Ischemia/reperfusion (I/R) is a common problem encountered in a variety of clinical situations, particularly organ transplantation. I/R results in damage that may affect cell viability and lead to organ failure. I/R injury involves a complex cascade of events, including loss of energy, derangement of the ionic homeostasis, production of ROS and cell death. There is ample evidence demonstrating that ROS play an important role in producing lethal cell injury associated with heart ischemia/reperfusion. The mechanisms for the enhanced ROS generation and cellular and subcellular target of ROS attack are not well established. One of the main sources of ROS in cardiomyocytes during ischemia and early reperfusion may be a defect in the mitochondrial ETC. activity, particularly at the level of complexes I and III, the main sites of ROS production. As major source of ROS production mitochondria could be major target of ROS attack. Given that ROS are a highly reactive and short-lived species, their effect should be greatest in an immediate area surrounding the locus of ROS production. It is conceivable that mitochondrial membrane constituents, including the ETC. complexes and phospholipid constituents could be the major target of ROS attack. Alterations in the activity of respiratory chain complexes in mitochondria isolated from ischemic and ischemic/reperfused rat heart have been reported [32, 35, 36, 107].
Several recent publications present evidence that melatonin has significant protective action against the cardiac damage and altered physiology that occur during ischemia/reperfusion injury [108–110]. This protective effect of melatonin has been also demonstrated in ischemia/reperfusion of rat liver . Our studies have demonstrated that melatonin, at pharmacological concentrations, strongly protects against ischemic/reperfusion myocardial damage . This protective effect of melatonin appears to be produced through its action at mitochondrial level. In fact, melatonin treatment of ischemic/reperfused rat heart, significantly lowered the degree of lipid peroxidation, counteracted the reduction in state 3 respiration and the associated decrease in the respiratory control ratio and prevented the loss in complex I and complex III activities in isolated rats heart mitochondria. In addition, melatonin treatment largely prevented the increase in the H2O2 production, the loss in CL content and the increase in its level of peroxidation. The protective effect of melatonin against these mitochondrial bioenergetic alterations was also demonstrated in ‘in vitro’ experiments on isolated rat heart mitochondria. The protection afforded by melatonin against mitochondrial dysfunction that occurs during cardiac ischemia/reperfusion injury was associated with an improvement of postischemic hemodynamic function of the heart as reflected in the greater LVDp and lower LVEDp. The mechanism of protection is likely to be due, at least in part, to the antioxidant efficacy of melatonin preventing CL peroxidation. Together, these data demonstrate that melatonin, at pharmacological concentrations, strongly protects against the alterations to oxidative metabolism, thus limiting cardiac reperfusion injury.
The lipophicity of melatonin allows it to accumulate in the inner mitochondrial membrane, where it scavenges reactive species generated during respiration. Thus, in addition to preventing CL peroxidation, melatonin may also protect directly mitochondrial ETC. complexes from oxidative damage, and this may contribute to protect mitochondrial function. Melatonin has also been shown to increase, in vitro, the activity of the mitochondrial respiratory complexes I and IV [91, 92]. Therefore, the ability of melatonin to directly influence the activity of these respiratory complexes could also contribute to the observed protective effect of this indoleamine against mitochondrial dysfunction associated with heart ischemia/reperfusion.
Cardiolipin, melatonin and mitochondrial permeability transition
Under physiological condition, the mitochondrial inner membrane is impermeable to almost all metabolites and ions. However, under conditions of high matrix Ca2+, especially when associated with enhanced ROS production, high phosphate, and low adenine nucleotide concentrations, an unspecific pore opens that allows passive diffusion of any molecules of <1.5 KDa, thus disrupting the permeability barrier of the inner mitochondrial membrane. This leads to the disruption of ionic homeostasis and uncoupling of oxidative phosphorylation causing irreversible damage to mitochondria resulting in necrotic cell death [112, 113]. The molecular structure of the mitochondrial permeability transition pore (MPTP) remains still uncertain. However, the cyclofillin D, the ANT and the voltage-dependent anion channel (VDAC) appear to be key structural components, while other proteins such as benzodiazepine receptors, exokinase and creatine kinase, Bax and Bcl2 may play a regulatory role .
