ANTIOXIDANTS & REDOX SIGNALING
Volume 10, Number 3, 2008
© Mary Ann Liebert, Inc.
DOI: 10.1089/ars.2007.1892

Forum Review
Mitochondria-Targeted Cytoprotective Peptides for
Ischemia–Reperfusion Injury
HAZEL H. SZETO

ABSTRACT
It is now recognized that oxidative injury and mitochondrial dysfunction are responsible for many clinical
disorders with unmet needs, including ischemia–reperfusion injury, neurodegeneration, and diabetes. Mitochondrial dysfunction can lead to cell death by apoptosis or necrosis. As mitochondria are the major source
of intracellular reactive oxygen species (ROS), and mitochondria are also the primary target for ROS, the
ideal drug therapy needs to be targeted to mitochondria. A number of approaches have been used for targeted delivery of therapeutic agents to mitochondria. This review will focus on a novel class of cell-permeable
small peptides (Szeto–Schiller peptides) that selectively partition to the inner mitochondrial membrane and
possess intrinsic mitoprotective properties. Studies with isolated mitochondrial preparations and cell cultures
show that these SS peptides can scavenge ROS, reduce mitochondrial ROS production, and inhibit mitochondrial permeability transition. They are very potent in preventing apoptosis and necrosis induced by oxidative stress or inhibition of the mitochondrial electron transport chain. These peptides have demonstrated
excellent efficacy in animal models of ischemia–reperfusion, neurodegeneration, and renal fibrosis, and they
are remarkably free of toxicity. The pharmacology of the SS peptides in models of ischemia—reperfusion will
be the focus of this review. Antioxid. Redox Signal. 10, 601–619.

INTRODUCTION

M

entered center stage as the
major regulator of life and death in the cell. In addition
to their major role as energy providers for cellular processes,
mitochondria are also the major producers of intracellular reactive oxygen species (ROS) (6, 103). Cells die from necrosis
when they cannot maintain adequate ATP levels (43, 48). On
the other hand, oxidative damage to mitochondria can result in
cytochrome c release into the cytosol and activation of the caspase cascade, leading to apoptosis (72, 73, 77). Intracellular
ATP serves as a switch between apoptosis and necrosis because
apoptosis involves energy-requiring steps (48, 56). Cell death
induced by mitochondrial oxidative damage plays an important
role in numerous clinical disorders, including ischemia—reperfusion injury, neurodegenerative disorders, diabetes, inflammatory disorders, drug-induced toxicity, and age-related deITOCHONDRIA HAVE RECENTLY

generative diseases (7, 11, 37, 44, 110). Many clinically approved drugs, including doxorubicin and paclitaxel, act directly
on mitochondria to induce apoptosis (86). Minimizing mitochondrial oxidative stress and protecting mitochondrial function can potentially provide therapeutic benefits to this vast
group of diseases with unmet needs. There are several hurdles
in the quest for mitoprotective drugs. The difficulties include
delivery of drugs to mitochondria, minimization of adverse effects, and delivering drugs across the blood-brain barrier. This
review will summarize some of the approaches that have been
used for targeted delivery of conventional therapeutic agents to
the mitochondrial matrix. The major focus of this review, however, will be on a novel class of cell-permeable small peptides
(Szeto—Schiller peptides) that selectively partition to the inner
mitochondrial membrane and possess intrinsic mitoprotective
properties (98, 99). This review will summarize results from
cell culture studies that show the extraordinary potency of these

Department of Pharmacology, Joan and Sanford I. Weill Medical College of Cornell University, New York, New York.

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SS peptides in preventing apoptosis and necrosis induced by
oxidative stress or inhibition of mitochondrial respiration. A
summary of results from ex vivo and in vivo animal models will
also be presented, with special emphasis on ischemia–reperfusion injury. The efficacy of these peptides as neuroprotective
agents was recently reviewed (99).

MITOCHONDRIA AS GATEKEEPER OF
CELL SURVIVAL
Long considered the powerhouses of our cells, mitochondria
are responsible for the enormous task of supplying intracellular ATP via oxidative phosphorylation. Mitochondria produce
more than 90% of our cellular energy under normal conditions.
Energy production results from the tricarboxylic acid (TCA)
cycle and the electron transport chain (ETC). The TCA cycle
provides reducing equivalents in the form of NADH and FADH
that then enter into the ETC. As the electrons donated from
NADH and FADH flow from Complex I through Complex IV
of the ETC, protons are pumped into the intermembrane space
to establish a protonomotive force, which is used by Complex
V to phosphorylate ADP to ATP by the F0F1 ATP synthase.
Oxygen normally serves as the ultimate electron acceptor in
the ETC and is reduced to water. However, electron leak to
oxygen through complex I and complex III can generate superoxide anion (O2иϪ)[see reviews, (6, 103)]. The rate of O2иϪ
production is affected by mitochondrial metabolic state and increases when the electron carriers harbor excess electrons, either from inhibition of oxidative phosphorylation, or from excessive calorie consumption (2). The location of O2иϪ within
mitochondria is important because O2иϪ does not diffuse readily across membranes. Recent studies suggest that complex I
produces O2иϪ into the matrix, while complex III can release
O2иϪ into the matrix as well as the intermembrane space (62).
The disposition of mitochondrial ROS was reviewed recently
(6). Superoxide anion can be eliminated by dismutation carried
out by the mitochondrial matrix enzyme Mn superoxide dismutase (MnSOD), or by the CuZnSOD in the intermembrane
space, to H2O2. Although H2O2 may freely diffuse out of mitochondria, it can be readily inactivated by catalase or glutathione reductase. The inner membrane also contains many redox-active transition metals such as iron and copper that can
convert H2O2 to the highly reactive hydroxyl radical (OHи) via
the Fenton reaction. OHи may further react with bicarbonate to
yield the very reactive carbonate radical anion. O2иϪ can also
react with another free radical nitric oxide formed by mitochondrial nitric oxide synthase to generate the highly reactive
peroxynitrite (ONOOϪ).
Mitochondria are normally protected from oxidative damage
by a multilayer network of mitochondrial antioxidant systems,
including glutathione peroxidase, thioredoxin, glutaredoxin,
and catalase [see review (6)], but they can undergo oxidative
damage when ROS production exceeds the antioxidant capacity of mitochondria. ROS can initiate damage to nucleic acids,
proteins, and lipids in mitochondria. Mitochondrial DNA is particularly susceptible to oxidative damage because they lack histones and have much limited base excision repair mechanisms
compared to nuclear repair mechanisms (82). Protein oxidation

SZETO
and nitration result in altered function of many enzymes in the
mitochondrial ETC, including NADH dehydrogenase, NADH
oxidase, cytochrome c oxidase, and ATPase (12, 66), while oxidation of the adenine nucleotide translocator impairs the influx
of ADP into the matrix for ATP synthesis (59).
Besides reduced function of mitochondrial enzymes, mitochondrial ROS can also cause lipid peroxidation that can lead
to mitochondrial dysfunction. Cytochrome c is normally bound
to the inner mitochondrial membrane by association with cardiolipin. Peroxidation of cardiolipin leads to loss of cardiolipin
on the inner membrane and dissociation of cytochrome c (77,
93). This will lead to compromised function of cytochrome c
oxidase, reduced ATP production, and increased ROS generation (14). Cytochrome c, once dissociated from the inner membrane, can then be released through the outer mitochondrial
membrane into the cytosol (93).The mechanisms by which cytochrome c may be released through the outer membrane,
termed mitochondrial outer membrane permeability (MOMP),
was reviewed recently (27). One mechanism is believed to involve mitochondrial permeability transition (MPT). The MPT
pore is a high conductance channel that is believed to be formed
by the apposition of the voltage-dependent anion channel
(VDAC) on the outer membrane and the adenine nucleotide
translocator (ANT) on the inner membrane (19). Opening of
the MPT pore causes a sudden increase in permeability of the
inner mitochondrial membrane. This will result in swelling of
the mitochondrial matrix, rupture of the outer membrane, and
release of cytochrome c (44, 74). Mitochondrial permeability
transition can be induced by calcium (Ca2ϩ) overload and high
concentrations of inorganic phosphate (Pi), conditions commonly associated with ischemia (see later section on ischemia–reperfusion). ROS can further promote MPT by causing
oxidation of thiol groups on the ANT (104). Cytochrome c release may also occur via MPT-independent mechanisms, and
may involve an oligomeric form of Bax (42, 87). Certain death
signals activate the pro-apoptotic protein Bax in the cytoplasm,
resulting in translocation to mitochondria, and oligomerization
of Bax on the outer membrane leads to the release of cytochrome c into the cytoplasm.
Cytochrome c in the cytosol binds to apoptotic protease activating factor-1 (Apaf-1) to initiate the formation of an apoptosome, which then binds pro-caspase-9 (54). The oligomerization of caspase-9 on the apoptosome activates the protease.
The active caspase-9 cleaves two “executioner” caspases, caspase-3 and caspase-7, that then go on to cleave key substrates
within the cell (29, 54, 121). This has been termed the intrinsic mitochondrial pathway of apoptosis. Mitochondrial dysfunction may therefore lead to necrosis or apoptosis (Fig. 1).
Necrosis is characterized by cell swelling and disruption of the
cell membrane, leading to release of cellular contents, especially proteolytic enzymes, which may result in destruction of
neighboring cells. Apoptosis, on the other hand, is defined as
programmed cell death wherein the organism eliminates senescent, abnormal cells without affecting surrounding cells, and is
deemed to be preferable for the survival of the organism since
it eliminates dying cells by phagocytosis. The decision step between death by apoptosis or necrosis appears to be dependent
on intracellular ATP content (48). Apoptosis involves energyrequiring steps, especially in the formation of the apoptosome
complex between Apaf-1 and cytochrome c (56, 121). Thus in

