REVIEW ARTICLE
The mitochondrial permeability transition from in vitro
artifact to disease target
Paolo Bernardi
1
, Alexandra Krauskopf
1,
*, Emy Basso
1
, Valeria Petronilli
1
, Elizabeth Blalchy-Dyson
2
,
Fabio Di Lisa
3
and Michael A. Forte
2
1 Department of Biomedical Sciences and CNR Institute of Neurosciences, University of Padova, Italy
2 Vollum Institute, L474, Oregon Health and Sciences University, Portland, OR, USA
3 Department of Biological Chemistry and CNR Institute of Neurosciences, University of Padova, Italy
Introduction
The mitochondrial permeability transition (PT) is an
increase of mitochondrial inner membrane permeabil-
ity to solutes with molecular masses up to  1500 Da.
Under the conditions used in most in vitro studies, PT
is accompanied by depolarization, matrix swelling,
depletion of matrix pyridine nucleotides (PN), outer
membrane rupture and release of intermembrane pro-
teins, including cytochrome c [1,2]. The occurrence of
swelling in isolated mitochondria, its stimulation by

of in vitro artifact to that of effector mechanism of cell death. We then
cover recent results based on genetic inactivation of putative permeability
transition pore components, and discuss their meaning for our understand-
ing of pore structure. Finally, we discuss evidence indicating that the per-
meability transition pore plays a role in pathophysiology, with specific
emphasis on in vivo models of disease.
Abbreviations
AAF, 2-acetylaminofluorene; ANT, adenine nucleotide translocator; CNS, central nervous system; CRC, Ca
2+
retention capacity; CsA,
cyclosporin A; CyP, cyclophilin; Dp, proton electrochemical gradient; Dw
m
, mitochondrial membrane potential; MMC, mitochondrial
megachannel; PARP, poly(ADP-ribose) polymerase; PBR, peripheral benzodiazepine receptor; PN, pyridine nucleotides; PT, permeability
transition; PTP, permeability transition pore; TNF-a, tumor necrosis factor; ROS, reactive oxygen species; Ub0, ubiquinone 0; VDAC, voltage-
dependent anion channel.
FEBS Journal 273 (2006) 2077–2099 ª 2006 The Authors Journal compilation ª 2006 FEBS 2077
conditions for isolation, storage and incubation of
mitochondria have been intentionally (albeit empiric-
ally) designed to minimize its occurrence.
The typical mitochondrial isolation and storage solu-
tions are based on K
+
-free mannitol and ⁄ or sucrose.
Incubation in these media promotes H
+
–K
+
exchange
and matrix acidification, which in turn potently inhib-

had
a physiological role in steroidogenesis. These authors
showed that Ca
2+
induces a ‘transformation’ of adre-
nal cortex mitochondria, allowing extramitochondrial
PN to gain access to the otherwise impermeable mat-
rix, and that NADPH entering in this way supports
the 11-b hydroxylation of deoxycorticosterone [19–21].
These findings matched those of Vinogradov et al.,
who documented a Ca
2+
-dependent release of matrix
PN through the otherwise impermeable inner mem-
brane in liver mitochondria [16]. The term ‘permeabil-
ity transition’, however, was introduced by Haworth &
Hunter, who carried out a detailed characterization of
its basic features in heart mitochondria. These authors
provided a key insight that the PT was caused by
reversible opening of a proteinaceous pore in the inner
mitochondrial membrane – the permeability transition
pore (PTP) – and proposed that it may serve an unde-
fined physiological role [22–25].
It is fair to say that this proposal was not met by
enthusiasm. In part, at least, this was an indirect conse-
quence of the general acceptance of the chemiosmotic
hypothesis, which had just been fully recognized with
the award of the Nobel Prize in Chemistry to Peter
Mitchell in 1978 [26]. As already noted [1], studies of
mitochondrial ion transport were mostly carried out in

