Protective role of melatonin in mitochondrial dysfunction
and related disorders
Giuseppe Paradies · Valeria Paradies ·
Francesca M. Ruggiero · Giuseppe Petrosillo
Received: 14 January 2015 / Accepted: 9 February 2015
© Springer-Verlag Berlin Heidelberg 2015
through which melatonin exerts its protective role in mito-
chondrial dysfunction and related disorders are reviewed.
Keywords Melatonin · Mitochondria bioenergetics ·
Cardiolipin · Oxidative stress · Physiopathology
Melatonin (N-acetyl-5-methoxytryptamine) is a highly
conserved molecule derived from tryptophan that is found
in all organisms from unicells to vertebrates (Hardeland
; Hardeland et al.
; Tan et al.
Melatonin and its metabolites can function as endogenous
free radical scavengers and broad-spectrum antioxidants
(Tan et al.
; Reiter et al.
and amphiphilic nature, melatonin can reach numerous
cellular and subcellular compartments, particularly mito-
chondria (Menendez-Pelaez and Reiter
), raising the
possibility of functional significance for this targeting with
involvement in situ in mitochondrial bioenergetic processes
(Leon et al.
; Paradies et al.
). Most of the ben-
eficial consequences resulting from melatonin administra-
tion may depend on its effect on mitochondrial physiology
(Acuña-Castroviejo et al.
Mitochondrial dysfunction is considered an important
contributing factor in a variety of physiopathological situ-
ations such as neurodegenerative and cardiovascular dis-
eases, diabetes, various forms of hepatic disorders, skeletal
muscle disorders, as well as in aging (Beal
; Schon et al.
). Alterations in mitochondrial
function such as defects in the electron transport chain
(ETC) activity and oxidative phosphorylation (OXPHOS)
have all been suggested as the primary causative factors in
the pathogenesis of these disorders.
Abstract Mitochondria are the powerhouse of the eukar-
yotic cell through their use of oxidative phosphorylation to
generate ATP. Mitochondrial dysfunction is considered an
important contributing factor in a variety of physiopatho-
logical situations such as aging, heart ischemia/reperfusion
injury, diabetes and several neurodegenerative and cardio-
vascular diseases, as well as in cell death. Increased forma-
tion of reactive oxygen species, altered respiratory chain
complexes activity and opening of the mitochondrial per-
meability transition pore have been suggested as possible
factors responsible for impaired mitochondrial function.
Therefore, preventing mitochondrial dysfunction could be
an effective therapeutic strategy against cellular degenera-
tive processes. Cardiolipin is a unique phospholipid located
at the level of inner mitochondrial membrane where it
plays an important role in mitochondrial bioenergetics, as
well as in cell death. Cardiolipin abnormalities have been
associated with mitochondrial dysfunction in a variety of
pathological conditions and aging. Melatonin, the major
secretory product of the pineal gland, is a well-known
antioxidant agent and thus an effective protector of mito-
chondrial bioenergetic function. Melatonin was reported to
prevent mitochondrial dysfunction from oxidative damage
by preserving cardiolipin integrity, and this may explain,
at least in part, the beneficial effect of this compound in
mitochondrial physiopathology. In this article, mechanisms
G. Paradies (*) · V. Paradies · F. M. Ruggiero
Department of Biosciences, Biotechnologies
and Biopharmaceutics, University of Bari, Bari, Italy
G. Petrosillo (*)
Institute of Biomembranes and Bioenergetics, National Research
Council, Bari, Italy
Mitochondria are considered the main intracellular
source of reactive oxygen species (ROS), as well as the
major target of free radical attach. ROS are generated at
very low levels during mitochondrial respiration under
normal physiological conditions, while the level of these
oxidants increases in several pathological conditions and
aging. ROS produced by the mitochondrial ETC attach
mitochondrial constituents including proteins, lipids and
mitochondrial DNA (mtDNA). ROS-induced alterations to
mitochondrial membrane constituents may lead to a decline
of the mitochondrial bioenergetic function, and this may
contribute to the etiology of a variety of pathological con-
ditions, including heart ischemia/reperfusion (Chen and
), aging and age-related cardiovascular and
neurodegenerative diseases (Boveris and Navarro
DiMauro and Schon
Recent studies have demonstrated that melatonin plays
an effective role in preserving mitochondrial homeosta-
sis (Martín et al.
; López et al.
; Paradies et al.
; Acuña-Castroviejo et al.
), which may explain the protective effect of this
molecule in several physiopathological conditions includ-
ing neurological (Patki and Lau
; Pandi-Perumal et al.
; Cardinali et al.
) and cardiovascular disorders
(Dominguez-Rodriguez and Abreu-Gonzalez
), all of which are associated with mitochon-
drial dysfunction. This protective effect of melatonin may
be explained, at least in part, on its antioxidant and free
radical scavenging properties, thus preserving the stability,
integrity and function of mitochondrial membranes (García
; Reiter et al.
Cardiolipin (CL), a unique phospholipid located at the
level of the inner mitochondrial membrane (IMM), has
been shown to play a central role in the mitochondrial func-
; Houtkooper and Vaz
; Ren et al.
). Abnormalities in CL not only
alter fluidity and folding of the IMM, but can profoundly
alter the organization and function of the respiratory chain
complexes and/or their organization in supramolecular
structure, such as supercomplexes (Musatov and Robinson
). In particular,
oxidation and depletion of CL have been associated with
mitochondrial dysfunction in several metabolic and degen-
erative diseases (Chicco and Sparagna
; Paradies et al.
). Recently, melatonin was reported to preserve
mitochondrial CL from oxidative damage, and this may
explain, at least in part, the beneficial effect of this mol-
ecule in mitochondrial dysfunction and associated disor-
ders (Petrosillo et al.
; Paradies et al.
). In this
review, we discuss the several mechanisms through which
melatonin can exert its protective role in mitochondrial
dysfunction and related disorders.
Mitochondrial function and ROS generation
Mitochondria contain multiple copies of circular genome
known as mtDNA as it has been characterized in humans
(Anderson et al.