Multiple studies point to a role of the MPTP in the early stages of the apoptotic or necrotic pathway of cell death [114–116]. Release of cytochrome c, as well as other proteins, from the mitochondria to the cytosol, appears to be a central event in the induction of the apoptotic cascade that ultimately leads to the programmed cell death. Cytochrome c, a component of the respiratory chain, is normally bound to the outer surface of the inner mitochondrial membrane primarily to CL molecules . Oxidation of CL promotes the detachment of cytochrome c from mitochondria [118, 119]. It is now accepted that cytochrome c release during apoptosis occurs via a two-step process involving first, the dissociation of the hemoprotein from the outer surface of the inner mitochondrial membrane, followed by permeabilization of the outer mitochondrial membrane and the release of cytochrome c into the extramitochondrial space [41, 114]. CL peroxidation may be involved in the permeabilization of the outer mitochondrial membrane, probably through its association with Bcl2 family proteins such as Bax and Bid [39, 114, 120].
Recently, we reported that oxidized CL, added exogenously to heart mitochondria, in the presence of Ca2+, promotes the mitochondrial permeability transition pore opening and the release of cytochrome c . These effects seem to involve an interaction of these two agents with the ADP/ATP carrier, which is considered a component of the MPTP. The involvement of oxidized CL in MPTP opening is further supported by our recent study showing that oxidation of intramitochondrial CL molecules results in MPTP induction . In fact, peroxidation of intramitochondrial CL molecules after exposure of mitochondria to t-BuOOH, in the presence of low concentrations of Ca2+, results in matrix swelling, ΔΨ collapse, release of preaccumulated Ca2+, and release of cytochrome c. All these events are inhibited by cyclosporine A, indicating that peroxidation of CL molecules, in the presence of Ca2+, induces MPTP opening and cytochrome c release. Melatonin, at micromolar concentration, inhibited the Ca2+/t-BuOOH MPTP induction as shown by its protective effect on matrix swelling, ΔΨ collapse and release of preaccumulated Ca2+. Moreover, melatonin inhibited CL peroxidation in Ca2+/tBuOOH-treated mitochondria. These results indicate that melatonin inhibits MPTP opening and this effect is most likely related to its ability to prevent CL peroxidation. Inhibition of MPTP opening by melatonin has been also reported in other studies [123–125]. In addition, our results demonstrate that the release of cytochrome c from mitochondria, after treatment of these organelles with Ca2+/t-BuOOH, is almost completely prevented by melatonin. This effect of melatonin can be explained on its ability to inhibit CL peroxidation, thereby preventing both cytochrome c detachment from the mitochondrial inner membrane and MPTP induction. Together, our results demonstrate that peroxidation of mitochondrial CL in the presence of Ca2+ induces MPTP opening and cytochrome c release from mitochondria. Melatonin, at micromolar concentrations is able to prevent CL peroxidation in mitochondria and this effect seems to be responsible for the protection afforded by this agent against the Ca2+/peroxidized CL -dependent MPTP opening and mitochondrial cytochrome c release. This effect of melatonin may have important implications in those physiopathological situations characterized by alterations of Ca2+ homeostasis and accumulation of peroxidized CL in mitochondria, such as ischemia/reperfusion, aging and other degenerative diseases.
Melatonin, cardiolipin and MPTP in heart ischemia/reperfusion
Growing evidence is available supporting a crucial role of mitochondrial MPTP in cardyomyocites cell death occurring during ischemia/reperfusion (I/R) [126, 127]. It has been suggested that MPTP remains closed during the ischemic period, because of the lactate-induced acid conditions. At reperfusion, there is an influx of Ca2+ into mitochondria, a burst of ROS production and rapid correction of acidosis. All these factors contribute to increase the probability of MPTP opening in the early minutes of reperfusion, following a prolonged period of ischemia. Identification and characterization of agents and interventions that can protect the heart from the damaging effect of MPTP are of considerable importance in attenuating I/R induced cardiac dysfunction. The use of antioxidants to prevent cardiac I/R injury via MPTP inhibition appears to be a logical measure to improve myocyte survival. It is now known that available antioxidants have not proven to be especially effective against I/R damage. It is possible that most antioxidants are not freely accessible to mitochondria, the main source of ROS production. The search for mitochondrial target antioxidants is now increasing. As mentioned earlier, melatonin is an antioxidant that can easily reach mitochondria where it seems to accumulate in high concentrations.
