;Jiang et al., 2014).
However, although a number of cohort studies support the correlation between flavonoid intake and a lower risk of mortality associated with cardiovascular diseases, many variability factors emerge, hindering the interpretation of data, such as food composition and variability in flavonoid content (Hertog et al., 1993b; Arts and Hollman, 2005). A further aspect of variability are the different effects of specific flavonoids, because they are very differences in physio-chemical properties, bioavailability and bioactivity. In fact, even if almost all flavonoids have antioxidant properties, some of them have specific pharmacodynamic profiles (Peterson et al., 2012).
Black tea consumption, for example, is related to reduction of coronary heart disease and myocardial infarction in epidemiological studies; although there is an evident heterogeneity of effects across countries, perhaps because of differences in the tea dose from country to country (Peters et al., 2001).
Epidemiological studies on wine consumption suggest a consistent dose-response cardiovascular preventive effect (Di Castelnuovo et al., 2002), such an evidence is stronger and more homogeneous than that seen with black tea, at least in part because of more accurate intake measurement of wine consumption (Demrow et al., 1995).
2.2 Classification and sources of flavonoids
Flavonoids are a family of phenolic compounds almost ubiquitous in plants. More than 5000 distinct flavonoids have been identified. Chemically, flavonoids consist of a benzopyran heterocycle linked to a benzene ring. They can be divided in different groups depending on the degree of oxidation of the C in position 4, the hydroxylation pattern and the substitution of the C3 position. The main subclasses of flavonoids are six: flavonols, flavones, flavanones, flavan-3ols, anthocyanidins and isoflavones (Figure 2).
Among these, flavonols are the most widespread in food and the most prominent are quercetin and kaempferol. The richest food sources of flavonols are onions, broccoli, apples and blueberries; red
wine and tea also contain significant amounts of flavonols (Hertog et al., 1992, 1993b; Manach et al., 2004).
Flavones are much less present than flavonols in fruits and vegetables; the most prominent are luteolin and apigenin, specially present in parsley and celery (Erdman et al., 2007).
Flavanones, such as naringenin and hesperetin, are present in high concentrations almost exclusively in citrus fruits. Main sources of isoflavones (genistein, daidzein, glycetein), structurally similar to estrogen hormons, are soy and soybean-derived products (Murphy et al., 1999).
Finally, flavan-3-ols are present in many fruits such as grape products, tea, cacao and chocolate; they exist either as monomers (epicatechin) or oligomers (proanthocyanidins). When polymerized, they are called condensed tannins and are responsible for the astringency of various fruits. These flavan-3-ols can be also found in certain seeds of leguminous plants, and the anthocyanins are present in the “red fruits”; indeed they own the colours to the anthocyanins that are pigments (an example is the black grape or the berries) (Mazza, 1995).
3 SPECIFICAL CARDIOPROTECTIVE PROPERTIES OF FLAVONOIDS
A number of experimental studies have examined the effects of each flavonoid class on several animal models of myocardial I/R injury and found protective activity in vitro or in vivo over a variety of treatment protocols. Protective effects of different flavonoids in I/R include improved cardiac functional recovery, increased coronary flow, decreased oxidative damage and protection from cell death. Typically, the protective effects of flavonoids on the cardiovascular system are attributed to their antioxidant effect, including their direct free scavenger property and their metal chelating activity (Frei et al., 2003). Besides these “aspecific” effects, protection against I/R may also be exerted through interaction with specific cell-signaling pathways (figure 1).