). The majority of mitochondrial pro-
teins needed for normal bioenergetic processes are encoded
by nuclear DNA, while some proteins essential for ETC
and OXPHOS are encoded by mtDNA. Human mtDNA
encodes for 13 polypeptides of subunits of complexes I,
III and IV and ATP synthase, 22 tRNA and two ribosomal
Mitochondria play a central role in energy-generating
processes within the cell through the ETC, the primary
function of which is ATP synthesis through the OXPHOS
process. The ETC, which is located in the IMM, comprises
a series of electron carriers grouped into four enzyme com-
plexes, namely complex I (CI) (NADH-ubiquinone reduc-
tase), complex II (CII) (succinate-ubiquinone reductase),
complex III (CIII) (ubiquinol-cytochrome c reductase)
and complex IV(CIV) (cytochrome c oxidase) (Lenaz and
). The electrons are transferred to molecular
oxygen via the electron transport complexes, resulting in
the reduction of oxygen to water at complex IV. During this
process, protons (H
) are pumped by CI, CIII and CIV into
the intermembrane space to form an electrochemical gra-
dient, which is the major contributor to the mitochondrial
inner membrane potential. Complex V (CV) (ATP syn-
thase) utilizes the stored energy of this proton gradient to
drive the formation of ATP from ADP and inorganic phos-
phate. ATP formed is then transferred by the ADP/ATP car-
rier (ANT) to the intermembrane space in exchange with
It is estimated that around 0.2–2 % of the oxygen taken
up by the cell is converted by mitochondria to ROS, mainly
through the production of O
(Boveris and Chance
OXPHOS, however, comes with an additional cost, the pro-
duction of potentially harmful ROS. Mitochondrial ETC is
considered the main source of ROS production. The pri-
mary ROS generated into the mitochondria is superoxide
), which is then converted to hydrogen peroxide
) by spontaneous dismutation or by superoxide dis-
mutase (SOD). Hydrogen peroxide in turn is broken down
into water by glutathione peroxidase or catalase; other-
can undergo Fenton’s reaction in the presence
of divalent cations such as iron to produce hydroxyl radical
(·OH), which can be even more harmful to the mitochon-
drial biomolecules. The sites of superoxide anion produc-
tion along the ETC have been the subject of many studies
(for a recent review, see Murphy
). The two major sites
production are complex I and complex III. Mito-
chondria can produce O
, predominantly from complex
I, when the matrix NADH/NAD+ ratio is high, leading to
a reduced FMN site on complex I, and when they have a
high proton-motive force and a reduced coenzyme Q pool,
leading to reverse electron transport. The site of superoxide
production at complex III is probably the unstable ubisem-
iquinone molecules (Turrens et al.
) or cytochrome b
). The production of O
at complex I
is believed to occur at the matrix site of the IMM. At com-
plex III, O
is released to both the matrix and the cytosolic
sides of the IMM. The relative contributions of complex I
and III to ROS production appear also to be dependent on
types of tissues, species and experimental conditions. The
rate of ROS production is also affected by mitochondrial
metabolic conditions. O
production is highest under state
4 respiration; when oxygen consumption is low, the proton-
motive force is high and ETC complexes are in reduced
; Korshunov et al.
). ROS are
also produced to a lesser extent outside of the mitochon-
drion. Examples of extra-mitochondrial ROS producing
reactions include xanthine oxidase, D-amino oxidase, the
P-450 cytochromes and proline and lysine hydroxylase.
Mitochondria can also produce nitric oxide (NO) from
mitochondrial nitric oxide synthase (Ghafourifar and Rich-
; Giulivi et al.
-arginine. NO can be then converted to various reactive
nitrogen species (RNS) such as nitroxyl anion (NO
) or the
toxic peroxynitrite (ONOO
). The oxidizing reactivity of
is generally considered equivalent to that of
NO strongly interferes with components of the ETC, in
particular with cytochrome c oxidase (Mander and Brown
not only interferes
with respiratory complexes, but can also trigger free radi-
cal-mediated chain reactions that in turn damage proteins,
lipids and DNA molecules (Rubbo et al.
; Levine and
). Damage to the mitochondrial respiratory
chain can cause a collapse of membrane potential with fur-
ther generation of free radicals, triggering a vicious cycle
that ultimately leads to cell death.
Mitochondrial antioxidant defense systems
Mitochondria are equipped with an intricate array of enzy-
matic and nonenzymatic antioxidant defense systems
poised to detoxify the ROS/RNS production. Nonenzy-
matic components of the system include hydrophilic and
lipophilic radical scavengers, such as cytochrome c, alpha-
tocopherol, ascorbate, ubiquinone, glutathione and mela-
tonin. Another specific mitochondrial defense mechanism
is the mild uncoupling that prevents marked increase in
membrane potential and hence O
components of the antioxidant systems include manganese-
superoxide dismutase (Mn-SOD), catalase, glutathione
peroxidase and phospholipid hydroperoxide glutathione
peroxidase. Within the mitochondrial matrix, Mn-SOD
, which can be further metabolized
by glutathione peroxidase (Gpx I) and peroxiredoxine
(Prx III) or diffuse from the mitochondria into the cytosol.
Part of the O
produced by the mitochondrial ETC can be
released into the inner membrane space where it can be
converted to H
by Cu–Zn SOD. The O
present in the
intermembrane space could be scavenged by the oxidized
form of the cytochrome c or diffuse into the cytosol through
the voltage-dependent anion channel (VDAC; Madesh and
may also react with nitric oxide
(NO) to form highly reactive ONOO
. Glutathione (GSH)
and multiple GSH-linked antioxidant enzymes exert also
an important mitochondrial antioxidant protection. Among
GSH-linked enzymes involved in mitochondrial antioxi-
dant defense are Gpx 1 located predominantly in the cyto-
sol and Gpx 4, also known as phospholipid hydroperoxide
glutathione peroxidase, which is associated with contact
sites of the two mitochondrial membranes. These enzymes
catalyze the reduction of H
and of lipid hydroperoxides.
Gpx 4 reduces hydroperoxide groups on phospholipids,
lipoproteins and cholesteryl esters. Gpx 4 is considered to
be the primary enzymatic defense mechanism against oxi-
dative damage to cellular membranes.