MITOCHONDRIA-TARGETED CYTOPROTECTIVE PEPTIDES

FIG. 1. A scheme showing mitochondrial ROS production
resulting in cell death by necrosis or apoptosis. Protein oxidation and nitration impairs the function of many enzymes in
the mitochondrial electron transport chain (ETC) and decreases
ATP production. Peroxidation of cardiolipin leads to loss of
cardiolipin on the inner membrane and dissociation of cytochrome c. This will lead to compromised function of cytochrome c oxidase, further reduction in ATP production, and
increased ROS generation. Cytochrome c, once dissociated
from the inner membrane, can be released through the outer mitochondrial membrane into the cytosol. Mitochondrial outer
membrane permeability (MOMP) can be elicited by mitochondrial permeability transition (MPT) or may involve an
oligomeric form of Bax forming a pore on the outer membrane.
Cytochrome c in the cytosol binds to Apaf-1 to initiate the formation of an apoptosome, followed by activation of the caspase
cascade to cause cell death by apoptosis. In the event of significant cellular ATP depletion, death can only occur by necrosis because ATP is required for the apoptosis pathway.

the event of significant cellular ATP depletion, death can only
occur by necrosis. The inhibition of ATP-dependent ion pumps
in the plasma membrane can lead to the opening of a “death
channel” that is selectively permeable to anions, resulting in cytoplasmic membrane swelling and rupture (69). Thus caspases
not only mediate apoptosis but also protect against necrosis.

THERAPEUTIC TARGETS FOR
MINIMIZING CELL DEATH INDUCED BY
MITOCHONDRIAL OXIDATIVE DAMAGE
As proposed in Fig. 1, a number of cellular targets are available for therapeutic development to prevent cell death caused
by mitochondrial oxidative damage. Caspase inhibitors can reduce apoptosis, but would merely switch the morphology of
cell death to necrosis, which in the whole organism would be
a worse outcome (80, 85). On the other hand, PARP [poly(ADP-ribose)-polymerase] inhibitors will help to preserve ATP
and switch the mode of cell death from necrosis to apoptosis
(85). To reduce total cell death, it is necessary to target upstream of cytochrome c release. This may be achieved with
MPT inhibitors that directly inhibit the formation of the pore.
MPT inhibitors such as cyclosporin A and trifluoperazine have

603

been shown to reduce MPT, decrease intracellular ROS, and reduce cell death in cultured cells exposed to oxidative stress such
as tert-butylhydroperoxide or inhibitors of complex I of the
electron transport chain (46, 49). Unfortunately, side effects associated with these MPT inhibitors limit their therapeutic potential. It is possible to target even further upstream by reducing mitochondrial oxidative stress since ROS play an important
role in MPT pore opening. The endogenous antioxidant proteins such as SOD or catalase do not penetrate cell membranes
and are therefore ineffective against intracellular ROS. Vitamin
E or coenzyme Q (CoQ) are very lipophilic and tend to be retained in cell membranes and fail to achieve significant intracellular concentrations. N-acetylcysteine (NAC), a thiol-antioxidant, can replenish the endogenous antioxidant glutathione
and is very effective in reducing oxidative cell death but must
be given in mM concentrations in cultured cells. In vivo, however, NAC failed to provide significant antioxidant effect, presumably due to its low lipid solubility and tissue distribution
(17). To overcome the limitations of natural antioxidants, a
number of low molecular weight catalytic antioxidants, generally referred to as SOD mimetics, have been developed [see review (21)]. Several of these SOD mimetics have been reported
to be effective in blocking oxidant stress in cell models, including protection against ionization radiation (47) and inhibition of staurosporine-induced apoptosis (79). Studies have
shown that these SOD mimetics can provide some protection
against ischemia—reperfusion injury (28) and neurodegenerative diseases such as amyotrophic lateral sclerosis (39), and several of these are now in clinical development [see review, (64)].
However, while these SOD mimetics are cell-permeable, they
do not selectively target mitochondria and they only act on superoxide. It should be mentioned that some of the spin trap
agents such as TEMPOL (2,2,6,6-tetramethylpiperidine-1-oxyl)
can have prooxidant activity and can be cytotoxic (IC50 ϳ 0.1
to 100 mM) (36, 70). Interestingly, it appears that TEMPOL
distributes to mitochondria and can inhibit complex I of the
ETC, resulting in reduced ATP production and increased ROS
generation (61).

TARGETED DELIVERY OF
THERAPEUTICS TO MITOCHONDRIA
Improved efficacy and reduced side effects can be achieved
with molecules that selectively target and concentrate in mitochondria. This is particularly important since superoxide is
formed by complex I and complex III on the mitochondria inner membrane, and peroxidation of cardiolipin on the inner
membrane is the leading step towards reduced ATP production,
MPT, and cytochrome c release (see Fig. 1). Thus in order to
prevent cell death caused by ROS-induced mitochondrial damage, it is important to deliver the treatment agent to mitochondria. This idea is reinforced by the recent report that overexpression of catalase in mitochondria increased lifespan by 20%
in mice, whereas overexpression of catalase in peroxisomes had
no significant effect (90). Specific targeting of the treatment
molecule to mitochondria can also minimize adverse effects.
For example, cyclosporin A has very high affinity for cyclophilin D and is a potent inhibitor of MPT. However, cy-

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closporin A targets at least eight other cyclophilins in the cell
which can result in unwanted side effects (105). These side effects would be minimized if cyclosporine could be targeted to
mitochondria, so that high drug levels can be attained at the site
of action while minimizing drug exposure elsewhere.
A number of approaches have been used to target molecules
to mitochondria. They can be divided into two general approaches: (i) mitochondrial potential-dependent methods; and
(ii) mitochondrial potential-independent methods.

Mitochondrial potential-dependent methods
The most common method for targeting compounds to mitochondria makes use of the potential gradient across the mitochondrial inner membrane. As a result of the proton gradient,
a negative potential of 150–180 mV is generated across the inner membrane. Depending on their charge, lipophilic cations
may accumulate 100–1000-fold in the mitochondrial matrix.

TPPϩ-conjugated antioxidants. Murphy and coworkers applied this approach by conjugating the triphenylalkylphosphonium cation (TPPϩ) to lipophilic antioxidants
such as CoQ (MitoQ). MitoQ is taken up into the mitochondrial matrix in a potential-dependent manner, and is effluxed out
of mitochondria upon depolarization. Within the mitochondria,
MitoQ is thought to be accumulated on the matrix side of the
inner membrane. The theoretical background on the development of these molecules and much of the work done to date
have been extensively summarized in a recent review [see review, (63)]. A series of TPPϩ-conjugates have been designed
to reduce superoxide (MitoSOD), hydrogen peroxide (MitoPeroxidase), ferrous ion (MitoTEMPOL), and lipid peroxidation (MitoQ and MitoE).
The accumulation of these lipophilic cations in the mitochondrial matrix can disrupt mitochondrial potential and inhibit
mitochondrial respiration and ATP production. As a result, the
therapeutic index of these molecules is rather low, with toxic
concentrations being only ϳ10-fold greater than effective concentrations. This may be even more of a problem with MitoTEMPOL considering the intrinsic mitotoxic effects of TEMPOL (36, 70). The utility of TPPϩ-conjugated antioxidants may
also be limited by their requirement of mitochondrial potential
for mitochondrial uptake. Diseased mitochondria are unlikely
to have normal mitochondrial potential. Furthermore, the uptake of these antioxidants are self-limiting in that uptake is reduced at concentrations Ͼ50 ␮M when the accumulated cations
in the matrix begin to depolarize mitochondria (96).
By preferentially accumulating in the mitochondrial matrix,
these TPPϩ-conjugated antioxidants are more potent than their
lipophilic counterparts in reducing intracellular ROS, preserve
reduced thiols, and reduce apoptosis in cultured cells [see review, (63)]. This enhanced potency of the TPPϩ-conjugated antioxidants was abolished in cells pretreated with the uncoupler
FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone), confirming their dependence on mitochondrial potential
for uptake. The conjugation of a nitroxide to TPPϩ (MitoCP)
effectively inhibited H2O2 and lipid peroxide-induced oxidative
stress and apoptosis while the unconjugated nitroxide was ineffective (23). However, TPPϩ-conjugated TEMPOL and
EUK-134 failed to inhibit staurosporin-induced apoptosis, and

SZETO
did not improve on the ability of the unconjugated compounds
to inhibit selenium-induced apoptosis (22).
In contrast to the extensive work that has been done on the
biochemistry of these compounds, there is relatively little data
to support their in vivo efficacy in animal models of clinical
disorders. Pretreatment of rats with MitoQ in their drinking water for 2 weeks led to protection of myocardial function in an
ex vivo myocardial ischemia—reperfusion model (3). However,
there is no evidence that MitoQ will be effective in an in vivo
model, especially without prolonged pretreatment prior to ischemia. A recent article reported that MitoE did not protect striatal medium spiny neurons in a model of perinatal ischemia—
reperfusion injury (18). Based on this finding, the authors
concluded that oxidative stress does not play an important role
in this model of ischemia—reperfusion. A more likely explanation is inadequate diffusion of MitoE across the blood—brain
barrier, or limited uptake of this lipophilic cation by compromised mitochondria. A study with Drosophila also showed that
MitoQ had no effect on the normal aging process (58). More
in vivo animal studies are needed to demonstrate the advantages
of this approach for mitochondrial targeting.