ANTINFLAMMATORY ACTIVITY- Myocardial damage due to reperfusion of ischemic tissue is also
such as tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) and interleukin 6 (IL-6). Infiltrating neutrophils also play a crucial role for the production of TNF-α in myocardial I/R injury. The rapid release of these factors contribute to transcriptional activation of intracellular adhesion molecule-1 (ICAM-1), besides the induction of NF-kB cascade and the activation of downstream pro-inflammatory mediators, including NO and expression of COX-2 (Zhang and Chen, 2008). Several reports have shown that flavonoids are able to decrease the expression of inflammatory signaling pathway, finally inhibiting the excessive release of NO, the COX-2 expression and the leukocyte activation. In particular, flavonoids reduce iNOS protein synthesis, through the prevention of NF-kB activation (Ha et al., 2008; Chen et al., 2001; Kim et al., 1999; Liang et al., 1999; Raso et al., 2001). The structural requirement is the planar conformation, typical of flavonoids with a keto group in position 4, and the 2,3 double bond; therefore flavone and flavonol classes exhibit higher antinflammatory effectiveness. The second main requisite is a sufficient lipophilicity, indeed flavonoids with many hydroxyl groups are only weakly active, while methoxy or acethoxy groups improves the activity. However, isoflavons act through the NF-kB pathway even if their structure do not include the 2,3 double bond and 4-keto group (van Acker et al., 1996). Some anthocyanins showed to inhibit TNF-α-induced VCAM-1, ICAM-1, and COX-2 levels, by means of an antagonism of NF-kB-dependent pathway (Kim et al., 2006b). At this regard, recently a paper on the potential anti-inflammatory property of malvidin, a natural pigment of anthocyanin class, has been published. The results showed that malvidin inhibited the TNF-α-induced ICAM-1 and VCAM-1 production, as well as antagonized NF-kB pathway (Huang et al., 2014a).
Again, a protection against I/R injury, by means of regulation of inflammatory cytokines, NF-kB and TNF-α, has been demonstrated for vitexin, a glycoside of apigenin. The authors reported that vitexin significantly reduced the elevation of the ST segment of ECG and myocardial infarct size, other than the activity of typical biochemical markers of myocardial damage (LDH e CK, Dong et al., 2011).
VASODILATOR ACTIVITY- Many flavonoids possess direct vasodilator actions, that can contribute
to a cardiac protection through an improvement of the blood flow in the injured tissue.
Flavone and flavanones are potent vasodilators, while flavanols lacking the keto group have a lower vasorelaxing activity. In general, an increased hydrophilicity due to many hydroxyl groups may abolish the vasodilator action, but the relationship between lipophilicity and vasodilating activity has not been fully proven (Duarte et al., 1993; Jeon et al., 2007; Xu et al., 2007; Dong et al., 2009) and the mechanism of vasodilator action has not been fully elucidated as yet. The presence of intact endothelium is not a requisite but may, at least slightly, enhance the activity of most flavonoids (Chan et al., 2000; Mishram et al., 2000; Ko et al., 1991). Of course, the endothelium-dependent relaxation effect is mediated by nitric oxide, interestingly, a part of the beneficial effects on ischemic-reperfused hearts of epigallocatechin 3-gallate and baicalein is mediated by an induction of eNOS (Potenza et al., 2007; Tan et al., 2014).
The mechanisms endothelium-independent are yet uncertain, but kinase proteins, phosphodiesterase or ion channels could to be involved . In this regards, Calderone and his colleagues demonstrated that a series of hydroxy derivatives belonging to flavone and flavanone classes, such as 5-hydroxyflavone or naringenin, were able to produce vasorelaxing effects by the opening of big conductance calcium activated and/or ATP-sensitive potassium channels (BKCa and KATP, respectively) (Calderone et al., 2004; Saponara et al., 2006; Qin et al., 2008); the blockage of extracellular Ca2+ influx or Ca2+-release from endoplasmic reticulum could be one of the possible mechanisms of flavonoid protection against a I/R-induced calcium-overload. Indeed, the ethanolic extract of Kaempferia parviflora, containing a variety of flavones, besides vasorelaxing activity, partly dependent of endothelium presence, had a cardiprotective effect on rat perfused heart subjected to I/R, whose exact action mechanism is also under investigation (Malakul et al., 2011). MITOCHONDRIOTROPIC ACTIVITY-Mitochondria play a crucial role in the cell fate, they can decide when and how a cell must die. Therefore, as above mentioned, the understanding of the mitochondrial pathways involved in the cardiac protection against I/R event is essential for
achieving an effective pharmacological strategy. The activity of some flavonoids on mitochondrial targets may be a further explanation for the cardioprotective effect, that it is made possible by ensuring the mitochondrial ATP production and the calcium homeostasis, preserving lastly the mPTP opening and subsequent cell apoptosis.
Such beneficial effects can result from action on multiple mitochondrial targets, among them ion channels, kinase proteins or oxidative phosphorylation; note that, very often, the involvement of more than one target emerges from available literature, highlighting the complexity of the mitochondrial mechanisms of protection.