The redox cycling in the mitochondria is very active and
serves to prevent significant loss of GSH. Melatonin pro-
motes de novo synthesis of GSH by stimulating the activ-
ity of the enzyme γ-glutamyl-cisteine synthase (Urata et al.
) and also through its effect on gene expression of
Gpx, SOD and catalase (Antolín et al.
; Reiter et al.
), thus favoring the recycling of GSH and maintain-
ing high GSH/GSSG ratio. These effects of melatonin may
have important implications in mitochondrial physiology
(Escames et al.
Melatonin and mitochondrial oxidative stress
Mitochondria are the most powerful intracellular source
of ROS and also the primary target for their damaging
effects. The interaction of ROS with mitochondrial compo-
nents impairs the function of these organelles and directly
affects cell viability and triggers cell death. ROS-induced
structural and functional modification of proteins is one of
the hallmarks of aging and several pathological disorders
in biological systems (Stadtman
). One important tar-
get of ROS is mtDNA, which encodes polypeptides that
are essential for ETC and ATP generation by OXPHOS.
MtDNA is particularly susceptible to attack by ROS
because of its proximity to the ETC and lack of protective
histones. MtDNA therefore represents a critical cellular tar-
get for oxidative damage that could lead to lethal cell injury
through the disruption of electron transport, mitochondrial
membrane potential and ATP generation. ROS-induced
mtDNA damage is probably a major source of mitochon-
drial genomic instability responsible for the mitochondrial
dysfunction. The instability of mtDNA is thought to be one
of the most important factors in aging (Wei and Lee
In addition to mtDNA and proteins, mitochondrial
membrane lipids are highly susceptible to oxidative dam-
age. Phospholipids are the most abundant lipid compo-
nents of the cellular and subcellular membranes, includ-
ing mitochondria. Phospholipids are essential structural
components of the mitochondrial membranes where they
play multiple roles. They define the essential membrane
permeability barrier and modulate the proper membrane
fluidity, which is required for the optimal functional activi-
ties of proteins and enzymes. Polyunsaturated fatty acids
(PUFAs) are essential components of mitochondrial phos-
pholipids. The presence of a methylene bridge between
two double bonds renders the PUFAs particularly sensitive
to ROS attack, enabling them to participate in long free
radical chain reactions generating hydroperoxides as well
as endoperoxides. These lipid peroxidation products can
undergo fragmentation to produce a broad range of reactive
intermediates, among them are malondialdehyde (MDA)
and the most reactive, 4-hydroxy-trans-2-nonenal (HNE).
Oxidation of membrane phospholipids is considered one of
the major causes of mitochondrial dysfunction in a variety
of physiopathological situations and aging. In fact, lipid
peroxidation alters the structural and functional organiza-
tion of the lipid bilayer, changing membrane fluidity and
permeability, thereby affecting respiration and OXPHOS
process, maintenance of mitochondrial membrane potential
and mitochondrial Ca
buffer capacity (Pamplona
Data accumulated in the last decade indicate that mela-
tonin plays an important role in antioxidant defense pre-
serving mitochondrial homeostasis, reducing free radical
generation and stimulating ETC complex activity (Martín
; López et al.
; Paradies et al.
Castroviejo et al.
; Navarro-Alarcón et al.
earliest evidence of the antioxidant capacities of mela-
tonin was reported in 1993 (Tan et al.
scavenges two molecules of
OH, and in the process, it is
converted to cyclic 3-hydroxymelatonin (Tan et al.
This later compound was detected by mass spectra analy-
sis and carbon and proton–nuclear magnetic resonance in
the urine of human and rats under oxidative stress condi-
tions and treated with melatonin (Tan et al.
). It was
reported that melatonin does not directly scavenge H
in vitro, while a direct interaction of melatonin with H
occurs only in the presence of traces of the transition metal
ions (Fowler et al.
), and this may have important
implications in vivo under stress condition. Besides its
effects on ROS, melatonin is also a powerful scavenger of
RNS. Nitric oxide is produced by several forms of NOS. In
the mitochondria, two NOS isoforms, namely constitutive
(c-mtNOS) and inducible (i-mtNOS), have been reported
(Ghafourifar and Richter
). NO strongly interferes
with components of the respiratory chain in particular
cytochrome c oxidase (Mander and Brown
). NO in
combination with ONOO
not only interferes with respira-
tory chain complexes, but can trigger free radical-mediated
chain reactions that in turn damage proteins, lipids and
DNA molecules (Rubbo et al.
Polyunsaturated fatty acids, the main constituents of
phospholipids (PLs), are particularly sensitive to peroxida-
tion. This process is considered to proceed via a sequence
of steps, including the abstraction of a hydrogen atom from
unsaturated fatty acids, forming an alkyl radical (PL
lowed by a rapid addition of oxygen to form the peroxyl
), and then formation of a hydroperoxide
(PLOOH) via abstraction of a hydrogen from another acyl
chain; as a consequence, the reaction is repeated and the
whole process continues in a free radical chain reaction.
Due to this auto-oxidative chain reaction, a single initiation
event could theoretically lead to the oxidation of all lipids
in a cellular organelle or in a cell. Other reactive species
which initiate lipid peroxidation include ONOO
. Because of the highly destructive struc-
tural and functional nature of lipid peroxidation, there is
great interest in identifying molecules which reduce the ini-
tiation and/or progression of the denaturation of PUFAs. It
has been well documented that melatonin and its derivatives
exert a protective effect against lipid peroxidation induced
by oxidative stress in mitochondrial membranes (Reiter
). The ability of melatonin to protect against lipid
peroxidation has been repeatedly documented either in ani-
mal or in plant tissues under various oxidizing conditions
such as ionizing radiation, heavy metal toxicity, and drug
metabolism. (García et al.
). The precise mechanism
by which melatonin and its derivatives affect lipid peroxi-
dation is not yet established. Melatonin has been shown
to scavenge the peroxyl radical PLOO
(Pieri et al.
Livrea et al.