soon followed by the demonstration that the inner
mitochondrial membrane is also endowed with a high-
conductance ( 1 nS) channel, the ‘mitochondrial
megachannel’ (MMC) [51,52]. The MMC is inhibited
by CsA [53], and possesses all the basic regulatory fea-
tures of the PTP [54,55], leaving little doubt that they
are the same molecular entity [56]. Electrophysiology
has greatly contributed to our understanding of the
MMC-PTP, and to the acceptance of the pore theory
of the PT [57].
A third contribution was the demonstration that the
PTP is controlled by the proton electrochemical gradi-
ent (Dp), the open–closed transitions being modulated
by the mitochondrial membrane potential (Dw
m
) and
by matrix pH [14,15,58]. These findings have been fully
confirmed by studies at the single channel level [59].
As the threshold voltage for PTP opening is affected
by a large variety of pathophysiological effectors
[60,61], PTP control by the Dp provided a conceptual
The mitochondrial permeability transition P. Bernardi et al.
2078 FEBS Journal 273 (2006) 2077–2099 ª 2006 The Authors Journal compilation ª 2006 FEBS
framework to accommodate a large number of individ-
ual agents known to induce or inhibit the PT [62–64].
The hypothesis that PTP opening could be a factor
in cell death, was put forward nearly 20 years ago [65].
A series of seminal studies published in the early 1990s
provided experimental support for this hypothesis in
hepatocytes subjected to oxidative stress [66,67], anoxia

on genetic and pharmacological strategies, and on the
study of relevant in vivo models of disease.
A Medline search identified close to 2000 publica-
tions on the PTP, a figure that demands a selection of
the primary references that can be quoted. We apolo-
gize in advance to those who could not find a place in
our reference list, and we refer the reader to recent
reviews discussing the possible role of the PTP in sev-
eral paradigms of disease for a more detailed coverage
[81–96].
Modulation of the PT
As mentioned above, the PT is most easily observed
after the matrix accumulation of Ca
2+
, and it is widely
believed to be caused by the opening of a regulated
channel, the PTP. The pore can be defined as a volt-
age-dependent, CsA-sensitive, high-conductance chan-
nel of the inner mitochondrial membrane. In the fully
open state, its apparent diameter is  3 nm, and the
pore open–closed transitions are highly regulated by
multiple effectors that may converge on a smaller set
of regulatory sites. We have classified factors that
affect the PT into matrix and membrane effectors, and
we refer the reader to a previous review for details [1].
Matrix effectors
Pore opening is favored by matrix Ca
2+
through a site
that can be competitively inhibited by other Me

pyrocarbonate) [14,15] and above pH 7.4 (through an
unknown mechanism). Histidyl residues (particularly
His126 of CyP-A and His87 of the FK506-binding
protein) have also been shown to play important roles
in ligand binding and peptidyl prolyl cis-trans iso-
merase catalysis by immunophilins [101]. Our recent
finding, that PTP modulation by matrix pH between
6.0 and 7.0 is identical in mitochondria from Ppif null
and wild-type animals, demonstrates that PTP-regula-
tory histidines are not located on CyP-D [47], as
already suggested based on the effects of diethylpyro-
carbonate [102]. It is important to stress that the over-
all effect of pH on the PTP can be dramatically
affected by energization, because an acidic pH does
not inhibit, but rather promotes, PTP opening in ener-
gized mitochondria owing to an increased rate of Pi
P. Bernardi et al. The mitochondrial permeability transition
FEBS Journal 273 (2006) 2077–2099 ª 2006 The Authors Journal compilation ª 2006 FEBS 2079
uptake, an effect that may worsen PTP-dependent tis-
sue damage in ischemic and postischemic acidosis
[103].
PTP regulation by matrix CyP-D will be discussed
below.
Membrane effectors
The inside-negative Dw
m
tends to stabilize the PTP in
the closed conformation [14]. We have postulated the
existence of a voltage sensor that decodes the changes
of both the transmembrane voltage and of the surface