MITOCHONDRIAL ION CHANNELS- Potassium ion channels, similar to those expressed on
sarcolemmal membrane, have been also described at mitochondrial level. In particular, BKCa and KATP channels have been recognized on inner mitochondrial membrane and have been called mitoBK and mitoKATP. Intense research permitted to highlight the pivotal role of mitoKATP and, more recently, mitoBK in myocardial I/R and the protective role played by activators of these channels. Indeed, mitochondrial potassium (mitoK) channels are viewed as end-effectors of cardioprotective pathway, downstream to kinase cascades and ending with the closure of mPTP and preservation of cytochrome c release (Testai et al., 2014).
Noteworthy, some flavonoids endowed with vasorelaxing property due to activation of sarcolemmal potassium channels, showed cardioprotective activity in I/R model on isolated rat hearts too. In particular, the presence of hydroxy group in position 5 accounted for significant protective effects against the myocardial injury induced by drastic conditions of I/R. In contrast, flavonoid derivatives not bearing the hydroxy group in position 5 failed to be cardioprotective in the same experimental conditions, highlighting that these effects are not indiscriminately shown by all flavonoid derivatives and leading to hypothesize the involvement of mitoK channels in cardioprotection (Testai et al., 2013a).
Really, Testai et al., have recently demonstrated the involvement of mitoBK in the cardioprotection of a citrus flavonoid naringenin (Testai et al., 2013b), likewise it has been demonstrated for puerarin
(Yao et al., 2010; Yang et al., 2008). The engagement of a mitoK channel has also been observed for epigallocatechin3-gallate and baicalein (Song net al., 2010; Chang et al., 2013).
Water soluble extracts of flavonoid-containing plants, such as Emblica officinalis, Bergenia crassifolia and Pentaphylloides fruticosa, produced a dose-dependent activation of mitoKATP channels, whose effect was abolished by the specific blocker, 5-hydroxydecanoate (5-HD; Mironova et al., 2008).
Besides mitoK channels, the mitochondrial Ca2+ uniporter shows to perform multiple roles in the regulation of ATP production, in the opening of the mPTP and subsequently in apoptosis. Worthy of note, Montero et al., reported that various natural flavonoids, among kaempferol, directly activated the mitochondrial Ca2+ uniporter, at concentrations compatible with a daily intake of flavonoid-rich foods, suggesting that a modulation of mitochondrial Ca2+ uptake may occur in vivo depending on the dietary intake (Montero et al.,2004).
OXIDATIVE PHOSPHORILATION/RESPIRATORY CHAIN-The main function of mitochondria
resides in capacity to produce energy for cell through the oxidative phosphorylation, then the maintenance of normal function of respiratory chain is fundamental to ensure it. Indeed, it is accepted that myocardial ischemia causes inhibition of complex I of the mitochondrial respiration chain, that together with the release of cytochrome c are the earlier events in ischemic heart damage. In this context, very recently Skemiene et al have demonstrated that anthocyanins, endowed with reductive capacity, may be a substrate for mitochondrial complex I, but only certain of these, cyanidin 3-O-glucoside and delphidin 3-O-glucoside, may sustain mitochondrial energetic functions damaged by ischemia and ensure the maintenance of normal functioning of complex I. These finding support the idea that anthocyanins possessing two or three hydroxyl groups showed a beneficial effect on oxidative phosphorylation, in contrast anthocyanins with only one hydroxyl group had very little effect (Skemiene et al., 2013a; 2013b; 2015).
Several papers reported that flavonoids influence the mitochondrial respiratory chain and modulate the intra-mitochondrial calcium concentration, preserving then the opening of mPTP. In particular,
Dorta et al. observed that a little number of selected flavonoids, structurally correlated, showed to influence mitochondrial functioning (Dorta et al., 2005), nevertheless their results are preliminary and further studies should be executed. Other studies find that flavonoids induce a partial uncoupling of oxidative phosphorilation in cardiac mitochondria, improving the mitochondrial respiration without irreversibly compromise it (Trumbeckaite et al., 2006).
PROTEIN KINASES- Complex kinase cascades represent a fundamental crossroad in the
mitochondrial pathways involved in myocardial I/R damage. Historically, the first survival kinase implicated in the IPreC and IPostC was the protein kinase C (PKC), known as the central mediator; however today a more confuse and complicate network among numerous kinases is emerging. It is known that, the activation of PKC, on one hand, is able to directly close mPTP and activate mitoKATP and mitoBK, on the other hand, it is able to interact with Akt and consequently with eNOS. In fact its activation involves the stimulation of Akt and enhances the eNOS and then COX expression; vice versa, inhibition of PKC down-regulates all downstream steps (Zhang et al., 2004; Simkhovich et al., 2013).