Choline esters of glutathione and N-acetyl-cysteine.
Glutathione (L-␥-glutamyl-L-cysteinylglycine) plays a very important role in detoxifying ROS and preventing thiol oxidation.
Glutathione is synthesized in the cytoplasm and transported into
mitochondria via specific carriers. Increasing mitochondrial glutathione can be very effective in preventing mitochondrial oxidative stress. N-acetyl-L-cysteine is often used to provide cysteine
for glutathione synthesis. Using a similar approach, Sheu and coworkers prepared choline esters of glutathione and N-acetyl-cysteine to enhance their uptake into mitochondria (92). Early studies show that they can protect against oxidative stress in cultured
cells, but in vivo animal studies are not yet available.

Liposome and liposome-like vesicles. Exploring
amphiphilic cations with a delocalized positive charge center
known to accumulate inside mitochondria, Weissig and coworkers selected dequalinium to prepare a colloidal delivery
system specific for mitochondria [see review, (109)]. Dequalinium is a dicationic compound, a symmetrical molecule
with two cationic charge centers separated by a hydrophobic
carbon chain. This molecule can self-assemble in aqueous
medium into stable vesicles, which have been termed DQAsomes. These DQAsomes penetrate cells via endocytosis, and
must subsequently be released from the endosomes before they
can deliver the “cargo” to mitochondria. Fortunately, cationic
lipids are known to exert a destabilizing effect on endosomal
membranes, and Weissig and co-workers have been able to deliver DNA into mitochondria using these DQAsomes (20).
However, most of the liposomes made from dequalinium
analogs are cytotoxic. This approach to deliver drugs or DNA
into mitochondria is still in early stages of development, and
further structure—activity studies will be required to optimize
a delivery system based on dequalinium.
Mitochondrial potential-independent methods
Nitroxide radicals conjugated to gramicidin S
peptides. Although nitroxides like TEMPOL have antioxi-

MITOCHONDRIA-TARGETED CYTOPROTECTIVE PEPTIDES

605

dant and electron scavenging properties, the required high concentrations (mM) limit in vivo efficacy. There have been attempts to improve the targeting of nitroxides to mitochondria
using peptide vectors such as gramicidin S (GS). Gramicidin
peptides are 15 residue peptides isolated from Bacillus brevis,
and GS is unique in that it has a cyclic peptide chain. Gramicidin peptides assemble as a ␤-helix inside lipid bilayers. Although the 15 residue peptides are not long enough to span the
membrane layer, they can dimerize to form an elongated channel that spans the membrane (5). Wipf and co-workers conjugated a pentapeptide sequence (Leu-D-Phe-Pro-Val-Orn) from
GS to 4-amino-TEMPO (4-AT) to enhance mitochondrial targeting. When tested at a concentration of 10 ␮M, the GS peptide-conjugated 4-AT significantly reduced intracellular superoxide generation, caspase activation, and DNA fragmentation
elicited by actinomycin D (112). In contrast, 4-AT itself had no
effect. A recent article showed that a modified peptide analog
(D-Phe-Pro-Val-Orn-Leu) is also effective in enhancing the delivery of 4-AT (38). At the effective concentration of 10 ␮M,
these GS-conjugated nitroxides had no effect on intracellular
ATP.
In addition to actinomycin D, uroepithelial cells were protected against radiation treatment if pretreated with 100 ␮M of
GS-conjugated 4-AT, whereas the unconjugated 4-AT was not
effective (40). In the first in vivo study, treatment with a GSconjugated TEMPOL derivative (XJB-5-131) (2 ␮mol/kg) prolonged survival of rats subjected to hemorrhagic shock (57).
This approach of using gramicidin peptides provides an alternative to the lipophilic cation approach for targeted mitochondrial delivery.

Mitochondria-targeted small peptides. In contrast
to the above approaches which all use a targeted delivery system to deliver conventional antioxidants to mitochondria, there
is a novel class of cell-permeable small peptides that selectively
partition into the inner mitochondrial membrane and possess
intrinsic mitoprotective activities. The rest of this review will
focus on the design, development, characterization, and evaluation of these unique molecules as mitoprotective and cytoprotective agents.

MITOCHONDRIA-TARGETED
CYTOPROTECTIVE PEPTIDES
Chemistry and cell uptake of SS peptides
These novel peptides were originally designed by Hazel H.
Szeto and Peter W. Schiller, hence they have been designated
SS peptides. They are small, water-soluble, peptides limited to
Ͻ10 amino acid residues. The chemical structures of some of
the SS peptides are shown in Fig. 2. The synthesis of these peptides using solid phase methods has been described in previous
publications (88, 89).
Common to all of these peptides is an alternating aromaticcationic motif, with the basic amino acid residues (such as Arg
and Lys) providing two positive charges. The free amine of the
N-terminus of these peptides provides a third positive charge
because the C-terminus has been amidated. This aromatic-

FIG. 2. Chemical structures of SS peptides. SS-02 (DmtD-Arg-Phe-Lys-NH2)l; SS-31 (D-Arg-Lys-Phe-NH2); SS20
(Phe-D-Arg-Phe-Lys-NH2). Dmt ϭ 2Ј,6Ј-dimethyltyrosine.

cationic motif allows them to freely penetrate cells despite carrying a 3ϩ net charge at physiologic pH (119). When intestinal epithelial cells (Caco-2) were incubated with [3H]SS-02, the
radiolabel was detected in cell lysate as early as 5 min, and
steady state levels were achieved by 30 min, suggesting that
the peptide was diffusing freely in and out of the cell. The rate
of uptake of [3H]SS-02 was found to be concentration dependent over a wide range of concentrations with no evidence of
saturability. Surprisingly, the uptake of SS-02 was not temperature dependent, and did not involve PEPT1, the peptide transporter that is highly expressed in Caco-2 cells (119). Similar
uptake of SS-02 was observed with a variety of cell types, including neuronal cells (SH-SY5Y and N2A cells), renal epithelial cells (MDCK), endothelial cells (HUVEC), and human
embryonic kidney cell line (HEK 293) (119). The specific
amino acid sequence does not appear to be important, as SS31 was also readily taken up by neuronal N2A cells (118). Cellular uptake of SS-02 was confirmed with the use of a fluorescent-labeled peptide and the microscopic image clearly showed
intracellular distribution (119, 120). Furthermore, SS-02 was
able to translocate across a monolayer of Caco-2 cells from the
apical side to the basolateral side (119). The apparent permeability coefficient of SS-02 was calculated to be 1.24 ϫ 10Ϫ5

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cm/s, which is comparable to that for the dipeptide Gly-Sar
(1.26 ϫ 10Ϫ5 cm/s) that is known to be taken up by PEPT1. In
contrast, this permeability coefficient is ϳ1000-fold greater
than for met-enkephalin, a pentapeptide that does not have any
positive charges. On the other hand, an arginine tetramer penetrates poorly, and the larger cationic peptides (such as Tat and
penetratin) enter by endosomal uptake (24, 81). The relative
ease with which SS-02 penetrates cell membranes is unprecedented for a 3ϩ net charge peptide, and it appears that the aromatic-cationic motif provides a unique and highly favorable
structure for membrane penetration.