-dependent pore open-
ing, irrespective of the inducing agent; and the inhibi-
tory effect of Ub0 and decylubiquinone (but not that
of CsA) can be relieved by pore-inactive quinones,
such as ubiquinone 5 [108]. High-throughput screening
of isolated mitochondria has recently identified a novel
PTP inhibitor, Ro 68–3400, which apparently interacts
with the same site as Ub0 [109]. This inhibitor will be
discussed in greater detail in relation to the possible
role of VDAC in PTP formation.
Consequences of pore opening
The only primary consequence of PTP opening is mito-
chondrial depolarization. Unless single channel events
are being recorded, however, PTP openings of short
duration may be undetectable. Indeed, for short
durations of the open time, repolarization follows, and
the depolarization–repolarization cycle may not be
detected by potentiometric probes. Furthermore, open-
ing events are not synchronized for individual mito-
chondria [110,111] and may be missed in population
studies as a result of probe redistribution among indi-
vidual mitochondria. The occurrence of PTP openings
of different durations in mitochondria in situ, and their
consequences on cell viability, have been addressed in
a series of specific studies to whom the reader is
referred for details [104,112,113].
For longer times of opening, depolarization can be
easily measured both in isolated mitochondria and
intact cells, and the PT may have consequences on res-
piration that depend on the substrates being oxidized.

that can be distinguished based on their redox interac-
tions with the outer and inner membrane electron
transfer systems [114]. About 15% of cytochrome c
can be reduced by outer membrane NADH-cyto-
chrome b
5
reductase, suggesting that it is located
within the intermembrane space; while 85% can only
be reduced by the inner membrane electron transfer
chain [114] and probably resides within the intercristal
compartment identified by tomographic reconstruction
of mitocondria after high-voltage electron microscopy
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2080 FEBS Journal 273 (2006) 2077–2099 ª 2006 The Authors Journal compilation ª 2006 FEBS
[115]. During proapoptotic stimulation, prominent cris-
tae remodeling occurs, which effectively increases the
communication between the two pools of cytochrome c,
and therefore the fraction that can be released through
BAX ⁄ BAK channels on the outer membrane [116].
In summary, a rigorous assessment of the occurrence
and of the consequences of PTP opening, in particular
mitochondrial depolarization, swelling and outer
membrane rupture, demands a careful measurement of
several variables that can only be deduced by indirect
means [78].
Whether the PTP has a physiological function other
than taking part in cell death remains a matter of spe-
culation. We, as well as others, have suggested that the
pore may serve as a mitochondrial Ca
2+

the ANT catalyzes Ca
2+
-dependent malate transport
that is inhibited by ADP and favored by atractylate
[124]. By using chromatography of mitochondrial
extracts on a CyP-D affinity matrix, specific binding of
the ANT has been demonstrated by two laboratories,
while there is a discrepancy as to whether VDAC is
also essential for reconstitution of PTP activity
[125,126]. Although intriguing, we think that the rele-
vance of these observations to PTP regulation remains
unclear. Indeed, in the work of Woodfield et al. [125],
CyP-D also bound many other proteins besides ANT,
some of them with high affinity and in a CsA-inhibita-
ble manner. Moreover, CyP-D bound equally well to
ANT purified from rat liver or from yeast, despite the
fact that the PT is not inhibited by CsA in yeast mito-
chondria [127]. In the work of Crompton et al., CyP-D
column eluates containing both ANT and VDAC con-
ferred CsA-sensitive permeabilization to proteolipo-
somes that had been treated with Ca
2+
plus Pi, yet it
is difficult to exclude that permeabilization was caused
by other species represented less than the abundant
ANT and VDAC [126].
Conclusive evidence that the ANT is not essential
for PTP formation was obtained in a detailed analysis
of mitochondria lacking all ANT isoforms, which
revealed that a Ca