Very recently, we have demonstrated that melatonin protects against heart I/R injury by inhibiting MPTP opening . Melatonin treatment significantly improves the functional recovery of Langendorff hearts on reperfusion, reduces the infarct size and decreases necrotic damage, as shown by the reduced release of lactate dehydrogenase [22, 128]. All these effects were accompanied by the inhibition of the MPTP opening detected in situ by the mitochondrial release of NAD+. Furthermore, mitochondria isolated from melatonin-reperfused heart were less sensitive than mitochondria from untreated reperfused heart to Ca2+-induced MPTP opening, as assessed by the CRC (calcium retention capacity). CRC is a sensitive and quantitative measure of the ability of mitochondria to open the MPTP in response to accumulation of Ca2+. Together, these results demonstrate that melatonin protects against heart I/R injury by inhibiting MPTP opening.
The possible mechanism involved in the inhibition of MPTP opening during I/R by melatonin treatment was also investigated . Although Ca2+ overload is considered an important factor responsible for MPTP opening during I/R, it is now accepted that additional factors may contribute to the MPTP opening during heart I/R. As described earlier, oxidized CL behaves as an inducer of MPTP in isolated rat heart mitochondria. Thus, it is possible that an increased level of oxidized CL may increase the probability of MPTP opening during I/R. The mitochondrial content of oxidized CL increases upon reperfusion, while melatonin treatment completely counteracts this effect. The fact that melatonin treatment inhibits both MPTP opening and CL peroxidation following I/R suggests a possible link between these two processes. On this basis, it is plausible that an increased level of peroxidized CL, together with Ca2+ overload, synergistically contribute to MPTP opening during reperfusion and that melatonin treatment inhibits MPTP opening by preventing the oxidative damage to CL. The inhibitory effect of melatonin on mitochondrial CL peroxidation during reperfusion can be reasonably explained on the ability of this indoleamine to inhibit the oxidation of linoleic fatty acid constituents of this phospholipid, as demonstrated in a model system in vitro .
In addition to its inhibitory effect on MPTP opening, melatonin inhibits also the release of cytochrome c from mitochondria following I/R. This effect of melatonin can be explained on its ability to inhibit the oxidation of CL thereby preventing cytochrome c detachment from mitochondrial membrane, an event that is considered the initial step in the process of cytochrome c release from mitochondria [39–41]. Thus, the inhibition of cytochrome c release by melatonin treatment may contribute, together to the inhibition of MPT opening, to the protective effect exerted by this compound against mitochondrial dysfunction associated with I/R injury. The powerful mitochondrial protection provided by melatonin against I/R injury, reinforces its therapeutic potential to combat a variety of oxidative stress–induced mitochondrial dysfunctions, as well as mitochondrial-mediated apoptosis in various diseases.
Melatonin, cardiolipin and electron transport chain in aging
Aging is a natural, complex and multifactorial biological process associated with impairment of bioenergetic function, increased oxidative stress, attenuated ability to respond to stresses and increased risk of contracting age-associated diseases. According to the free radical theory of aging, ROS, generated as by-products of biological oxidations, cause random and cumulative oxidative damage to macromolecules, leading to cellular dysfunction with age and eventually cell death . Mitochondria seem to be intimately involved in the aging process because these organelles are considered the main intracellular source of ROS, as well as the major target of free radicals’ attack. According to the mitochondrial theory of aging, ROS produced by the mitochondrial respiratory chain attack mitochondrial constituents including proteins, lipids and mitochondrial DNA [130, 131]. Accumulation of mtDNA mutations may lead to impairment of the respiratory chain complexes, leading to increased mitochondrial ROS production and subsequent accumulation of more mitochondrial DNA mutations . This vicious cycle has been proposed to account for the increased oxidative damage during aging, which leads to the progressive decline of cellular and tissue function as a result of insufficient supply of energy and/or increased susceptibility to apoptosis [133, 134]. The age-related increase in oxidative damage to DNA, lipids and proteins has been well documented  along with evidences supporting increased mtDNA deletions  and mitochondrial dysfunction with aging [137, 138].