), which is produced during lipid peroxi-
dation and being sufficiently reactive to propagate the chain
reactions. The efficacy of melatonin to function as a PLOO
scavenger was evaluated by measuring inhibition of metal
ion-, radiation- or human macrophage-mediated oxidation
of human low-density lipoprotein (Abuja et al.
atonin was shown to be more effective than vitamin E in
and inhibiting lipid peroxidation (Pieri
). Another major contributor to lipid per-
oxidation is ONOO
, which is a powerful initiator of lipid
breakdown. Due to the ability of melatonin to neutralize
, this is another means whereby melatonin may
protect membrane lipids (Cuzzocrea et al.
sumed that melatonin inhibits lipid peroxidation by inter-
fering with the radicals that initiate this process, especially
OH and ONOO
, and by positioning itself in a superfi-
cial location in membrane lipids layers near the polar heads
of these molecules (Ceraulo et al.
). Its small molecu-
lar size and its amphiphilic properties facilitate melatonin’s
penetration into subcellular compartments. In vitro assays
showed that melatonin inhibits lipid peroxidation in rat
brain homogenates, brain and liver microsomes and mito-
chondria treated with an ascorbate-Fe
lipid peroxidation in many experimental models (Maharaj
; Parlakpinar et al.
Mitochondrial membranes, which are rich in phospho-
lipids containing PUFAs, are fluid structures, and optimal
membrane fluidity is required for their proper function.
When PUFAs are oxidized, mitochondrial membranes
become more rigid; thus, their protection from oxidation
is essential for optimal function of these organelles. Since
the degree of lipid breakdown in mitochondrial membranes
generally correlates with the fluidity of these organelles, it
could be predicted that melatonin, by preventing lipid per-
oxidation, could preserve the proper fluidity and function of
the membranes. Aging is characteristically associated with
elevated cell membrane rigidity. Depressed level of mela-
tonin naturally occurring with aging leads to elevated levels
of lipid peroxidation and to more rigid cellular membranes
(Reiter et al.
). Likewise, treatment
of senescence-accelerated prone mice with melatonin pre-
serves mitochondrial membranes in a more fluidity state
(García et al.
Melatonin and mitochondrial function
Melatonin is highly lipophilic molecule that crosses cell
membranes to easily reach cellular compartments including
mitochondria, where it seems to accumulate in high con-
centration (Acuña-Castroviejo et al.
). Several stud-
ies have shown that melatonin can influence mitochondrial
homeostasis by stabilizing mitochondrial inner membrane,
thereby improving ETC activity and mitochondrial func-
tion (López et al.
was reported to increase the activity of C I and C IV in a
time-dependent manner in mitochondria isolated from rat
brain and liver tissues, while having no effect on C II and
C III (Martín et al.
prevented the inhibitory effect of ruthenium red on C I and
C IV activities as well as on GPx enzyme in rats (Martín
). Melatonin (1 nM) significantly increases the C
I and C IV activities in rat liver mitochondria, while higher
concentrations of this compound are required to stimulate
the activity of these complexes in rat brain mitochondria.
The effects on C I were also studied using BN-PAGE his-
tochemical procedure to measure change in its activity
induced by melatonin. This study documented an increase
in C I activity following melatonin treatment. Melatonin
also counteracted cyanide-induced inhibition of C IV,
as shown by the increase in the ETC activity coupled to
OXPHOS and ATP synthesis, both either in normal or in
rat brain and liver mitochondria depleted of ATP by cya-
nide treatment (Martín et al.
). The stimulatory effect
of melatonin on the C I and C IV activities does not only
rely on the antioxidant role of this indoleamine. Because
of its high redox potential (0.94 V; Tan et al.
atonin may interact with the ETC complexes by donating
and accepting electrons, thereby increasing electron flux,
an effect not shared by other antioxidants.
Recently, another effect of melatonin on mitochondrial
bioenergetic parameters was described (López et al.
Experiments carried out in vitro with normal mitochondria
demonstrate that this indoleamine protected the mitochon-
drial function from oxidative damage, decreasing oxygen
consumption in the presence of ADP in a concentration-
dependent manner and reducing the membrane potential,
thereby inhibiting the production of O
tory control ratio, the efficiency of the OXPHOS and ATP
synthesis, while enhancing the activity of ETC complexes
(mainly C I, C III and C IV). These effects of melatonin
probably depend on a direct interaction of this indoleamine
with mitochondria, as shown by the fact that mitochondria
take up melatonin in a time- and concentration-dependent
manner, and thus, the effects of melatonin were due to its
presence within the mitochondria. The ability of mitochon-
dria to accumulate melatonin is of great pharmacological
interest because it means that, following exogenous admin-
istration in vivo, melatonin enters the mitochondria and
exerts its beneficial action on mitochondrial function.
Cardiolipin and mitochondrial function
Cardiolipin is commonly referred to as the signature phos-
pholipid of mitochondria. This phospholipid is associated
with membranes designed to generate an electrochemi-
cal gradient that is used to produce ATP, such as bacterial
plasma membrane and the IMM (Ren et al.
association between CL and energy transducing mem-
branes suggests an important role for CL in bioenergetic
processes (Schlame et al.
; Ren et al.
). In fact,
CL has been shown to interact with a number of IMM pro-
teins including, among others, the ETC complexes involved
in OXPHOS (Musatov and Robinson
; Schlame et al.
; Houtkooper and Vaz
and the anionic substrates carriers (Klingenberg
Indeed, CL is required for optimal activity of C I (Sharpley
). Crystallographic stud-
ies have shown the presence of a few tightly bound CL
molecules in each of the crystal structures of the C III, C
IV and ADP/ATP carrier (Lange et al.
; Ozawa et al.
). These results suggest that CL is an
integral component of these proteins, the presence of which
is critical for their proper folding. CL seems to facilitate the
association and stabilization of respiratory chain complexes
into supercomplexes (Zhang et al.
Such supercomplexes formation is thought to improve the
efficiency of OXPHOS by eliminating the need for diffu-
sion of substrates and products between individual ETC
component (Genova and Lenaz
). CL is also required
for the interaction between ADP/ATP carrier proteins and
respiratory supercomplexes (Claypool
Another important role of CL is its participation in the
process of apoptosis in animal cells through the interac-
tion with a variety of death-inducing proteins, including
cytochrome c (Gonzalvez and Gottlieb
; Ott et al.