Indeed, PKC is connected upstream and downstream with other survival kinases, such as MAPKs, PKG, PKA, PI3K/Akt, converging in the activation of mitoK channels. In particular, it is well known that three subgroups of MAPKs (JNK, ERK and p-38) play distinct and complementary roles: when activated at myocardial infarction, ERK mediates cytoprotective effects, vice versa p-38 MAPK and JNK exert a detrimental effect. In agreement, the cardioprotection observed administering baicalin, glycoside of baicalein, for 7 days was correlated with an increased expression of ERK and a decreased of JNK and p-38 MAPK (Liu et al., 2013). Noteworthy, Choerospondias axillaris, medicinal plant used in Mongolia for cardiovascular diseases and rich in total flavones, produces beneficial effects against the myocardial ischemia through the involvement of MAPK signaling pathway (Li et al., 2014).
Rhamnetin, or 7-methoxy quercetin, has been also demonstrated to act as cardioprotective agent by phosphorylation of Akt and MAPK pathways in H2O2-injured H9c2 cells; since specific inhibitors
of these ways were able to attenuate rhamnetin-mediated cytoprotection (Park et al., 2014). Moreover, 3′,4′-dihydroxyflavonol (DiOHF), administered during ischemia just before reperfusion, inhibited mPTP opening and effectively preserved cardiac mitochondrial function, improving function of complex I, through a mechanism likely to be independent of its antioxidant activity, of direct effect on the mPTP and on respiration in isolated mitochondria. These beneficial actions on mitochondrial function could be due to inhibition of p-38 MAPK and JNK (Lim et al., 2013; Woodman et al., 2014).
It has been also reported that orientin, glycoside of luteolin, prevented I/R-induced mPTP opening and protected H9c2 cardiomyocytes against I/R injury by inhibiting the mitochondrial apoptotic pathway in a PI3K/Akt-dependent manner (Lu et al., 2011); in fact, PI3K/Akt cascade is deeply involved in the myocardial protection, as suggested from the evidence that its activation is an event upstream of PKC and of mPTP inhibition. Moreover, baicalein, flavonoid of Scutellaria baicalensis, increased Akt phosphorylation, as well as decrease JNK phosphorylation, in H2O2-exposed cardiac cells, suggesting that mechanism of baicalein cardioprotection can reside in this mitochondrial effects (Huang et al, 2014b).
Finally, a less investigated signal transduction pathway is JAK/STAT, implicated mainly in delayed IPreC, since that its activation conveys signals from cell membrane to the nucleus, where gene expression is modulated. At this regard, the knowledge about a flavonoid-induced cardioprotection through this target are really scares, an unique paper reported an increased JAK2/STAT3 phosphorylation by which isoliquiritin, a chalcone, protects the heart against I/R injury (Zhang et al., 2013).
The experimental studies carried out on the more significant flavonoids and proving the cardioprotective activity versus ischemic insult are reported below, focusing the attention on the specific mitochondrial targets summarized in figure 3.
- Quercetin-induced cardioprotective effects
Quercetin possesses beneficial effects on rat hearts submitted to I/R period; indeed its administration, either 15 min before ischemia or during the entire reperfusion period, improved the post-ischemic recovery of functional parameters (Bartekova et al., 2010). In another paper, quercetin and also rutin showed, on in vivo model of coronary occlusion, cardioprotective property either on normal or on streptozotocin-induced type I diabetic rats; such an effect emerged from the evaluation of infarct size and the oxidative stress markers (Annapurna et al., 2009).
Besides the papers attributing the cardioprotection of quercetin to its antioxidant properties, Jin and his colleagues have more recently demonstrated that the antiischemic effects of this flavonoid are mediated by attenuation of the expression of inflammatory mediators, such as TNF-α, IL-10 and cytokines (Jin et al., 2012).