fhydryl reagents is not observed in mitoplasts, suggest-
ing that inner membrane permeability changes require
the outer mitochondrial membrane as well [34]. Several
lines of evidence suggest that the outer membrane
protein involved in PTP formation may be VDAC,
namely that (a) purified VDAC incorporated into pla-
nar phospholipid bilayers forms channels with a pore
diameter of 2.5–3.0 nm whose electrophysiological
properties are strikingly similar to those of the PTP
[131,132], (b) the VDAC channel properties are modu-
lated by the addition of NADH, Ca
2+
, glutamate
P. Bernardi et al. The mitochondrial permeability transition
FEBS Journal 273 (2006) 2077–2099 ª 2006 The Authors Journal compilation ª 2006 FEBS 2081
[133–135] and by binding of hexokinase [136–138], all
conditions that also modulate the activity of the PTP
[17,97,139], and (c) as mentioned above, chromato-
graphy of mitochondrial extracts on a CyP-D affinity
matrix allowed purification of VDAC and the ANT,
which in the presence of CyP-D catalyzed CsA-sensi-
tive permeabilization of liposomes to solutes [126].
The inner and outer mitochondrial membranes
appear to interact in specialized regions called the
‘contact sites’, which are enriched in ANT and VDAC
bound to cytosolic hexokinase I [140], leading to the
idea that the PTP may be formed by interacting ANT
and VDAC molecules at these sites. Fractions of deter-
gent-solubilized mitochondria containing hexokinase
activity were assessed by western blotting and found to

correspond (closely or not) to properties of the PTP
observed in mitochondria, therefore VDAC must be part
of the PTP. This generalization has been embellished in
most recent reports on the involvement of VDAC in
the PTP. Thus, data demonstrating the VDAC activity
in bilayers is modulated by ruthenium red and La
3+
[134], arsenic trioxide [144], hexokinase [145], protein
cross-linkers [146] and fluoxetine [147] and may reflect
in vitro alterations in VDAC activity by these treat-
ments, but cannot formally be extended to reflect the
involvement of VDAC in the PTP, either in mitochon-
dria or in a cellular context. These considerations
obviously do not exclude a possible role for VDAC in
apoptotic pathways not dependent on the PTP.
As been demonstrated in the case of CyP-D, poten-
tially the most convincing data on the role of VDAC
in PTP formation could be generated through an
examination of PTP activity in mitochondria prepared
from tissues in which VDAC has been genetically elim-
inated. The difficulty with these studies stems from the
fact that in mammals three genes encode VDAC iso-
forms (for a review of the genetics of VDAC see ref.
148) and each VDAC isoform is able to form channels
when incorporated into planar bilayers, albeit with
somewhat different characteristics [149]. Mice have
also been created in which genes encoding individual
VDAC isoforms have been eliminated by ‘knockout’
strategies. Mice missing VDAC1 and VDAC3 are
viable, but show isoform-specific phenotypes [150],

advanced genetic approaches [e.g. loxP versions of
individual VDAC genes or the use of small interfering
The mitochondrial permeability transition P. Bernardi et al.
2082 FEBS Journal 273 (2006) 2077–2099 ª 2006 The Authors Journal compilation ª 2006 FEBS
(siRNA)] may allow the generation of more reliable
data. Alternatively, the development of pharmacologi-
cal agents that specifically target all VDAC isoforms
with high affinity may also allow the involvement of
VDAC in the PTP to be convincingly established.
Until such studies are available, the involvement of
VDAC in the PTP remains unproven.
Role of the PBR
The PBR is an 18 kDa, highly hydrophobic protein
located in the outer mitochondrial membrane [153]
and was initially identified as a binding site for ben-
zodiazepines in tissues that lack 4-aminobutyrate
receptors, the clinical target of benzodiazepines in the
central nervous system (CNS). The PBR shares no
amino acid homology with CNS 4-aminobutyrate
receptors, and can be distinguished pharmacologically
from CNS receptors by its binding to a variety of
high-affinity ( nm), PBR-specific ligands [154,155],
notably the benzodiazepine, Ro5-4864, and the iso-
quinoline carboxamide, PK11195. These, and a num-
ber of other compounds, have been used extensively in
the biochemical and physiological characterization of
the PBR in vivo and in vitro [156]. The PBR is found
in a wide variety of tissues at varying levels and is
especially abundant in cells producing steroid hor-
mones, such as adrenal cortex and Leydig cells of the