). This hemoprotein is believed to acts as per-
oxidase, which reacts quite specifically with CL, causing
oxidation and then hydrolysis of the product CL hydrop-
eroxide (Kagan et al.
). The consequence is that the
cytochrome c is released into the intermembrane space,
while the oxidized CL is translocated to the outer mito-
chondrial membrane and participates in the opening of the
mitochondrial permeability transition pore (Petrosillo et al.
). The opening of this pore facilitates the release of
several proapoptotic factors, including cytochrome c, from
mitochondria into the cytosol where they trigger apopto-
sis. CL appears to play an important role in mitochondrial
morphology and dynamics including fusion and fission
processes (DeVay et al.
; Ban et al.
), as well as
in the protein insertion and assembly into the mitochondria
(Marom et al.
). Given the role played by CL in mito-
chondrial bioenergetic processes as well as in apoptosis
and in mitochondrial membrane stability and dynamics, it
is conceivable that abnormalities in CL structure, content
and acyl chains composition may have important implica-
tions in mitochondrial dysfunction associated with several
physiopathological states and diseases (Chicco and Spar-
; Paradies et al.
). Alterations in mito-
chondrial CL profile may occur mainly as a consequence
of: (1) loss of the CL content due to the changes in the CL
synthase activity; (2) changes in acyl chain composition
due to altered CL remodeling and (3) CL oxidation due to
Melatonin and cardiolipin oxidation
Oxygen free radicals lead to primary reaction and damage
in the immediate surroundings of where they are generated,
due to their high chemical reactivity. Therefore, the effect
of these reactive species should be greatest at the level of
mitochondrial membrane components such as phospho-
lipid molecules particularly rich in PUFAs. Among phos-
pholipids, CL molecules are particularly sensitive to oxi-
dation, either because of their location in IMM near to the
site of ROS production, or because of their high content of
PUFAs. In fact, CL molecules are rich in unsaturated fatty
acyl chains, particularly linoleic acid in heart and liver,
or docosahexanoic and arachidonic acids in brain tissue
mitochondria. In addition, CL molecules are located in the
mitochondrial membrane near to the site of ROS produc-
tion, mainly represented by the respiratory chain complexes
I and III to which CL molecules are associated.
As described above, melatonin and its derivatives exert
a protective effect on lipid peroxidation in mitochondrial
; García et al.
; Reiter et al.
). Recently, we have studied the ability of melatonin
to inhibit CL oxidation in isolated mitochondria (Petrosillo
). CL oxidation in mitochondria was induced
by treating these organelles with t-butylhydroperoxide
(t-BuOOH), a lipid-soluble hydroperoxide that closely
resembles endogenous lipid hydroperoxides generated dur-
ing oxidative stress. Treatment of rat heart and brain mito-
chondria with t-BuOOH in the presence of micromolar
concentrations of copper ions resulted in a loss of CL con-
tent and in an increase in the level of oxidized CL, the lat-
ter being detected by a normal phase HPLC technique with
UV detection at 235 nm, indicative of conjugated dienes.
Melatonin, at micromolar concentration, was able to pre-
vent the oxidation/depletion of CL. This inhibitory effect of
melatonin on CL oxidation in mitochondria can be reason-
ably explained on the ability of this indoleamine to inhibit
the peroxidation of linoleic acyl chains, which are the main
constituents of CL molecules. In fact, previous results
have demonstrated the antioxidant effect of melatonin on
linoleate oxidation initiated by HO
free radical generated
by water gamma radiolysis (Mekhloufi et al.
linoleate micelles as lipid model, two index of peroxidation
have been measured, i.e., conjugated dienes and hydroper-
oxides. The results obtained in this study demonstrate that
melatonin displays strong in vitro lipid peroxyl radicals
) scavenging properties, as shown by its inhibitory
effect on the radiation-induced peroxidation of linoleate.
Emerging insights have linked CL oxidation/depletion
to mitochondrial dysfunction associated with a variety of
diseases and physiopathological settings including heart
ischemia/reperfusion, diabetes, aging and age-associated
disorders (Chicco and Sparagna
; Paradies et al.
). CL oxidation is also emerging as a key player in
the regulation of several of the mitochondrial steps of cell
death and in mitochondrial dynamics (Ott et al.
Paradies et al.
). Therefore, the ability of melatonin
to prevent CL oxidation in mitochondria may have impor-
tant implications in mitochondrial dysfunction and related
Cardiolipin and MPTP
Mitochondrial permeability transition pore (MPTP) is
defined as the sudden increase in IMM permeability to
low molecular weight metabolites (
˂1.5 kDa) in response
to many stimuli, including high levels of Ca
; Leung and Halestrap.
ing of the MPTP promoted by elevated matrix Ca
associated with high phosphate, low adenine nucleotide
concentrations and oxidative stress, induces the collapse
of transmembrane ion gradients, resulting in membrane
depolarization and uncoupling of OXPHOS. This causes
irreversible damage to mitochondria, resulting in cell death
predominantly through necrosis. A number of molecules
were accepted as key structural components of the MPTP,
including, Cyp-D in the matrix, ANT and phosphate car-
riers in the IMM and VDAC (also known as porin) in the
outer membrane (Halestrap
). More recently, dimers
of the F
ATP synthase were suggested to be new puta-
tive components of the MPTP. In fact, reconstituted dimers
ATP synthase, incorporated into lipid bilayers, form
-activated channels with properties similar to those of
the mitochondrial mega-channel, the electrophysiological
equivalent of the MPTP (Giorgio et al.
Release of cytochrome c from mitochondria into cyto-
sol is considered a central event in the induction of apop-
totic cascade that ultimately leads to cell death (Ott et al.
). Cytochrome c is normally bound to the outer
surface of the IMM primarily to CL molecules (Rytomaa
). Oxidation of CL promotes the detachment of
cytochrome c from mitochondrial membrane and its release
into the extramitochondrial space (Petrosillo et al.