Mitochondrial uptake of SS peptides
The first evidence of mitochondrial uptake of these SS peptides came from confocal microscopic studies with a fluorescent-labeled SS-02 analog (SS-19) (120). Intestinal epithelial
cells (Caco-2) were incubated with SS-19, and fluorescence was
detected in the cytoplasm of all cells within 15 min, but the
peptide was entirely excluded from the nucleus. The fluorescent label showed a perinuclear distribution that resembled the
intracellular distribution of Mitrotracker TMRM, suggesting
that the peptide was localized to mitochondria. Mitochondrial
uptake of SS-02 and SS-31 were confirmed using isolated

SZETO
mouse liver mitochondria, and uptake of [3H]SS-02 and [3H]SS31 were rapid with maximal levels reached within 2 min (118,
120). The fraction of peptide partitioned to mitochondria was
estimated to be 1000–5000-fold compared to extramitochondrial concentration. Even though these are cationic peptides,
mitochondrial fractionation studies revealed that the peptides
are localized to the inner mitochondrial membrane rather than
in the matrix (Fig. 3) (120). Contrary to MitoQ and MitoE, the
uptake of these aromatic-cationic peptides into mitochondria is
not dependent on mitochondrial potential, as the extent of uptake was only reduced by ϳ10–15% in mitochondria that were
depolarized with FCCP (120). Because these positive-charged
peptides are not delivered into the mitochondrial matrix, their
uptake is not limited to mitochondria with normal potential.
This is a major advantage when dealing with diseased mitochondria with compromised mitochondrial potential.

Tyrosine-containing SS peptides can scavenge
ROS and inhibit lipid peroxidation
The structure of SS-02 (Dmt-D-Arg-Phe-Lys-NH2; Dmt ϭ
2Ј,6Ј-dimethyltyrosine) and SS-31 (D-Arg-Dmt-Lys-Phe-NH2)
provide free radical scavenging abilities to these peptides. Tyrosine-containing analogs can dose-dependently scavenge

FIG. 3. Targeted delivery of compounds to mitochondria. MitoQ [Coenzyme Q conjugated to TPPϩ (triphenylphosphonium cation)] is targeted into the mitochondrial matrix in a potential-driven manner. SS-31 and other SS peptide analogs target
the inner mitochondrial membrane and concentrate more than 1000-fold. The mitochondrial uptake of SS peptides is not dependent on mitochondrial potential.

MITOCHONDRIA-TARGETED CYTOPROTECTIVE PEPTIDES
H2O2, OHи, and ONOOϪ (Fig. 4). The specific location of the
tyrosine or dimethyltyrosine (Dmt) residue did not seem to be
important as SS-31 was found to be as effective as SS-02 in
scavenging all three ROS. However, replacement of tyrosine
with phenylalanine (SS-20; Phe-D-Arg-Phe-Lys-NH2) eliminated all scavenging ability, suggesting that the phenolic group
on tyrosine mediates the scavenging activity (120). Tyrosine is
known to scavenge oxyradicals, forming relatively unreactive
tyrosyl radicals, which can be followed by radical—radical coupling to give dityrosine, or react with superoxide to form tyrosine hydroperoxide (111). Dimethyltyrosine was found to be
more effective than tyrosine in scavenging H2O2, and Dmt-containing peptides were more effective than the tyrosine counterparts. By scavenging OHи, SS-31 and SS-02, but not SS-20,
also inhibited lipid peroxidation, as demonstrated by the reduction in conjugated dienes formed from linoleic acid (Fig. 4)
(120).

607

SS peptides reduce mitochondrial ROS production
By targeting the inner mitochondrial membrane, the SS peptides are ideally located to reduce mitochondrial oxidative
stress. Indeed, spontaneous H2O2 production by isolated guinea
pig cardiac mitochondria was dose-dependently reduced by SS31 and SS-02 (Fig. 5). Similar findings were obtained with
mouse liver mitochondria (120). Mitochondrial ROS production can be prevented by mitochondrial depolarization with the
use of uncouplers, but none of the SS peptides decreased mitochondrial potential in isolated mitochondria as measured with
Mitotracker TMRM (99). Furthermore, SS-31 and SS-02 can
reduce mitochondrial H2O2 production in response to antimycin
A (120). Antimycin A binds to complex III and inhibits the oxidation of ubiquinol, thereby generating large amounts of superoxide, which can then undergo dismutation by SOD to H2O2.
Although SS-20 also produced a statistically significant de-

FIG. 4. Antioxidant properties of SS peptides. SS-31 (black bar) and SS-02 (gray bar) dose-dependently scavenge hydrogen peroxide (H2O2), peroxynitrite (ONOOϪ), and hydroxyl radical (OHи). Hydrogen peroxide was measured by luminol chemiluminescence in the presence of horseradish peroxidase (HRP). Peroxynitrite was generated by thermal decomposition of SIN-1
and measured by luminal chemiluminescence. The hydroxyl radical was generated by the Fento reaction in the presence of deoxyribose, H2O2, and FeCl2. Linoleic acid peroxidation was induced by 2,2Ј-azobis(2-amidinopropane) and detected by the formation of conjugated dienes measured by absorbance at 234 nm. SS-20 (cross-hatched bar) does not scavenge ROS and does
not inhibit lipid peroxidation. *p Ͻ 0.05 compared to control.

608
crease in H2O2 generation, the magnitude of effect was much
less compared to SS-31 or SS-02 (Fig. 5). Since SS-20 does not
scavenge H2O2 (Fig.4), these results suggest that SS20, and
probably the other SS peptides, may have an independent action on reducing mitochondrial ROS production.

SS peptides inhibit mitochondrial
permeability transition
Mitochondrial ROS are known to contribute to MPT and mitochondrial swelling elicited by high Ca2ϩ and Pi. In turn, mitochondrial Ca2ϩ overload stimulates ROS production. SS-02
and SS-31, by reducing mitochondrial ROS, also effectively reduced mitochondrial swelling in isolated guinea pig cardiac mitochondria induced by high Ca2ϩ and Pi (Fig. 5). Similar findings were obtained with isolated mouse liver mitochondria

SZETO
(120). The inhibition of Ca2ϩ and Pi-induced MPT by SS peptides prevented cytochrome c release into the cytosol (120), and
this is relevant to the ability of these SS peptides in ischemia
protection (see later section on Ischemia—reperfusion injury).
Interestingly, SS20 was also able to inhibit mitochondrial
swelling induced by high Ca2ϩ and Pi. Although SS20 does
have some minimal ability to reduce mitochondrial ROS production, it is unlikely that the effect would be sufficient to inhibit MPT. Furthermore, other antioxidants known to inhibit
mitochondrial ROS, such as trolox and N-acetylcysteine, are
unable to prevent Ca2ϩ-induced mitochondrial swelling (99). It
is not known whether the TPPϩ-conjugated antioxidants, such
as MitoQ, are able to inhibit MPT, but higher concentrations
of MitoQ were reported to induce mitochondrial depolarization
(41, 96).
In addition to MPT induced by Ca2ϩ overload, SS peptides
can inhibit MPT elicited by inhibitors of the mitochondrial electron transport chain. MPPϩ (1-methyl-4-phenylpyridium), the
active metabolite of MPTP (1-methyl-4-phenyl-1, 2, 3, 6tetrahydropyridine), is known to inhibit mitochondrial complex
I activity, reduce O2 consumption, increase ROS production,
and cause mitochondrial depolarization and swelling (13).
MPPϩ-induced inhibition of mitochondrial O2 consumption
was significantly attenuated by addition of either SS-31 (50
␮M) or SS-20 (100 ␮M) (K.S. Zhao and H.H. Szeto, unpublished results). In addition, SS-31 and SS-20 prevented MPPϩinduced swelling in isolated mouse liver mitochondria (K.S.
Zhao and H.H. Szeto, unpublished results). Since SS-20 does
not scavenge ROS, these findings suggest that SS20 and the
other SS peptides directly inhibit MPT. This idea is further supported by the finding that SS20 can protect cell viability during ischemia (see later section on Ischemia—reperfusion injury)

SS peptides protect mitochondrial function and
inhibit cell death in cultured cells

FIG. 5. SS peptides reduce spontaneous H2O2 production
and inhibit swelling in isolated guinea pig cardiac mitochondria. Hydrogen peroxide was measured by luminol
chemiluminescence in the presence of horseradish peroxidase
(HRP). Mitochondrial swelling was induced by high Ca2ϩ and
Pi and measured by absorbance at 540 nm. All three SS peptides were able to inhibit Ca2ϩ/Pi induced mitochondrial
swelling. *p Ͻ 0.05.