mitochondria underwent PTP-dependent swelling and
cytochrome c release following treatment with platelet
activating factor. Here, swelling was inhibited by
pretreatment of the mitochondria with Ro5-4864 or
PK11195, as well as by treatment with CsA or the
platelet activating factor inhibitor, BN50730 [168].
Also, rat heart mitochondria underwent a decrease in
phosphorylation rate when treated with H
2
O
2
, but
were restored to their normal rate when treated with
Ro5-4864 [169].
While these apparently contradictory effects of
PBR ligands can be rationalized on the basis of dif-
ferences in cell type, additional confusion arises from
studies showing that individual PBR ligands can have
different effects on the same cell. For example, in
U937 cells, Ro5-4864 counteracted TNF-a-mediated
apoptosis, while PK11195, in a similar concentration
range, enhanced apoptosis. Indeed, the addition of
Ro5-4864 could overcome the effect of PK11195
[165]. This study also showed that Jurkat T cells,
which contain little or no PBR, became more sensi-
tive to cell death caused by TNF-a after they had
been transfected with a gene expressing the PBR pro-
tein. As would be predicted from the studies men-
tioned earlier, Ro5-4864 protected the transfected
cells from apoptosis [165].

being between 5 and
8nm [175–177]. Early indications that CyP-D is
involved in modulation of the PTP affinity for Ca
2+
(and conversely that Ca
2+
modulates the efficacy of
PTP inhibition by CsA) included the demonstration
that Ca
2+
displaced CsA from high-affinity binding
sites in rat liver mitochondria [176] and that higher
concentrations of CsA were required to inhibit spread-
ing of the PTP to a population of mitochondria when
the Ca
2+
load was increased [54].
The immunosuppressive effects of CsA are caused
by the Ca
2+
-calmodulin-dependent inhibition of cal-
cineurin (a cytosolic phosphatase) by the complex of
the drug with cytosolic CyP-A [178]. In turn, this pre-
vents dephosphorylation and nuclear translocation of
nuclear factor of activated T cells and other transcrip-
tion factors that are essential for the activation of T
cells [179]. Available evidence suggests that calcineurin
is not involved in the effects of CsA on the PTP
because CsA derivatives have been described that bind
CyP-D and desensitize the pore, but do not inhibit cal-

CyP-D is a regulator, but not a component, of the
PTP, whose structure is unlikely to be altered by the
absence of CyP-D. A further implication of these
results is that the effect of CsA is best described as
‘desensitization’ rather than inhibition, of the PTP,
because its effects (similarly to the lack of CyP-D) can
be overcome by a moderate increase of the mitochond-
rial Ca
2+
load [54]. We would like to stress that, at
variance from the conclusions of recent influential
reviews [183,184], the in vivo studies on Ppif
– ⁄ –
mice
can only be interpreted in terms of the role of CyP-D,
not of the PTP, in cell death. Indeed, all studies agree
that the PTP can form and open in the absence of
CyP-D, provided that a permissive Ca
2+
load is accu-
mulated [46–49]. Furthermore, these results cannot be
used to conclude that the PTP only plays a role in nec-
rotic, rather than apoptotic, responses [183,184]
because it should not be surprising that matrix CyP-D
does not play a role in cytochrome c release by tBID
and BAX added to isolated mitochondria [46], a proto-
col that directly permeabilizes the outer mitochondrial
membrane. Thus, the inference that PTP opening does
not take place because CyP-D is absent has not been
documented in vivo, an issue that questions the conclu-