Ott et al.
). It has been proposed that the release of
cytochrome c from the mitochondria takes place by a two-
step process involving, first, the dissociation of this hemo-
protein from the IMM, followed by permeabilization of
the outer membrane probably through its association with
Bcl2 family proteins, such as Bax and Bid, and/or through
the MPTP opening (Ott et al.
). CL oxidation may be
involved in the permeabilization of the outer mitochondrial
membrane, probably through its association with Bcl2 fam-
ily proteins such as Bax and Bid (Kagan et al.
; Jiang et al.
We have shown that exogenously added oxidized CL to
mitochondria sensitizes these organelles to Ca
MPTP opening (Petrosillo et al.
). This synergistic
effect of Ca
and oxidized CL on the induction of MPTP
opening suggests that both these compounds could play a
coordinated role in this process by interacting with compo-
nents of the MPTP, probably with adenine nucleotide car-
rier and/or with ATP synthase dimers. The involvement of
oxidized CL in MPTP opening is further demonstrated by
our recent study, showing that oxidation of intramitochon-
drial CL molecules results in MPTP induction (Petrosillo
). Interestingly, the induction of MPTP opening
by oxidized CL and Ca
is associated with the release of
cytochrome c from mitochondria.
Melatonin and MPTP
Studies carried out in our and other laboratories have dem-
onstrated a role of melatonin in the modulation of MPTP
opening (Andrabi et al.
; Hibaoui et al.
rosillo et al.
). A direct MPTP inhibition by
melatonin has been reported (Andrabi et al.
tonin diminished MPTP current with an IC
of 0.8 μM,
a concentration which would require accumulation of
melatonin within mitochondria. Indeed, it has been dem-
onstrated that melatonin due to its amphiphilic nature can
be actively accumulated by mitochondria (Messner et al.
; López et al.
). The direct MPTP inhibition by
melatonin should be interpreted on the basis of a low affin-
ity binding site. This effect may contribute to the anti-apop-
totic properties of melatonin.
Our studies have shown that melatonin, at micromolar
concentrations, inhibited both the Ca
CL peroxidation and MPTP opening, as indicated by the
protective effect this indoleamine on matrix swelling, ΔΨ
collapse and release of preaccumulated Ca
). These results suggest that melatonin, by pre-
venting endogenous CL peroxidation, inhibits MPTP open-
ing. In addition, our results demonstrate that the release of
cytochrome c from mitochondria associated with the MPTP
opening induced by oxidative stress 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 IMM
and MPTP opening. The ability of melatonin to prevent
MPTP opening may have important implications in those
physiopathological situations characterized by alterations
homeostasis and accumulation of peroxidized CL
in mitochondria, such as heart ischemia/reperfusion, aging
and other degenerative diseases.
Melatonin’s action in preventing MPTP opening induced
by oxidative stress caused by t-BuOOH was shown in
another study carried out in primary skeletal muscle cul-
tures (Hibaoui et al.
). Melatonin (1–100 µM) fully
prevented myotube death induced by t-BuOOH as assessed
by acid phosphatase, caspase 3 activities and cellular
morphological changes. Using fluorescence imaging, it
was shown that the mitochondrial protection provided by
melatonin was associated with an inhibition of t-BuOOH-
induced ROS generation. In isolated mitochondria, mel-
atonin desensitized the MPTP to Ca
t-BuOOH-induced mitochondrial swelling, pyridine nucle-
otide and GSH oxidation. The inhibition of MPTP opening
by melatonin was suggested as an explanation for the pro-
tective action of this indoleamine against oxidative stress in
myotubes (Hibaoui et al.
MPTP may occur in the cell through two modes, the
transient MPTP and the prolonged MPTP, having different
outcomes of survival or death. The transient MPTP protects
mitochondria, whereas the prolonged MPTP triggers the
“point of no return” for apoptosis or necrosis. It has been
shown that melatonin targets mitochondrial Ca
MPTP for protection during mitochondrial Ca
ated apoptosis in astrocytes (Jou
). With the applica-
tion of fluorescence laser scanning imaging microscopy, it
was demonstrated that melatonin does not inhibit the MPT
pore, rather it preserves the pore in its protective mode of
transient MPT during mitochondrial Ca
stress. In addi-
tion, the melatonin-preserved transient MPT allowed mito-
chondria to release the toxic overloaded Ca
levels, thus preventing Ca
-mediated fission of mitochon-
-dependent prolonged MPT and possibly improv-
-dependent ATP synthesis through activation of
mitochondrial dehydrogenases. This unique melatonin-
dependent modulation of MPTP has been suggested to have
important therapeutic potential in the treatment of mito-
-mediated astrocyte-associated neurodegen-
erative disorders (Jou
Melatonin and mitochondrial dysfunction in heart
Ischemia/reperfusion (I/R) leads to myocardial dysfunction
and irreversible damage characterized by cardiomyocyte
hypercontracture, reduction of left ventricular pressure and
elevated incidence of ventricular fibrillation. Mitochon-
dria are known to be involved in the processes that lead to
cell death following I/R and are therefore potential target
for protective intervention (Camara et al.
). ROS are
recognized as an important factor in producing lethal cell
injury associated with cardiac I/R (Chen and Zweier
Mitochondria isolated from I/R rat heart exhibit altered
bioenergetic function, associated with CL abnormalities.
In fact, results obtained in our laboratory have shown that
mitochondria isolated from I/R rat heart exhibit decreased
rate of mitochondrial oxygen consumption, reduced activ-
ity of C I and CIII and increased basal rate of H
duction (Petrosillo et al.
; Paradies et al.
changes in the mitochondrial bioenergetic parameters were
associated with an oxidation/depletion of CL. The defect in
the respiratory chain complexes activity observed in mito-
chondria isolated from I/R rat heart has been ascribed, at
least in part, to ROS-induced oxidation of CL, a phospho-
lipid required for the optimal activity of these enzymatic
complexes. Melatonin treatment had strong protective
effect against I/R-associated mitochondrial bioenergetic
alterations (Petrosillo et al.