Mitochondrial membrane phospholipids play a causal role in aging and longevity by modulating oxidative stress and molecular integrity. Because of their high content of unsaturated fatty acids and to their location in the inner mitochondrial membrane near to the site of ROS production, CL molecules are particularly prone to peroxidative attack by ROS. It is conceivable that CL may represent an important target of ROS attack in aging. Oxidation/depletion of CL with aging, would negatively impact the biochemical function of mitochondrial membranes leading to cellular dysfunction and eventually cell death. The content of mitochondrial CL was found to decrease in heart  and brain tissues  of aged animals, associated with an increase in the level of oxidized CL.
A widely recognized experimental fact concerning aging and energy transduction is that a decreased electron transport activity is observed in mitochondrial membranes isolated from rats and mice tissues upon aging, although there is little agreement regarding changes in various electron transport complexes activities with age. Of the five respiratory chain complexes, complex I and IV show a selectively reduced enzymatic activity in mitochondria isolated from different tissues of animals upon aging [68, 69, 139, 140]. Attempts to identify some of the mechanisms responsible for these age-related declines in activity indicate that one possible factor responsible for the reduction in complex I and IV activity might be the oxidation/depletion of mitochondrial CL [68, 69]. This assumption is supported by the finding that CL -liposomes, added exogenously to mitochondria from aged rats, almost completely restore the activity of these enzyme complexes to the value of young control animals. This effect of CL could not be replaced by other major mitochondrial phospholipid constituents such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) nor by peroxidized CL. Altogether, these results clearly indicate that the defect in complex I and IV activities, observed in mitochondrial from aged rats, could be ascribed, at least in part, to oxidative damage to mitochondrial CL, because of ROS attack to its linoleic fatty acid constituents.
Several properties of melatonin indicate that this compound may be beneficial in aging. Serum levels of melatonin significantly decrease in aged animals compared with young animals [141, 142]. In humans, the total antioxidative capacity of serum correlates well with its melatonin levels. Thus, the reduction in melatonin with age may be the factor in the elevated oxidative damage observed in the elderly. Recent studies, using the senescence-accelerated mouse (SAM), investigated the effects of chronic administration of physiological doses of melatonin on mitochondrial oxidative stress and function . The results of this study show an age-dependent oxidative damage in the heart of SAM mice, which was accompanied by a reduction in the electron transport chain complexes activity and in ATP levels. Chronic melatonin administration between 1 and 10 months of age normalized the redox and the bioenergetic status of the mitochondria and increased ATP levels. These results support the beneficial effect of chronic pharmacological intervention with melatonin, which reduces the deteriorative and functional oxidative changes in heart mitochondria with aging .
A large body of experimental evidence suggests that mitochondrial decay is a major contributor to brain tissue alterations associated with aging . A potential role of melatonin in mitigation of changes associated with brain aging has been described [144, 145]. In addition, numerous investigations have identified melatonin as a potential mitochondria-targeted protector to defend against various oxidative stress–associated brain diseases. In a recent study, we demonstrated a decline in the complex I activity and in state 3 respiration and an increase in H2O2 production in brain mitochondria isolated from aged rats . The molecular basis of this decline was ascribed to oxidation/depletion of CL. The alterations to mitochondrial bioenergetic parameters associated with rat brain aging were counteracted by long-term melatonin administration. This protective effect of melatonin appears to be because of melatonin’s ability to directly prevent CL oxidation/depletion in mitochondria, as also supported by results of ‘in vitro’ experiments .