Moreover, quercetin was also able to mimic the IPostC phenomena, acting on the PI3K/Akt signaling pathway and influencing the mitochondrial expression of Bcl2/Bax proteins (Wang et al., 2013). Noteworthy, Brookes et al. demonstrated that quercetin had in rats a good bioavailability after oral administration; in fact it promoted cardioprotection after treating for 4 days with oral low dosage (equivalent to drinking 1-2 glasses of red wine). In this study, the authors observed an improvement of heart functional parameters, together with significant protection of mitochondrial function; suggesting that a daily use of quercetin-rich foods may ensure a myocardial protection against I/R events (Brookes et al., 2002).
- Luteolin-induced cardioprotective effects
Liao’s group have published a paper reporting the luteolin-induced cardioprotection, where it was administered 15 minutes before occlusion of the coronary artery, in order to mimic the IPreC. Luteolin, significantly suppressed the incidence and duration of ischemic arrhythmias, decreased blood LDH levels, an indicator of cellular damage, and reduced the infarct injury. Moreover, the authors found lower levels of malonyldialdehide, an oxidative stress marker, hypothesizing that the
antioxidant activity played a pivotal role in cardioprotection (Liao et al., 2011). Furthermore, protective effects of luteolin have been also demonstrated on adult rat cardiomyocytes underwent to I/R, where it reduced apoptotic and necrosis markers (Qi et al., 2011). Fang et al., suggested that luteolin-induced myocardial protection was at least partly mediated by the activation of PI3K/Akt pathway, since the treatment with specific inhibitors abolished the its effect (Fang et al., 2011). However, today the exact mechanism needs further investigation; since the involvement of other kinases, such as MAPK, JAK/STAT have been considered, but not yet clarified (Xu et al., 2012). Again, in 2013 it has been published that luteolin-mediated cardioprotection is due to mechanisms different from the antioxidant ones, involving the preservation of the integrity of mitochondrial membranes (Madesh and Vaiyapuri, 2013).
- Green tea flavonoids-induced cardioprotective effects
Epigallocatechin-3-gallate, the major flavonoid in green tea, attenuates myocardial I/R injury in several animal species (Hirai et al., 2007), through the mitoKATP channel activation. Indeed, either the non-selective KATP channel blocker, glibenclamide and the selective mitoKATP channel blocker, 5-HD, abrogated the epigallocatechin-3-gallate-induced cardioprotection (Song et al., 2010). A similar mechanism of protection is exhibited by theaflavin, another polyphenol typical of green tea, it protects the rat heart against I/R injury through the opening of mitoKATP channel and the inhibition of mPTP (Ma et al., 2011).
Suzuki group found also another specific mechanism of epigallocatechin-3gallate, that can account for the protective effect on myocardial ischemia, involving the activation of STAT1 (Darra et al., 2007).
Recently, another catechin present in cacao, (-)epicatechin, has been reported for its cardioprotective effects on a rodent model of I/R injury. The results showed a significant reduction in infarct size and such an effect was accompanied by activation of the reperfusion injury salvage kinase (RISK) pathway, involving Akt and ERK proteins (Yamazaki et al., 2008; Hausenloy and
Yellon, 2007). Moreover, (-)epicatechin could confer cardioprotection in a permanent coronary occlusion model, by an unclear and unrelated to Akt or ERK activation mechanism (Yamazaki et al., 2010).
Noteworthy, in 2010 a novel target of (-)epicatechin, accounting for its cardioprotection, has been discovered: the activation of delta-opioid receptor and the related kinase signaling cascade (Panneerelvam et al., 2010).
- Puerarin-induced cardioprotective effects
Puerarin is an isoflavone endowed with cardioprotective property, by means specific effects at the mitochondrial level. Indeed, in mitochondria isolated from hearts pretreated with puerarin, a significant inhibition of the calcium-induced swelling was observed, and this effect was attenuated by 5-HD, demonstrating the involvement of mitoKATP channels. Moreover, puerarin prevented the ischemia-induced apoptosis and atractyloside (mPTP activator) inhibited this effect (Gao et al., 2006).
Therefore, the same research group reported the involvement of another mitochondrial potassium channel, the mitoBK one, in the cardioprotection of puerarin, it, like the well-known BK channel opener NS1619, attenuated the mitochondrial depolarization induced by H2O2-stress and inhibited the calcium-induced swelling. This effect was antagonized by paxilline, selective blocker of BK channels (Yang et al., 2008).