). In fact, melatonin
administration to I/R rat heart counteracted the reduction in
C I and C III activity and the associated decrease in state
3 respiration in isolated rat heart mitochondria as well as
CL alterations. Similarly, melatonin prevented alterations
to C I and C III as well as to CL in in vitro experiments
on isolated rat heart mitochondria subjected to oxidative
stress conditions. The melatonin’s protective effect against
I/R-induced mitochondrial dysfunction could be ascribed
to its ability to preserve CL integrity and/or to directly
improve the activity of the respiratory chain complexes.
This effect was associated with an improvement of post-
ischemic hemodynamic function of the heart. These results
emphasize that melatonin-induced mitochondrial adaptive
changes are likely of great value for the cardioprotective
actions of the indoleamine (Dominguez-Rodriguez et al.
; Yang et al.
A large body of experimental evidence supports a cru-
cial role of MPTP in cardiomyocyte cell death occurring
with I/R (Halestrap
; Ong et al.
suggested that MPTP remains closed during the ischemic
period, because of the low pH due to lactate accumula-
tion. At reperfusion, there is an uptake of Ca
chondria, a burst of ROS production and rapid correction
of acidosis, all events that contribute to increase the like-
lihood of MPTP opening. Pharmacological intervention
aimed to protect the heart from the damaging effect of
MPTP opening are of considerably importance in attenu-
ating mitochondrial dysfunction associated with I/R injury.
Recent studies have shown that melatonin and several of its
metabolites have significant protective actions against car-
diac damage and altered physiology during I/R contributing
to the rehabilitation of the heart contractile function dur-
ing reperfusion (Sahna et al.
; Petrosillo et al.
Paradies et al.
; Dominguez-Rodriguez et al.
Lochner et al.
). Other studies demonstrated that mel-
atonin plays a role in the mitochondrial adaptive changes
and that cytochrome c is a significant mediator of this
process (Giacomo and Antonio
). Very recently, we
have demonstrated that melatonin protects against mito-
chondrial dysfunction associated with heart I/R injury by
inhibiting MPTP opening (Petrosillo et al.
tonin 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. All these
effects were accompanied by the inhibition of the MPTP
opening as detected in situ by the mitochondrial release of
. Furthermore, melatonin desensitizes mitochondria
isolated from melatonin-reperfused heart to Ca
MPTP opening, as assessed by the CRC (calcium reten-
tion capacity), a sensitive and quantitative measure of the
ability of mitochondria to open the MPTP in response to of
uptake. Together, these results demonstrate that mela-
tonin protects against heart I/R injury by inhibiting MPTP
opening, thus improving the post-ischemic hemodynamic
function of the heart. The possible mechanism underlying
the inhibition of MPTP opening during I/R by melatonin
treatment was also investigated (Petrosillo et al.
is now accepted that, in addition to Ca
factors may contribute to the MPTP opening during heart
I/R. As described above, oxidized CL, together with Ca
promotes the induction of MPTP in isolated rat heart mito-
chondria, suggesting that an increased level of oxidized
CL may increase the probability of MPTP opening during
I/R. We found an increased level of oxidized CL in mito-
chondria isolated from rat heart subjected to I/R, which
was prevented by melatonin treatment. Thus, it is plausi-
ble that CL oxidation, together with Ca
gistically contribute to MPTP opening during I/R and that
melatonin treatment inhibits MPTP opening by preserv-
ing CL integrity by ROS attack. Melatonin was reported
to inhibit linoleate peroxidation in a model system in vitro
(Mekhloufi et al.
). 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 which is the
main constituent of heart CL.
It has been shown that CL binds cytochrome c to the
outer surface of the IMM (Rytomaa et al.
tion of CL results in the detachment of this hemoprotein
from mitochondrial membrane and this event is considered
an important initial step in the cytochrome c release from
mitochondria (Petrosillo et al.
; Ott et al.
Our results have shown that, in addition to inhibit MPTP
opening, melatonin prevents also the release of cytochrome
c from mitochondria upon I/R. This effect of melatonin can
be explained on the ability of this indoleamine to inhibit CL
oxidation, thus preventing cytochrome c detachment from
IMM. The inhibitory effect of melatonin on cytochrome c
release and MPTP opening may contribute to the protective
effect exerted by this indoleamine against mitochondrial
dysfunction associated with I/R. These beneficial effects
of melatonin reinforce the therapeutic potential of this
compound to combat a variety of oxidative stress-induced
oxidative dysfunctions as well as mitochondrial-mediated
apoptosis in various cardiovascular disorders.
Melatonin and mitochondrial dysfunction in aging
Aging is a multifactorial process, which is genetically
determined and influenced epigenetically by environment
). This biological process is characterized
by impairment of bioenergetic functions, increased oxi-
dative stress and increased risk of contracting age-associ-
ated diseases. Mitochondria are intimately involved in the
aging process because these organelles are recognized as
the main intracellular source of ROS as well as the major
target of their oxidative attack. According to the mitochon-
drial theory of aging, ROS produced by the mitochondrial
respiratory chain, attack mitochondrial constituents includ-
ing proteins, lipids and mitochondrial DNA (mtDNA; Har-
; Miquel et al.
; Pak et al.
). As mtDNA
encodes essential components of OXPHOS and protein
synthesis machinery, accumulation of mtDNA mutations
may lead to impairment of either the assembly or the func-
tion of the respiratory chain, leading to further ROS gen-
eration and subsequent accumulation of more mtDNA
mutations. This triggers a vicious cycle which leads to the
progressive decline of mitochondrial and cellular bioener-
getics functions as results of insufficient supply of energy
and/or increased susceptibility to cell death (Judge and
; Paradies et al.
). Although there
is a large consensus on the mitochondrial free radical the-
ory of aging (Harman
), recent findings, obtained in
particular in Caenorhabditis elegans and in rodents, suggest
that ROS generation may not be the main factor involved in
the aging process (Hekimi et al.
A number of studies have shown a decreased electron
transport activity in mitochondria isolated from rat and
mouse tissues upon aging (Navarro and Boveris
Judge and Leeuwenburgh
). Of the five respiratory chain complexes, C I and C
IV show a selectively reduced enzymatic activity in mito-
chondria isolated from various tissues of rats and mice
upon aging (Lenaz et al.