By targeting and concentrating Ͼ1000-fold in the mitochondrial inner membrane, the tyrosine-containing SS peptides
(SS-02 and SS-31) are extremely potent in preventing oxidative cell death. Exposure of neuroblastoma N2A cells to H2O2
resulted in necrotic cell death that was completely prevented by
1 ␮M SS-31 (75). SS-31 was also very effective in preventing
necrotic cell death in G93A Cu,Zn SOD1-transfected N2A cells.
About 20% of cases of familial amyotrophic lateral sclerosis
carry this mutation (83), and motor neuron-like cells carrying
this mutation have increased ROS production, increased lipid
peroxidation, increased cytochrome c release, and reduced cell
viability (34). Furthermore, G93A SOD1 mutant cells are more
sensitive to the cytotoxicity of H2O2. This enhanced sensitivity is apparently due to increased free radical generation in response to H2O2 rather than abnormal SOD activity (114). SS31 was able to protect G93A Cu,Zn SOD1-transfected cells
from H2O2-induced cell death at 1 ␮M (75). It was apparent
from this study that doses lower than 1 ␮M of SS-31 would
have been effective in protecting against oxidative cell damage.
In contrast, an earlier study reported increased cell viability in
G93A SOD1 mutant-expressing cells with 20 mM of N-acetylcysteine or 25 mM of DMPO, a highly effective spin trapping
agent (55). At the present time, SS-31 is by far the most potent
cytoprotective agent against oxidative stress. Mice overex-

MITOCHONDRIA-TARGETED CYTOPROTECTIVE PEPTIDES
pressing G93A SOD1 mutation show motor neuron disease similar to amyotrophic lateral sclerosis. Daily treatment of these
transgenic mice with SS-31 (5 mg/kg, i.p.) delayed the onset of
neurological signs and prolonged survival (75). Histopathologic
studies revealed significant protection of spinal cord motor neurons in mice receiving daily SS-31 (75). A much larger dose of
DMPO (5 mg/mouse, i.p.) was required to delay neurological
symptoms and prolong survival in these mice (55), while vitamin E only delayed disease onset without increasing survival
time (33).
Tert-butylhydroperoxide (tBHP) is a membrane-permeant
prooxidant compound that can induce cell death via apoptosis
or necrosis. SS-31 was extraordinarily potent in reducing intracellular ROS and preventing apoptosis in neuronal cells after tBHP treatment, with EC50 in the ϳ0.1 nM (118) In contrast, most antioxidants require at least 100 ␮M to reduce
oxidative cell death (4, 68). MitoQ was able to block H2O2-induced apoptosis at 1 ␮M, but Ͼ10 ␮M caused cytotoxicity (41).
The remarkable potency of SS-31 in whole cells can be accounted for by its Ͼ1000-fold concentration in the inner mitochondrial membrane. Thus local concentration of SS-31on the
inner mitochondrial membrane may be Ͼ1 ␮M when extracellular concentration is only 1 nM. In addition to its extraordinary potency, SS-31 is not cytotoxic in normal cells even at 1
mM, resulting in a therapeutic window in excess of a millionfold.
tBHP-induced apoptosis appears to be triggered by MPT, and
trifluoperazine (5 ␮M), an inhibitor of MPT, was able to reduce cell death in hepatocytes treated with tBHP (68, 78). Treatment of neuroblastoma N2A cells with tBHP resulted in mitochondrial depolarization and decreased mitochondrial function
(118). SS-31 dose-dependently inhibited tBHP-induced mitochondrial depolarization and preserved mitochondrial function
at 1 nM (118). The much greater potency of SS-31 compared
to trifluoperazine may be partly due to the selective partitioning of SS-31 to the inner mitochondrial membrane, and partly
due to its antioxidant properties. It has been shown that deferoxamine (0.6 mM), an inhibitor of iron-catalyzed hydroxyl radical scavenger, is more effective than trifluoperazine in preventing tBHP-induced cell death, suggesting that ROS
contribute significantly to the induction of MPT by tBHP. It is
clear that SS-31 is the most potent compound reported that can
inhibit cell death caused by tBHP. The prevention of MPT by
SS-31 will minimize MPT-induced ROS accumulation and reduce further oxidative damage on mitochondria. By preventing
MPT, SS-31 was able to prevent phosphatidylserine translocation, caspase-9 activation, and nuclei condensation associated
with apoptotic cell death (118).
Other studies have shown that SS peptides can prevent cell
death induced by inhibitors of the electron transport chain. In
one study, SS-02 prevented mitochondrial depolarization induced by 3-nitroprionic acid in Caco-2 cells (120). 3-Nitroprionic acid is an irreversible inhibitor of the complex II enzyme
succinate dehydrogenase. In another study, SS-31 and SS-20
dose-dependently prevented cell death elicited by MPPϩ in
SN4741 dopamine cells. MPPϩ is known to inhibit complex I
activity, reduce O2 consumption, increase ROS production, and
leads to mitochondrial depolarization and swelling (13). Studies with isolated brain mitochondria showed that SS-31 and SS20 (50–100 ␮M) can both prevent the MPPϩ-induced reduc-

609

tion in O2 consumption and mitochondrial swelling (K.S. Zhao
and H.H. Szeto, unpublished results). However, a significant
reduction in apoptotic cells caused by MPPϩ was observed with
just 1 nM of either SS-31 or SS-20. The large discrepancy between the effective peptide concentrations required for isolated
mitochondria studies versus whole cell studies supports the
tremendous ability of these peptides to concentrate in the inner
mitochondrial membrane. Thus the targeting of SS-31 to inner
mitochondrial membrane allows the use of a very low concentration of SS-31 for selective scavenging of mitochondrial H2O2
without affecting cytoplasmic or extracellular H2O2. For example, dopamine causes cell cycle arrest in proliferating lymphocytes via extracellular H2O2 (60). While this action of dopamine was readily inhibited by extracellular catalase, it was
not blocked by SS-31 at concentrations that have been shown
to prevent mitochondria-mediated apoptosis (60).

ROLE OF MITOCHONDRIAL
DYSFUNCTION AND OXIDATIVE
DAMAGE IN ISCHEMIA—REPERFUSION
INJURY
Ischemia is associated with lack of oxygen and substrate delivery to the tissue and results in impaired energy metabolism
that, if prolonged, can result in cell death. Myocardial ischemia
and cerebral ischemia are two leading causes of death in the
western world. Persistent ischemia will lead to cell death. With
brief periods of ischemia, the depressed cellular energy state
can be fully reversed on reperfusion, and cells remain viable
and functional. Coronary reperfusion utilizing thrombolytics,
coronary angioplasty, or by-pass surgery can, if performed in
a timely fashion, partially rescue the ischemic myocardium and
limit the size of the infarct. However, reperfusion itself, especially after prolonged ischemia, may also lead to increased cell
death, a phenomenon known as “reperfusion injury.” Reperfusion injury is also encountered in organ transplantation after the
organ has been subjected to prolonged ischemic storage.
Progressive ischemia leads to inhibition of several components of the mitochondrial electron transport chain, including
complex I, complex V, and the adenine nucleotide translocator, and decrease in ATP production [for review, see (51)] . In
addition, ischemia decreases cardiolipin and cytochrome c content in cardiac mitochondria (50, 52), both of which will inhibit
cytochrome c oxidase activity (115). There is also evidence to
support ROS generation during ischemia (53), and this leads to
cardiolipin peroxidation and further inhibition of cytochrome c
oxidase activity (50) (Fig. 6).
With the decrease in mitochondrial ATP production, the cardiac cell strives to maintain ATP production by increasing glycolysis. However, glycolysis is much less efficient in ATP generation, and during ischemia, the F0F1 ATPase begins to
actively hydrolyze ATP rather than synthesize ATP, resulting
in a dramatic reduction in ATP and corresponding increase in
Pi (Fig. 6). The activation of glycolysis also leads to accumulation of lactic acid and decrease in intracellular pH. Intracellular pH can decrease to Ͻ6.0 after only 10 min of ischemia.
In an attempt to correct this Hϩ accumulation, the Naϩ/Hϩ exchanger results in increased intracellular Naϩ (45). The inhibi-

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SZETO

FIG. 6. A scheme showing mitochondrial dysfunction and oxidative damage during cardiac
ischemia—reperfusion. Progressive ischemia
leads to inhibition of the mitochondrial electron
transport chain (ETC) and decrease in ATP production. The cardiac cell strives to maintain ATP
production by increasing glycolysis, resulting in
further reduction in ATP, increase in Pi, and decreased intracellular pH. Activation of the
Naϩ/Hϩ exchanger, and inhibition of Naϩ/Kϩ
ATPase, results in increased intracellular Naϩ.
Intracellular Ca2ϩ becomes elevated via the
Naϩ/Ca2ϩ exchanger, resulting in mitochondrial
Ca2ϩ overload and mitochondrial depolarization.
In addition, ROS production and cardiolipin peroxidation further contributes to loss of inner
membrane integrity, inhibition of the ETC, and
mitochondrial depolarization. Restoration of
blood flow will help to restore ATP levels, but
the damaged mitochondria generate enormous
amounts of ROS during reperfusion, leading to cardiolipin peroxidation which would further inhibit oxidative phosphorylation
and promote mitochondrial permeability transition (MPT). Opening of the MPT pore results in cytochrome c release into the cytoplasm, activation of the caspase cascade, and apoptosis.