preformed pore, but rather the result of oxidative dam-
age to membrane proteins [187]. In the model of He
& Lemasters, conductance through these misfolded
protein clusters would be normally blocked by chaper-
The mitochondrial permeability transition P. Bernardi et al.
2084 FEBS Journal 273 (2006) 2077–2099 ª 2006 The Authors Journal compilation ª 2006 FEBS
one-like proteins, including CyP-D, and it would be
modulated by Ca
2+
in a CsA-sensitive manner. When
protein clusters exceed chaperones available to block
conductance, opening of ‘unregulated’ pores would
occur, which would no longer be sensitive to CsA
[186]. While interesting, the model fails to account for
PTP regulation by the voltage and by matrix pH,
which is not easy to reconcile with a permeability
pathway created by a heterogeneous set of denatured
proteins. Furthermore, Ro 68-3400 inhibits the PTP in
the submicromolar range without affecting CyP-D
activity, and under conditions of full pore inhibition it
binds to a 32 kDa protein rather than to the large set
of proteins that would be reasonable to find based on
this model of the PTP [109]. Finally, it should be men-
tioned that Ca
2+
-dependent, CsA-insensitive PT-like
activities have been described that are formed or acti-
vated by fatty acids [188,189] or 3-hydroxybuty-
rate ⁄ polyphosphate [190].
Irrespective of the molecular nature of the pore, a

mation of the PTP and that intermembrane factors
could also play a role, possibilities that are omitted for
clarity). In the absence of outer membrane interac-
tions, the PTP could flicker between the closed state
(panel 2) and the open state (panel 3) under the effect
of inner membrane and matrix modulators such as the
Dw
m
, pH, CyP-D, Ca
2+
and PN. Stabilization of the
open conformation could lead to the rearrangement of
cristae structure and, eventually, to outer membrane
rupture (panel 4). This scheme is meant as an example
of how the outer membrane could confer regulatory
features to the PTP without necessarily providing a
permeability pathway for solute diffusion, but it
should by no means be taken literally. As a matter of
fact, and despite our detailed knowledge of PTP regu-
lation, with the exception of CsA it is currently
impossible to assign any pore effectors to a particular
site, a key issue that will have to await PTP identifica-
tion. We are developing new tools for the identification
of pore component(s) through screening of chemical
libraries, a program that is well underway and should
soon provide novel clues about PTP structure and
function.
The PT in pathophysiology
Occurrence of a PT has been amply documented in a
variety of cell culture models. The number of such

.
m.o
.m.i
.m.o
.m.i
.
m.o
.m.i
1 2 3 4
P
T
P nepO
Fig. 1. Model for permeability transition pore (PTP) regulation by
outer membrane proteins. Hypothetical model of inner membrane
(i.m.) PTP modulation by interaction with outer membrane (o.m.)
proteins, which could be the target of cytosolic effector molecules.
Broken lines denote outer membrane rupture following PTP open-
ings of long duration. For explanation see the text.
P. Bernardi et al. The mitochondrial permeability transition
FEBS Journal 273 (2006) 2077–2099 ª 2006 The Authors Journal compilation ª 2006 FEBS 2085
advances have been made in understanding the role of
CyP-D, and to some extent of the PTP, in organ and
in vivo models of disease. In this section we will focus
mostly on systems where an involvement of the PTP
has been investigated in vivo.
Myocardial ischemia-reperfusion
The relevance of mitochondrial dysfunction to the
onset of irreversible injury of the heart has prompted
numerous studies aimed at defining the involvement of
the PTP, especially in the setting of myocardial ische-