Petrosillo et al.
). One possible factor respon-
sible for the age-related impairment of C I and C IV activ-
ity might be the oxidation/depletion of mitochondrial CL as
supported by the following experimental observations. The
content of normal CL declines, while the level of oxidized
CL increases with aging (Petrosillo et al.
Cardiolipin molecules are specifically bound to C I and
C IV of the respiratory chain and required for their func-
tional activity (Lange et al.
; Musatov and Robinson
). In addition, mitochondrial-mediated ROS genera-
tion affects C I and C IV activity through CL peroxidation
in beef heart submitochondrial particles (Paradies et al.
). These results suggest that the age-associated
defects in mitochondrial C I and C IV activities could be
ascribed, at least in part, to ROS-induced oxidative damage
to mitochondrial CL. Complex I is considered a rate-limit-
ing factor in the mitochondrial respiratory chain and also
the main source of ROS during the aging process. Thus, the
impairment of mitochondrial C I activity, in addition to that
of C IV, may increase the electron leak from the ETC, gen-
erating more superoxide radical, triggering a cycle of oxi-
dative damage that ultimately leads to mitochondrial bio-
energetic decay in aging (Judge and Leeuwenburgh
Paradies et al.
). The age-associated impair-
ment of mitochondrial C I e C IV activity observed in heart
and brain tissues may have important implications in the
etiopathology of age-associated cardiovascular and neuro-
degenerative disorders and may represent an important tar-
get for the development of potential therapeutic strategies
Growing evidence indicate that the individual compo-
nents of the mitochondrial ETC may exist as large macro-
molecular assemblies, or so-called supercomplexes (Zhang
; Genova and Lenaz
general role for CL in respiratory supercomplexes forma-
tion and stability in mammalian mitochondria has been
suggested (Zhang et al.
; Bazán et al.
age-associated destabilization of rat heart mitochondrial
supercomplexes has been reported (Gómez and Hagen
). Due to the oxidation/depletion of CL with aging, it
is possible that abnormalities in CL might be involved in
the destabilization and dysfunction of mitochondrial respir-
atory supercomplexes, thus contributing to mitochondrial
bioenergetics decay with aging.
Several properties of melatonin indicate that this com-
pound may have beneficial effects in aging. Serum levels of
melatonin significantly decrease in aged animals compared
with young animals (Reiter et al.
). In humans,
the total antioxidative capacity of serum correlates well
with its melatonin levels. Thus, the decreased level of mel-
atonin in aging has been associated with the increased oxi-
dative damage observed in the elderly. The mechanism of
aging process can be studied in experimental animal mod-
els like the senescent accelerated mouse (SAM; Takeda
). Recently, using SAM mice, the effects of chronic
administration of physiological doses of melatonin on
mitochondrial oxidative stress and mitochondrial function
in heart tissue were investigated (Rodríguez et al.
Mitochondrial oxidative stress was determined by measur-
ing the levels of lipid peroxidation, GSH and GSSG and the
activities of GPx and GRd. The activities of several mito-
chondrial bioenergetic parameters including ETC com-
plexes and ATP levels were also evaluated. The results of
these studies showed an age-dependent mitochondrial oxi-
dative damage in the heart tissue of SAM mice, which was
associated with a reduction in the ETC complexes activity
and in ATP levels. Chronic melatonin administration nor-
malized these age-associated alterations in mitochondrial
bioenergetic parameters and increased ATP level. Moreo-
ver, melatonin treatment had beneficial effect on longev-
ity of SAM mice (Rodríguez et al.
). Together, these
studies indicate that melatonin treatment counteracts age-
related oxidative damage and mitochondrial dysfunction in
heart tissue of SAM mice by improving mitochondrial bio-
energetic function as reflected by increase in ATP produc-
tion and prolonged longevity.
Experimental evidence indicates that mitochondrial
decay is a major contributor to brain tissue alterations asso-
ciated with aging (Navarro and Boveris
). Aged mam-
malian brain has a decreased capacity to produce ATP by
OXPHOS and it is considered that this decreased capacity
for energy production becomes limiting under physiologi-
cal conditions in aged individuals. The current knowledge
indicates that the impairment of brain mitochondrial func-
tion in aging is mainly due to decreased electron transfer
rates by C I and C IV, among other decreased mitochon-
drial activities (Navarro and Boveris
; Paradies et al.
). Impaired mitochondrial respiration with NAD-
dependent substrates has been consistently observed in
brain mitochondria isolated from aged rats and mice (Nav-
arro and Boveris
; Petrosillo et al.
). Given the
brain’s high energy requirements, the impairment in brain
ETC complexes activity could have a significant impact on
brain function in aging and on the etiology of age-associ-
ated neurodegenerative disorders (Beal
A potential role of melatonin in mitigation of mitochon-
drial decay in brain aging has been described (Bondy et al.
; Bondy and Sharman
). Moreover, melatonin
has been identified as a potential mitochondria-targeted
protector against several oxidative stress-associated brain
disorders (Caballero et al.
laboratory have shown that brain aging is associated with
an impairment in C I and C IV activity, decrease in oxygen
consumption and membrane potential and with an increase
in mitochondrial ROS production (Petrosillo et al.
Paradies et al.
). These age-related mitochondrial bio-
energetic alterations were largely attenuated by melatonin
administration. Melatonin administration did not affect
these bioenergetic parameters when administered to young
rats, suggesting that the observed protective effects of this
indoleamine are related to changes produced by aging. The
melatonin’s ability to prevent the age-related alterations of
mitochondrial bioenergetic parameters in rat brain could be
ascribed, at least in part, to its protective effect against CL
peroxidation (Paradies et al.
) as also supported by in
vitro experiments on isolated rat brain mitochondria (Pet-
rosillo et al.
Other studies carried out in brain mitochondria iso-
lated from male and female SAM mice indicated that there
was significant age-dependent mitochondrial dysfunction
with a reduced efficiency of the ETC and diminished ATP