tion of Naϩ/Kϩ ATPase further increases intracellular Naϩ and
results in increased intracellular Ca2ϩ via the Naϩ/Ca2ϩ exchanger (101). Increased cytosolic Ca2ϩ leads to mitochondrial
Ca2ϩ overload driven by the potential gradient across the inner
membrane and via the Ca2ϩ uniporter, resulting in mitochondrial depolarization (25). In addition, cardiolipin peroxidation
further contributes to loss of inner membrane integrity and mitochondrial depolarization (76).
Restoration of blood flow will help to limit infarct size, but
recovery of mitochondrial function depends on the duration of
ischemia. With prolonged ischemia, there is evidence that the
rate of oxidative phosphorylation continues to deteriorate despite reperfusion, suggesting that additional mitochondrial dysfunction can take place (51). The damaged mitochondria generate enormous amounts of ROS during reperfusion, and there
is an excess of hydroxyl radicals as a result of iron overload
following ischemia. This will lead to cardiolipin peroxidation
and further inhibit oxidative phosphorylation and promote
MPT. The amount of ROS generation upon reperfusion is dependent on the extent of mitochondrial injury suffered during
ischemia.
Although the conditions of excess Pi, elevated mitochondrial
Ca2ϩ, and ROS all increase the probability of MPT pore opening during ischemia, most evidence suggest that the pore remains closed during ischemia and does not open until reperfusion takes place (31). The increase in mitochondrial Ca2ϩ flux
with the restoration of mitochondrial respiration during reperfusion promotes the opening of the MPT pore (35). It has been
proposed that ischemia is the MPT priming period, while reperfusion is the MPT trigger period, and the duration of ischemia
would determine the susceptibility of myocytes to MPT upon
reperfusion (108). Unless the pore closes rapidly, the cell would
not be able to survive, and the cell dies by necrosis as a result
of loss of plasma membrane integrity. If the pores close quickly,
maintenance of ATP will switch from necrosis to apoptosis. In
addition, the reactivation of mitochondrial respiration upon
post-ischemic reperfusion generates a burst of superoxide that

cannot be readily eliminated. ROS production is also exacerbated by mitochondrial Ca2ϩ that accumulates during reperfusion. This oxidative burst during reperfusion serves to promote
Ca2ϩ-induced MPT and apoptosis, adding to the ischemic damage.
The mitochondrial permeability transition pore and ROS are
widely considered the most promising targets for cardioprotection in ischemia—reperfusion injury. But Fig. 6 clearly shows
that inhibition of cardiolipin peroxidation, maintenance of electron transport chain function, and preserving mitochondrial potential can all contribute to cardioprotection. Because of the
multiple pathways by which the SS peptides may limit ischemia—reperfusion damage, these peptides have been studied in
several animal models of ischemia—reperfusion injury (see below).

EVALUATION OF SS PEPTIDES IN
MODELS OF ISCHEMIA—REPERFUSION
INJURY
SS peptides protect contractile function in
myocardial ischemia—reperfusion—ex vivo
studies
The SS peptides were first evaluated in ischemia—reperfusion injury in ex vivo guinea pig hearts undergoing retrograde
perfusion in a Langendorff apparatus. The isolated hearts were
subjected to 30 min global ischemia, followed by 90 min reperfusion. The SS peptides (1 nM) were introduced either upon
reperfusion (Fig. 7) or before ischemia and throughout reperfusion (Fig. 8). Despite restoration of flow after ischemia, the
buffer-treated hearts showed progressive deterioration of contractile force during the reperfusion period, reflecting a stunned
myocardium. Contractile force at 10 min and 90 min after onset of reperfusion was significantly improved if SS-31 was

MITOCHONDRIA-TARGETED CYTOPROTECTIVE PEPTIDES

611

FIG. 7. Post-ischemic treatment with SS-31 improves
myocardial contractile activity
after global ischemia in the ex
vivo heart. Isolated guinea pig
hearts were perfused in a retrograde manner with Krebs—
Henseleit buffer in a Langendorff
apparatus. After a 20 min stabilization period, global ischemia
was induced for 30 min, followed
by 90 min reperfusion with or
without 1 nM of SS-31 or SS-20.
Post-ischemic treatment with SS31 significantly improved contractile force, especially in the
late reperfusion period. SS-20
was not effective. The dashed
line shows mean contractile force
prior to global ischemia. *p Ͻ
0.05 compared to buffer alone.

given upon reperfusion only (Fig. 7). SS-20, on the other hand,
offered no protection if given after ischemia. Although a number of pharmacological agents have been shown to improve cardiac function if given prior to ischemia (preconditioning), SS31 is one of the very few agents that can improve contractile
force even when it is given upon reperfusion. A previous report showed that SS-02 was also able to prevent myocardial
stunning in the ex vivo heart when given upon reperfusion (113),
and this was later confirmed in vivo (see below) (97).
SS-31 and SS-02 are currently the most potent (nM) cardioprotective agents that can be used to reduce myocardial stunning without pre-ischemic treatment. These studies lend support to oxidative free radicals being the major cause of
myocardial stunning (9) because SS-20 was ineffective when
given upon reperfusion. By selectively partitioning to the inner

FIG. 8. Pre-ischemic treatment
with SS peptides improves myocardial contractile activity in the
ex vivo heart. Isolated guinea pig
hearts were perfused in a retrograde manner with Krebs—Henseleit buffer alone, or buffer containing either 1 nM SS-31 or SS-20, in
a Langendorff apparatus. After a
20 min stabilization period, global
ischemia was induced for 30 min,
followed by 90 min reperfusion
with or without 1 nM of SS-31 or
SS-20. Pre-ischemic treatment
with either SS-31 or SS-20 significantly improved contractile force
in the early reperfusion period, but
only SS-31 prevented myocardial
stunning in the late reperfusion period. The dashed line shows mean
contractile force prior to global
ischemia. *p Ͻ 0.05 compared to
buffer alone.

mitochondrial membrane, SS-31 and SS-02 can scavenge mitochondrial ROS and inhibit cardiolipin peroxidation, thus inhibiting the onset of MPT. Opening of the MPT pore in the
early phase of reperfusion is believed to underlie contractile
dysfunction in the stunned myocardium. Inhibition of MPT pore
opening at the onset of reoxygenation with cyclosporin A and
sanglifehrin A were reported to improve contractile function in
human atrial tissue (91). Recently, a novel SOD-mimetic MPT
inhibitor (HO-3538) was reported to significantly enhance the
recovery of mitochondrial energy metabolism and contractile
function (8). The SS peptides are at least 100-fold more potent
than these other agents in inhibiting MPT during ischemia—
reperfusion.
Surprisingly, even though SS-20 was ineffective during
reperfusion, pre-ischemic treatment with SS-20 significantly

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SZETO

improved contractile function in early reperfusion (Fig. 8).
However, the protective effect of SS-20 was not sustained during late reperfusion, perhaps due to overwhelming ROS production. These studies suggest that the SS peptides may improve mitochondrial function during ischemia. This idea is
supported by subsequent in vivo studies.

SS peptides reduce infarct size in model of
LAD occlusion
The ability of SS peptides to protect against myocardial damage during ischemia was further confirmed in an in vivo model
where the LAD (left anterior descending coronary artery) was
occluded for 60 min in rats. Rats received an intraperitoneal injection of either saline, SS-31 (3 mg/kg) or SS-20 (3 mg/kg) 30
min prior to occlusion of the LAD, and another dose 5 min before onset of reperfusion for another 60 min. Pre-ischemic
treatment with either SS-31 or SS-20 significantly improved
myocardial ATP content, decreased lipid peroxidation (as measured by 4-hydroxynonenal), reduced infarct size, and prevented ventricular arrhythmias during early reperfusion (Fig. 9)
(15). Although both peptides reduced infarct size compared
with saline control, interestingly the peptide without scavenging activity (SS-20) preserved the ischemic myocardium even
better than the scavenging peptide (SS-31). The study provided
some interesting information about SS-20. Even though SS-20
does not scavenge H2O2, nor does it inhibit lipid peroxidation
in vitro (120), it was able to reduce lipid peroxidation in the
ischemic myocardium. This suggests that SS-20 can reduce mitochondrial ROS production during ischemia—reperfusion,
most likely by improving mitochondrial bioenergetics during
ischemia. This is consistent with higher ATP content in the ischemia myocardium.

SS peptides reduce myocardial stunning in model
of LAD occlusion
In addition to improving mitochondrial bioenergetics during
ischemia, a recent study reported that pre-ischemic treatment with
SS-02 in a similar model of LAD occlusion in rats reduced reperfusion arrhythmias, shortened the time to left ventricular recovery, with significant improvement in ejection fraction and systolic wall thickening (97) (see Table 1). Thus, the SS peptides
not only reduce necrotic cell death (infarct size) in ischemia—
reperfusion, they can also reduce myocardial stunning in vivo.