Intraperitoneal injection of puerarin (120 mg/kg) up-regulated myocardial eNOS gene and protein expression, protein kinase B (Akt/PKB) phosphorylation and subsequently increased serum NO production in rats with myocardial infarction, which might be the possible mechanism underlying the therapeutic action of puerarin in coronary artery diseases (Zhang et al., 2008b).
Very recently, naringenin, a flavanone abundant in the Citrus genus, has been demonstrated to possess cardioprotective activity due to the opening the mitoBK channels. Indeed, the naringenin administration, before the onset of a global ischemia on isolated rat hearts, halved I/R injury size and ameliorated all parameters of myocardial functionality (Testai et al., 2013a). Moreover, in acute infarct model on anaesthetized rats, naringenin showed protective effects. On isolated cardiac mitochondria, it was able to produce all the typical effects of the mitoK channel openings, such as increase of entry of potassium ions, mild mitochondrial depolarization and decrease of calcium uptake. The involvement of mitoBK channel has been suggested by the clear inhibitory effect of selective blockers, paxilline and iberiotoxin (Testai et al., 2013b). More recently, in 2014, naringin, the glycoside of naringenin, has been demonstrated to exhibit protective effects on high glucose-damaged H9c2 cardiac cells through MAPK pathway (Chen et al., 2014); a such hypothesis, if confirmed also in vivo models (in which naringin is hydrolyzed in the aglycone) could mean that the recruitment of MAPK is an upstream event of mitoBK activation.
- Anthocyanin-induced cardioprotective effects
In 2008, Martin’s group treated for 7 days male Wistar rats with a special anthocyanin-rich diet, then the 7th day, the hearts were excised, perfused according to the Langendorff model and submitted to a 30 minutes of regional ischemia and reperfusion for 2 hours. Parallely, another group of treated-rats were anaesthetized and subjected to 30 minutes of coronary occlusion followed by 2 h of reperfusion; finally hearts from both ex-vivo and in vivo protocols were used to measure the extension of the infarct area. This paper, for the first time, provided evidence of an absorption of anthocyanin following oral diet, demonstrating that they are more bioavailability that previously thought, and that a dietary intake of plant-derived anthocyanins can produce cardioprotective effects (Toufektsian et al., 2008). The recent paper of Skemiene and colleagues is in agreement with this finding, furthermore they identify in mitochondrial respiratory chain the target of anthocyanin action (Skemiene et al., 2015).
5-CONCLUSIONS
In the light of the experimental results at today available, it is possible to conclude that, beside the antioxidant activity, the mitochondrial pathway can be a target of the cardioprotection played from certain flavonoids; therefore the mitochondrial pharmacology can be viewed as a new challenging paradigm to understand the numerous properties of flavonoid classes.
As this regard, innovative and challenging drug delivery strategies have been proposed, in order to ameliorate the selective entry of flavonoids in the mitochondria and minimize eventual aspecific and undesired effects. The first strategy consistsin developingflavonoid derivative conjugated with a lipophilic cation triphenylphosphonium (TPP+), that rapidly and extensively accumulated into mitochondria, driven by the mitochondrial membrane potential (about -180 mV; Porteous et al., 2010); a such strategy has been attempted with quercetin, that can then increase its effectiveness at mitochondrial level (Sassi et al., 2012). Another strategy aimed at ameliorating the selective accumulation into mitochondria of a drug consists in the use of nanoparticles. This approach has been recently published by Ghosh et al., the oral treatment with nanoencapsulated quercetin may play a protective role against the oxidative damage in neural I/R induced on young and aged rats (Ghosh et al., 2013).
Both these innovative strategies can contribute to improve the selectivity of action and the poor pharmacokinetic profile of flavonoids, which is often an “Achilles heel”, representing an insurmountable limit for clinical applications.
6-LEGENDS
Figure 1
Diagram showing principal mechanisms through which flavonoids can play protection on myocardium underwent to ischemia-reperfusion injury. Specific effects are emphasized by an uppercase character, moreover in the light blue box, protective functions involving mitochondrial pathways are highlighted.
Figure 2
Chemical structures of the flavonoid classes.
Figure 3
Schematic representation of kinase-dependent mechanisms by means several flavonoids (discussed in the second part of paragraph 3) can play cardioprotective effects. Whole lines indicate that the cardioprotection is mediated by an activating effect played by flavonoid on the route; whereas dashed lines indicate an inhibiting/attenuating effect mediated by flavonoid treatment.
7- REFERENCES
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