[Ca
2+
] and Pi provides an ideal scenario for promoting
PTP opening, which would be further favored by the
overproduction of ROS and the recovery of neutral
pH.
Initial supporting evidence that the PTP plays a role
in reperfusion injury was obtained in experiments on
isolated cardiomyocytes and perfused hearts, where
CsA administration reduced the occurrence of con-
tractile impairment and irreversible damage [203,204].
More direct evidence was subsequently obtained by
methods allowing the detection of PTP opening in
intact cells and tissues [71,112,205,206]. Occurrence of
PTP opening was assessed through the (re)distribution
of molecules that are not able to cross the inner mito-
chondrial membrane unless a PT occurs. While calcein
has been utilized to investigate the relationship
between PTP and apoptosis in intact cells [78], the
mitochondrial uptake of the otherwise impermeant
6-phosphodeoxyglucose provided an elegant demon-
stration that PTP opening occurs in isolated hearts
only upon postischemic reperfusion [71,82].
We recently assessed PTP opening in heart ischemia-
reperfusion through the redistribution of an endog-
enous ‘probe’, the pool of mitochondrial PN, which
does not readily permeate the inner membrane unless
a PT occurs [16]. We demonstrated that in isolated
hearts subjected to ischemia-reperfusion, PN are
released from the mitochondrial matrix into the inter-

We have recently demonstrated that N-methyl-
N¢-nitro-N-nitrosoguanidine, the reference compound
utilized for activating PARP, causes PTP opening and
cell death that are prevented by CsA [210]. PARP acti-
vation may certainly hasten the progression towards
cell death by rapidly consuming the NAD
+
released
by mitochondria, yet it is unlikely to be a primary
cause of mitochondrial dysfunction.
The results obtained with both the deoxyglucose
and NAD
+
distribution techniques demonstrate that
myocyte viability is maintained when PTP opening is
prevented [71,206]. This causal relationship between
PTP-dependent mitochondrial dysfunction and cell
death rules out the alternative possibility that PTP
opening may be a secondary consequence of the mas-
sive intracellular Ca
2+
overload that follows sarcolem-
ma rupture. The relevance of these concepts to a
clinical setting has been substantially strengthened by
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2086 FEBS Journal 273 (2006) 2077–2099 ª 2006 The Authors Journal compilation ª 2006 FEBS
the observation that PTP inhibitors limit the loss of
viability even when administered at the time of reper-
fusion [211,212]. Of note, CsA derivatives that lack
immunosuppressive activity were also effective, indica-

The hepatoprotective effects of CsA have been tested
in several in vivo models of disease (i.e. treatment of
rats with ethanol [220], with lipopolysaccharide of
Streptococcus after liver sensitization with heat-inacti-
vated Propionibacterium acnes [221] or d-galactosamine
[222,223], or of cats with lipopolysaccharide alone
[224–226]; treatment of mice with anti-Fas Ig [227],
diclofenac [228] or acetaminophen [229,230]; in proto-
cols of liver ischemia-reperfusion [231,232]; in one ani-
mal model of a-1 antitrypsin deficiency with liver
injury and mitochondrial autophagy [233]; and in one
cohort of cases of fulminant viral hepatitis [234]). In
the animal models, the dose of CsA ranged between 5
and 100 mgÆkg
)1
body weight, which conferred vari-
able degrees of protection; and the time of administra-
tion relative to the hepatotoxic treatment ranged from
pretreatment with a single dose to repeated administra-
tions of the same dose at different time intervals. The
variability of dose and timing reflects the basic uncer-
tainty of whether the PTP is actually inhibited after
the administration of CsA in vivo, and of whether
more than a single dose is necessary for the inhibitory
effect to persist. We systematically investigated this
problem in the rat, and found that the PTP is maxi-
mally inhibited in the liver between 2 and 9 h of intra-
peritoneal (i.p.) injection of 5 mg CsAÆkg
)1
body