SS peptides reduce infarct size in model of
cerebral ischemia—reperfusion
SS-31 has been investigated in a mouse model of cerebral
ischemia—reperfusion achieved by transient occlusion of the
middle cerebral artery for 30 min, followed by reperfusion (71).
Oxidative stress significantly reduced glutathione levels in the
ipsilateral cortex and striatum. Treating mice with SS-31 (2
mg/kg or 5 mg/kg given intraperitoneally) immediately after
reperfusion, and at 6, 24, and 48 h after ischemia significantly
reduced glutathione depletion in the cortex and reduced infarct
size by 32% and 35%, respectively, in the two dose groups. Focal ischemia is normally associated with a local inflammatory
reaction that contributes to tissue damage after the ischemic insult. Treatment with SS-31 also significantly decreased hemi-

FIG. 9. SS-31 and SS-20 improved cardiac outcome after
transient occlusion of the LAD (left anterior descending
coronary artery). Rats were given either saline, SS-31 (3
mg/kg, ip) or SS-20 (3 mg/kg, ip) 30 min prior to occlusion of
the LAD, and another dose 5 min before onset of reperfusion
for another 60 min. Pre-ischemic treatment with either SS-31
or SS-20 significantly improved myocardial ATP content, decreased lipid peroxidation (as measured by 4-hydroxynonenal
(HNE)), reduced infarct size, and prevented ventricular arrhythmias during early reperfusion. *p Ͻ 0.05 compared to
saline control.

spheric swelling. The success of SS-31 in reducing infarct size
when given after onset of reperfusion is significant for clinical
applications, and is likely enhanced by the its rapid distribution
across the blood—brain barrier.

SS peptides preserve coronary flow in hearts
after prolonged cold ischemic storage and
warm reperfusion
Ischemia—reperfusion injury is also a necessary consequence of all organ and cell transplantation. Prolonged cold
ischemic storage results in mitochondrial damage, and transplantation of the ischemic organ is met with oxidative damage.
Cardiac transplantation is hindered by donor shortage and
preservation time. Cold ischemic storage of hearts for transplantation is currently limited to 4–6 h, with longer storage time

613

MITOCHONDRIA-TARGETED CYTOPROTECTIVE PEPTIDES
associated with compromised short-term and long-term outcomes. Endothelial injury is now recognized as the most important cause of poor outcome after cardiac transplantation (26).
The SS peptides have been investigated in a model of prolonged
cold ischemic storage—warm reperfusion of guinea pig hearts.
Isolated hearts were perfused in a Langendorff apparatus with
a cardioplegic solution alone, or cardioplegic solution containing SS-31 (1 nM) or SS-20 (100 nM), for 3 min, and then stored
in the same solution at 4oC for 18 h. After the storage time, the
hearts were re-mounted in the Langendorff apparatus and perfused with Krebs—Henseleit buffer (34oC) for 90 min. Coronary flow was reduced 70% at the end of warm reperfusion in
hearts stored in the cardioplegic solution alone compared to preischemic flow. In contrast, hearts stored with either SS-31 or
SS20 in the cardioplegic solution had significantly better coronary flow, with only 10–20% reduction compared to pre-ischemic values. The compromised coronary flow in the heart stored
for prolonged time in cardioplegic solution alone was due to
extensive apoptosis of the endothelial cells in the coronary vessels (Fig. 10). In contrast, endothelial apoptosis was absent in
the hearts stored with SS-31 or SS-20. Endothelial cell apoptosis is a significant problem in cold ischemic storage of other
organs as well, and caspase inhibition has been reported to improve ischemia—reperfusion injury after lung transplantation.
These results suggest that the SS peptides, by preventing endothelial apoptosis, may allow much longer cold ischemic storage time and significantly increase organ availability for transplantation.

SS peptides preserve pancreatic islet cells during
isolation and improves transplantation outcome
Stressors present during pancreas procurement, preservation,
isolation, and following transplantation contribute to islet cell
demise and poor transplant outcome. The islet isolation procedure disrupts islet cells from their extracellular matrix via an
enzymatic and mechanical process and subjects the islets to cytokine, oxidative, and ischemic stress (1, 10). Improved glucose
control was observed in a marginal islet mass model with islets
from mice overexpressing glutathione peroxidase, MnSOD, and
CuZnSOD, suggesting that oxidative stress plays a major role
in islet viability (65). Supplementation of isolation buffers with
1 nM SS-31 significantly increased islet yield and mitochondrial potential of the islet cells, and reduced percent of apoptotic cells from by 50% (102). In a marginal mass transplantation model in mice, 5 of 10 recipients of islets isolated in the
presence of 1 nM SS-31 remained normoglycemic post transplantation, whereas all of the animals who received control
islets remained diabetic (102). These findings suggest that SS31 can improve islet survival during the isolation process and
also improve graft survival during the immediate ischemic
phase after transplantation. SS-31 may serve to increase the
pool of eligible organ donors for treatment of type I diabetes.

injury. However, the pharmaceutical development of peptide
drugs has been fraught with difficulties. The disadvantages of
peptide molecules include solubility challenges, lack of stability, difficulty with transport across membranes (especially
blood—brain barrier), rapid clearance from body, and low oral
bioavailability. Surprisingly, preliminary studies have shown
that the SS peptides have excellent pharmacokinetic properties
in several animal species, making them extremely feasible for
pharmaceutical development. Furthermore, loss of cell viability was only observed at concentrations that are Ͼ1000-fold
higher than concentrations required to prevent oxidative cell
death, and no apparent toxicity has been observed in animal
studies even after chronic dosing.

Pharmacokinetic studies of SS peptides in
animal models
All three SS peptides illustrated in Fig. 2 bear 3ϩ net charge
at physiologic pH and are therefore readily soluble in water.
Stability studies have shown that these tetrapeptides are stable
in aqueous solution for as at least 6 months, even when stored
at 37oC. Because of their solubility in water, these peptides can
be administered via many routes, including intravenous, subcutaneous, intraperitoneal, intrathecal, and intracerebroventricular injections (15, 16, 75, 95, 97, 102, 117).
In vivo pharmacokinetic studies were made possible with the
development of highly specific and sensitive liquid chromatography—mass spectrometry methods (32, 106, 107). Early
pharmacokinetic studies of SS-02 were carried out in sheep utilizing continuous intravenous infusion (100). The calculated
pharmacokinetic parameters revealed an apparent volume of
distribution (ϳ60 ml/kg) and an elimination half-life of ϳ1.8
hours. The very small apparent volume of distribution is consistent with the highly polar character of these 3ϩ charge peptides, suggesting very little accumulation of these peptides in
adipose tissue. Yet, SS-02 is rapidly distributed to the brain and
other highly perfused organs including lung, heart, and kidneys.
Following an intravenous administration of [3H]SS-02 to mice,
radioactivity peaked in the brain and other organs before 5 min.
The rapid distribution of SS-02 across the blood—brain barrier
was unexpected for a polar compound, but is consistent with
its rapid transcellular uptake across an epithelial cell monolayer
(119). In addition, SS-02 has high affinity for opioid receptors
(116) and onset of analgesia was very rapid after subcutaneous
administration in mice, with peak analgesic effect observed between 30–45 min (67, 117). Extensive brain uptake of SS-02 is
supported by the finding that SS-02 was 36 times more potent
than morphine after subcutaneous administration (117). In contrast, other synthetic opioid peptides are generally not potent
TABLE 1. EFFECTS OF SS-02 ON LEFT VENTRICULAR
FUNCTION AFTER TRANSIENT LAD OCCLUSION
Saline

THERAPEUTIC POTENTIAL
OF SS PEPTIDES
The in vivo animal studies described above suggest that the
SS peptides may be beneficial against ischemia—reperfusion

Time to recovery (min)
Fractional shortening (%)
Ejection fraction (%)
Systolic wall thickening (%)
*p Ͻ 0.01.
Ͻ 0.05.

†p

10.5
32.1
32.3
33.5

Ϯ
Ϯ
Ϯ
Ϯ

2.2
9.1
13.7
7.7

SS-02
4.4
45.7
48.9
50.5

Ϯ
Ϯ
Ϯ
Ϯ

2.2*
2.4†
10.9†
8.4*

614

SZETO

FIG. 10. SS peptides prevent endothelial apoptosis in hearts subjected to prolonged cold ischemic
storage and warm reperfusion. Isolated guinea pig hearts
were perfused in a Langendorff apparatus with a cardioplegic solution
alone, or cardioplegic solution containing SS-31 (1 nM) or SS-20 (1 or
100 nM) for 3 min, and then stored
in the same solution at 4oC for 18
hours. After the storage time, the
hearts were re-mounted in the Langendorff apparatus and perfused
with
Krebs—Henseleit
buffer
(34oC) for 90 min. Hearts were then
set in paraffin blocks, sectioned, and
stained for apoptotic cells using the
TUNEL method with fluorescent
secondary antibody. Extensive
TUNEL-positive endothelial cells (green) were found in hearts stored in buffer (A) while storage with either SS-31 (B) or SS20 (C) prevented apoptosis.