Functional impairment of mitochondrial complex I has been also associated with a wide spectrum of age-related neurodegenerative disorders, in particular Parkinson’s disease [146, 147]. Moreover, it has been reported that complex I deficiency, which occurs in numerous neurodegenerative situations, sensitizes neurons to the action of death agonists such as Bax, through mitochondrial CL peroxidation . Thus, melatonin treatment, by preventing age-associated complex I deficiency, may improve mitochondrial physiology in brain aging and age-associated brain diseases.
The functional alterations to the mitochondrial respiratory chain complexes, observed in heart I/R and heart and brain tissues of old animals, because of oxidation/depletion of CL molecules, may increase the electron leak from the electron transport chain, generating more O2·− and perpetuating a cycle of oxygen radical-induced damage that ultimately leads to mitochondrial dysfunction. Melatonin, by inhibiting CL peroxidation, would prevent respiratory chain dysfunction and subsequent ROS production, thus interrupting this sequence of events (Fig. 1). This would have beneficial effects in mitochondrial physiopathology. Therefore, melatonin administration might represent a valid therapeutic strategy for combating neurodegenerative and cardiovascular disorders associated with heart and brain aging, in which, both mitochondrial respiratory chain deficiency and CL peroxidation could play a critical role.
Figure 1. Schematic diagram of the role of ROS and cardiolipin peroxidation in mitochondrial dysfunction associated with heart ischemia/reperfusion and heart and brain aging and possible mechanism of melatonin protection. Mel = melatonin; CL = cardiolipin; CLOOH = peroxidized cardiolipin; ROS = reactive oxygen species.
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Melatonin, cardiolipin and MPTP in aging
The mitochondrial permeability transition may be a factor in cardiac dysfunction associated with aging. Given the association between deficits of mitochondrial function and aging, an important question is whether MPTP becomes more inducible with aging. An increased susceptibility to Ca2+-induced MPTP opening has been shown in mitochondria isolated from different tissues such as liver, brain and heart of old animals [149–153], although the mechanisms underlying this age-related mitochondrial alterations are still not completely elucidated. As mentioned earlier, oxidized CL behaves as an inducer of MPTP in isolated rat heart mitochondria . This effect of oxidized CL on MPTP is also associated with the release of cytochrome c from mitochondria. It is thus conceivable that an increased level of oxidized CL with age could enhance the susceptibility to Ca2+-induced MPTP opening and promote the release of cytochrome c from mitochondria. In addition, melatonin inhibits CL peroxidation in rat heart mitochondria and prevents MPTP and cytochrome c release .
Very recently, we found that heart aging is associated with an increased susceptibility to Ca2+-induced permeability transition and to cytochrome c release from mitochondria (G.Petrosillo, N. Moro, V. Paradies, F.M. Ruggiero and G. Paradies, personal observations). These events may be related to the observed increased extent of apoptosis in aged heart . Long-term treatment with melatonin counteracts both these events. This protective effect of melatonin might be explained on the ability by this indoleamine to inhibit the age-related oxidation of CL, thereby preventing both the detachment of cytochrome c from the inner mitochondrial membrane and its release through the MPTP opening. Figure 2 summarizes the processes involved in melatonin’s actions in preventing ROS-mediated, mitochondrial-dependent cellular apoptosis via inhibition of CL peroxidation.
Figure 2. Role of ROS and cardiolipin peroxidation on MPTP opening and cytochrome c release from mitochondria and the effects of melatonin. Respiratory chain–mediated ROS production causes cardiolipin peroxidation which, in turn, promotes the detachment of cytochrome c from the inner mitochondrial membrane, the opening of the MPTP and the release of cytochrome c from the mitochondria. Melatonin prevents this cascade of events by inhibiting cardiolipin peroxidation. ETC. = electron transport chain; ROS = reactive oxygen species; cyt. C = cytochrome c; CL = cardiolipin; CLOOH = peroxidized cardiolipin; MPTP = mitochondrial permeability transition pore; Mel = melatonin.
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The fact that heart mitochondria from old rats are more susceptible to Ca2+-induced activation of MPTP and to cytochrome c release might have important implications in necrotic and apoptotic myocyte cell death associated with ischemia/reperfusion injury as well as with other age-related cardiovascular disorders. Pharmacological intervention with melatonin might help to ameliorate these age-related cardiovascular disorders.