which desensitizes the PTP and the liver mitochondrial
apoptotic pathway to TNF-a in vivo. The adaptive
response is of an epigenetic nature, and represents a
mitochondrial tumor-promoting event centered on the
PTP that may contribute to the selection of resistant
hepatocytes in the population of chemically trans-
formed cells [238].
An adaptive response of the PTP ex vivo has been
also demonstrated after bile duct ligation in rats [239].
Furthermore, it should be noted that hepatitis C virus
core protein localizes to mitochondria, where it inhibits
electron flow at Complex I, causing increased ROS
production and possibly increased PTP opening [240],
and that a similar sequence of events may take place
in liver chronic alcohol exposure [241]. Alcoholic liver
disease and chronic hepatitis C are leading causes of
hepatocarcinoma, and it appears worth considering
whether inhibition of liver apoptosis through PTP
P. Bernardi et al. The mitochondrial permeability transition
FEBS Journal 273 (2006) 2077–2099 ª 2006 The Authors Journal compilation ª 2006 FEBS 2087
adaptation also plays a role in the onset of liver cancer
in these high-prevalence conditions.
A promising application of CsA, based on PTP inhi-
bition, is liver preservation for organ transplants. It
has been shown that CsA provides partial protection
in liver cold preservation ⁄ warm reperfusion, suggesting
that the PTP may play an important role in organ
decay during storage, and that inhibition by CsA may
improve the function of the grafted liver after trans-
plantation [242]. However, it should also be considered

calcineurin) failed to protect mitochondria and neu-
rons of the dentate gyrus against hypoglycemic dam-
age [246] and (b) that in a rat model of transient focal
ischemia significant neuroprotection was afforded by
treatment with N-methyl–Val4–CsA [249], which inhib-
its the PTP, but not calcineurin [44].
Another interesting observation is that CsA pro-
longs the survival of mouse models of amyotrophic lat-
eral sclerosis [260,261], an effect that is also exerted by
minocycline through inhibition of the PTP [262]. Pro-
tective effects that could be traced to inhibition of the
PTP have also been reported for melatonin following
middle cerebral artery occlusion and reperfusion [263],
for promethazine in a mouse model of stroke [264],
and for topiramate in pilocarpine-induced epilepsy
[265]. Taken together, these promising results suggest
that the PTP is a viable pharmacological target in neu-
rological diseases.
Muscle diseases
The hypothesis that Ca
2+
-dependent mitochondrial
dysfunction could play a role in muscular dystrophies
was put forward 30 years ago [266]. An unexpected
and exciting development in this field has been the
demonstration that the PT plays a key role in the
pathogenesis of muscular dystrophy in a mouse model
of collagen VI deficiency [267]. Inherited mutations of
collagen VI genes cause two muscle diseases in
humans: Bethlem myopathy [268]; and Ullrich congen-

high-prevalence diseases. CsA proved essential for the
development of the field and for testing the role of
CyP-D (and to some extent of the PTP) in pathophysi-
ology in vivo. However, the demonstration that a PT
can occur in the absence of CyP-D must induce some
caution in interpreting experimental results in Ppif null
animals, in particular the conclusion that the PTP does
not play a role in apoptosis. Assessing this key point
The mitochondrial permeability transition P. Bernardi et al.
2088 FEBS Journal 273 (2006) 2077–2099 ª 2006 The Authors Journal compilation ª 2006 FEBS
will require the molecular definition of the PTP, a pro-
gram that is being actively pursued in collaboration
with Genextra S.p.A. (Milano, Italy). Screening of a
chemical library has allowed the identification of novel
high-affinity PTP inhibitors, which represent promising
tools towards the identification of the molecular com-
ponents of the PTP.
Acknowledgements
Research in our laboratories is supported by the Ital-
ian Ministry for the University, AIRC Grant 1293,
Telethon-Italy Grant GGP04113 and the National
Institutes of Health – Public Health Service (USA)
Grant GM69883.
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