when given systemically. Thus the aromatic—cationic nature
of the SS peptides allows them to readily cross the blood—
brain barrier.
The relatively long elimination half-life of SS-02 is highly
unusual for a peptide. The SS peptides were designed to minimize enzymatic degradation, with the incorporation of a Damino acid either in the first (SS-31) or second residue (SS02), and amidation of the carboxy terminus. These tetrapeptides
do not undergo degradation when incubated in whole blood for
2 hours (100). Being such polar molecules, the SS peptides
might be expected to undergo renal clearance. Surprisingly, the
plasma clearance of SS-02 from blood (23 ml/kg/h) was found
to be almost 10-fold slower than creatinine clearance (ϳ210
ml/kg/h), suggesting substantial tubular reabsorption of this
peptide, most likely via PEPT2, the peptide transporter present
on proximal tubular cells of the kidney (100).
The pharmacokinetics of SS-02, SS-31, and SS-20 have been
confirmed in rats following intravenous bolus administration
and their elimination half-lives ranged from 30 to 40 min. The
systemic absorption of these peptides following intraperitoneal
administration is rapid, with peak plasma levels observed before 20 min. Despite the relatively short half-lives, a single subcutaneous dose of SS-02 (1 mg/kg) was found to produce significant analgesic response for more than 12 h in mice (117).
Similarly, once a day dosing with intraperitoneal SS-31 (5
mg/kg) was sufficient to protect spinal cord motor neurons in
a transgenic mouse model of amyotropic lateral sclerosis (75).
Significant improvement in pancreatic islet transplantation was
also achieved with once a day dosing with SS-31 (102). Because these peptides are stable in aqueous solution at 37oC, they
can also be delivered using mini-osmotic pumps for sustained
subcutaneous delivery over 2–4 weeks in mice and rats.
Although peptides are generally considered to be poor candidates for oral administration, available data suggest that these
SS peptides may have reasonable oral bioavailability. Oral absorption can be predicted using Caco-2 cells (human intestinal
epithelial cell) grown in a monolayer. The minimum Papp (ap-

parent permeability coefficient) required to anticipate 100% absorption in humans has been estimated between 1 ϫ 10Ϫ6 and
6 ϫ 10Ϫ5 cm/s (30, 84). With a calculated Papp of 1.24 ϫ 10Ϫ5
cm/s for SS-02 (119), we may anticipate reasonably good oral
absorption for these peptides. Furthermore, oral administration
of SS-02 produced dose-dependent analgesia in mice (Szeto,
unpublished results), demonstrating that these SS peptides are
orally active.

Cytotoxicity studies in cultured cells
The SS peptides have been found to be relatively free of cytotoxicity in cultured cells. SS-02 and SS-31 have been shown
to be effective in preventing cell death induced by oxidative
damage in the concentration range of 0.1–100 nM (118, 120).
When given alone, these peptides had no effect on cell viability in epithelial cells (Hela, Caco-2, HKC-8, MDCK) or neuroblastoma cells (N2A, SH-SY5Y) even at 1 mM.

In vivo toxicity
The SS peptides have been studied extensively in animal
models. Because of its high affinity for opioid receptors, SS02 can cause constipation (67) and respiratory depression (94)
similar to morphine. In contrast, SS-31 and SS-20 have negligible affinity for opioid receptors and are therefore devoid of
opioid receptor-mediated effects at therapeutic doses. SS-31 has
been administered daily (5 mg/kg) for up to 5 months in a mouse
model of amyotrophic lateral sclerosis and no adverse effects
were observed while treatment was actually associated with significant protection of spinal cord motor neurons, delayed onset
of neurological deficits, and increased survival time (75). In
other studies, repeated dosing with higher doses of SS-31 (up
to 20 mg/kg) for 14 days in mice did not result in any apparent toxicity. While available studies suggest that these SS peptides appear to be free of toxic side effects, formal toxicological studies will need to be conducted to determine the relative

615

MITOCHONDRIA-TARGETED CYTOPROTECTIVE PEPTIDES

FIG. 11. Proposed sites and mechanisms of
action for the SS peptides in protecting
against
ischemia—reperfusion
injury.
Pre-ischemic treatment with any of the three
peptides (SS-02, SS-31, or SS-20) protects the
mitochondrial ETC and helps maintain ATP
levels during ischemia. All three peptides can
inhibit mitochondrial permeability transition
induced by Ca2ϩ/Pi. Upon reperfusion, there
is a massive burst of ROS activity, and only
the scavenging peptides (SS-02 and SS-31) can
effectively minimize mitochondrial permeability transition.

safety of these novel compounds for pharmaceutical development.

SUMMARY
It is now recognized that oxidative injury and mitochondrial
dysfunction are responsible for many clinical disorders with unmet needs. Mitochondrial dysfunction can lead to cell death by
apoptosis and necrosis. As mitochondria are the major source
of intracellular ROS, and mitochondria are also the primary target for ROS, the ideal drug therapy needs to be targeted to mitochondria. The mitochondria-targeted cytoprotective peptides
described in this review represent a novel class of mitoprotective agents that are selectively targeted to and concentrate in
mitochondria. Extensive data from in vivo animal studies support their clinical utility in ischemia—reperfusion injury. Figure 11 summarizes the mechanisms by which the SS peptides
can help minimize ischemia—reperfusion injury. These peptides can be divided into two major groups. Those containing
tyrosine or modified tyrosine residues can scavenge ROS, and
are particularly effective in disorders associated with significant ROS generation, such as reperfusion injury in post-ischemic tissues. Those that do not have scavenging capability are
still highly effective in preventing ischemic injury and can be
used for minimizing ischemia—reperfusion injury during angioplasty, coronary bypass surgery, cardiac surgery, and organ
transplantation. Animal studies to date indicate that these novel
mitochondria-targeted peptides have excellent pharmacokinetic
properties and are relatively free of toxicity, suggesting that
they may have enormous therapeutic potential.

ACKNOWLEDGMENTS
The author is grateful to the many collaborators who contributed to the work presented in this review. Majority of the
work presented in this review was supported by NIDA (PO1
DA08924), NINDS (R21 NS48295), and NIDDK (R01

DK073595). The SS peptides were developed in collaboration
with Dr. Peter W. Schiller of the Clinical Research Institute of
Montreal, Montreal, Quebec, Canada. All biochemical, cellular, and ex vivo studies mentioned in this article were carried
out by Drs. Kesheng Zhao, Guomin Zhao, Dunli Wu, and Yi
Soong (Department of Pharmacology, Weill Medical College
of Cornell University). Special thanks to Drs. Mun Hong,
Wooyuk Song, Janghyun Cho and Kyungheon Won (Department of Internal Medicine, Weill Medical College of Cornell
University) for the in vivo studies with SS peptides in myocardial ischemia—reperfusion; Dr. Sunghee Cho (Burke Research
Institute, White Plains, NY, and Department of Neurology/Neuroscience, Weill Medical College of Cornell University) for the
studies of SS-31 in cerebral ischemia—reperfusion; and Drs.
Dolca A. Thomas and Manikkam Suthanthiran (Departments of
Medicine and Transplantation Medicine, Weill Medical College of Cornell University) for the pancreatic islet transplantation studies.

ABBREVIATIONS
ANT, adenine nucleotide translocator; CO3иϪ, carbonate radical anion; CoQ, coenzyme Q; Dmt, 2Ј,6Ј-dimethyltyrosine;
ETC, electron transport chain; FCCP, (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone); LAD, left anterior descending coronary artery; MOMP, mitochondrial outer membrane
permeability; MPPϩ, 1-methyl-4-phenylpyridium; MPT, mitochondrial permeability transition; MPTP, 1-methyl-4-phenyl-1,
2, 3, 6-tetrahydropyridine; NAC, N-acetylcysteine; O2иϪ, superoxide anion; OHи, hydroxyl radical; ONOOϪ, peroxynitrite;
PARP, poly-(ADP-ribose)-polymerase; ROS, reactive oxygen
species; SOD, superoxide dismutase; SS peptides, Szeto—
Schiller peptides; SS-02, Dmt-D-Arg-Phe-Lys-NH2; SS-20,
Phe-D-Arg-Phe-Lys-NH2; SS-31, D-Arg-Dmt-Lys-Phe-NH2;
TEMPOL, 2,2,6,6-tetramethylpiperidine-1-oxyl; tBHP, Tertbutylhydroperoxide; TCA, tricarboxylic acid; TPPϩ, triphenylalkylphosphonium cation; VDAC, voltage-dependent anion
channel.

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SZETO

DISCLOSURE
Patent applications have been filed by Cornell Research
Foundation Inc (CRF) for the technology (SS peptides) described in this article. Hazel H. Szeto is the inventor. CRF, on
behalf of Cornell University, has licensed the technology for
further research and development to a commercial enterprise in
which CRF and Dr. Szeto have financial interests.

17.
18.

19.
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Address reprint requests to:
Hazel H. Szeto, M.D., Ph.D.
Department of Pharmacology
Weill Medical College of Cornell University
1300 York Avenue
New York, N.Y. 10021
E-mail: hhszeto@med.cornell.edu
Date of first submission to ARS Central, August 29, 2007; date
of final revised submission, September 9, 2007; date of acceptance, September 10, 2007.