Báo cáo khoa học: The mitochondrial permeability transition from in vitro artifact to disease target ppt

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

Ca
2+
, Pi and fatty acids, its inhibition by Mg
2+
, aden-
ine nucleotides and acidic pH, and its detrimental
effects on energy conservation, have been clearly
recognized since the early studies on isolated
mitochondria were carried out [3–13]. Ever since, the
Keywords
apoptosis; calcium; cancer; cell death;
degenerative diseases; drugs; mitochondria;
necrosis; permeability transition
Correspondence
P. Bernardi, Department of Biomedical
Sciences, University of Padova, Viale
Giuseppe Colombo 3, I-35121 Padova, Italy
Fax: +39 049 827 6361
E-mail:
*Present address
Center for Integrative Genomics, University
of Lausanne, 1015 Lausanne-Dorigny,
Switzerland
(Received 6 January 2006, revised 1 March
2006, accepted 3 March 2006)
doi:10.1111/j.1742-4658.2006.05213.x
The mitochondrial permeability transition pore is a high conductance chan-
nel whose opening leads to an increase of mitochondrial inner membrane
permeability to solutes with molecular masses up to  1500 Da. In this
review we trace the rise of the permeability transition pore from the status

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-

its the PT [14,15]. The presence of EGTA prevents
Ca
2+
accumulation during homogenization, but also
depletes the physiological pool of matrix Ca
2+
, which
is an essential permissive factor for PTP opening.
Finally, most in vitro studies have used succinate (in
the presence of rotenone) as the energy source. From
the bioenergetics point of view, this choice of substrate
tends to provide the most consistent results because
succinate oxidation does not require matrix PN, which
can be lost via the PT during storage or incubation
even when swelling is undetectable [16]. What was not
appreciated until recently is that rotenone makes PT
opening more difficult by blocking electron flux
through Complex I and by preventing the oxidation of
PN [17,18]. Having carefully selected conditions that
minimize its occurrence, it should not be too surprising
that the requirements to induce a PT in isolated mito-
chondria may appear ‘extreme’ in what obviously rep-
resents a circular argument. Yet, together with the
detrimental effects of the PT on energy conservation,
this state of affairs has significantly contributed to the
widespread feeling that the PT was an in vitro artifact
of little pathophysiological relevance.
A notable exception was the proposal by Pfeiffer
and coworkers that the ‘damaging’ effects of Ca
2+

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

the same laboratories involved in clarifying the mecha-
nisms of energy conservation; and they tended to
become tests of the predictions of the chemiosmotic
theory, in particular about the existence of a membrane
potential across the inner membrane. Given the view,
prevailing well into the 1980s, that mitochondria did
not possess cation channels [27] it is not too surprising
that the existence of a large pore in the inner membrane
appeared to contradict the basic tenets of chemiosmo-
sis. As a result, and with very few exceptions [28–37],
research in this area did not enjoy much popularity
until a set of major findings began to attract a larger
number of investigators.
A turning point was the discovery that the PT could
be inhibited by submicromolar concentrations of the
immunosuppressant drug, cyclosporin A (CsA) [38–
41]. It was later shown that CsA inhibits the PTP after
binding to matrix cyclophilin (CyP)-D, a peptidyl-
prolyl cis-trans isomerase whose enzymatic activity is
blocked by CsA in the same range of concentrations
required to inhibit the pore [42–45]. The recent charac-
terization of mitochondria from mice with genetic
inactivation of the Ppif gene encoding CyP-D has
allowed a better understanding of the role of CyP-D in
PTP regulation [46–49], and will be discussed in some
detail later in the review.
A second major finding was made possible by the
demonstration that mitochondria possess ion channels
that can be studied by electrophysiology using the
patch-clamp technique [50]. This seminal study was

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

[68] or treatment with ATP [69], and in cardiomyo-
cytes [70] and isolated hearts [71] exposed to ischemia
followed by reperfusion. The recent surge of interest in
the PT as an effector mechanism of cell death, how-
ever, only followed the demonstration that in the
course of apoptosis, cytochrome c is released into the
cytosol [72], together with apoptosis-inducing factor
[73] and a set of proteins involved in the effector phase
of apoptosis [74–77].
A rigorous test of whether a PT takes place in intact
cells, organs and living organisms remains a major
challenge. This is caused by the intrinsic complexity of
PTP modulation [1,2]; by the fact that occurrence of a
PT must still be deduced by indirect means, which in
turn generates major interpretative problems of the
experimental results [78]; by the lack of both selectivity
and persistence of action of CsA [79], which may yield
‘negative’ results even in conditions where the PTP is
actually involved; and by the lack of a defined struc-
ture for the channel itself, as should become clear from
the discussion of the most studied candidate proteins
[i.e. the adenine nucleotide translocator (ANT), the
voltage-dependent anion channel (VDAC) and the
peripheral benzodiazepine receptor (PBR)]. We have
already pointed out the many sources of artifacts that
have hampered research in this field [78], and we have
identified outstanding mechanistic problems on the
role of mitochondria in cell death that involve the PTP
[80]. Here we will provide an account of the status of
PTP research in the light of recent achievements based

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

2+
ions, such as Mg
2+
,Sr
2+
and Mn
2+
, and by Pi
through a still-undefined mechanism. Pore opening is
strongly promoted by an oxidized state of PN and of
critical dithiols at discrete sites [97,98], and both effects
can be individually reversed by proper reductants. The
dithiol–disulfide interconversions correlate with the
redox state of glutathione and can be blocked by
1-chloro-2,4-dinitrobenzene, suggesting that the dithiol
is in redox equilibrium with matrix glutathione. This
finding accounts easily for the PT-inducing effects of
both peroxides and redox-cycling agents, and for the
corresponding inhibition with monofunctional thiol
reagents, such as N-ethylmaleimide and monobromobi-
mane [1]. PTP modulation by these redox-sensitive
sites easily accommodates the inducing effects of
p66Shc, which directly oxidizes cytochrome c to pro-
duce superoxide anion and causes PTP-dependent cell
death [99,100].
The PT is strictly modulated by matrix pH. In
de-energized mitochondria, the pH optimum for open-
ing is 7.4, while the open probability decreases sharply
both below pH 7.4 (through reversible protonation of
critical histidyl residues that can be blocked by diethyl-

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

potential into changes of the PTP open probability
[60]. Such a sensor would easily account for pore
opening following depolarization as such, and for the
effects of a large variety of membrane-perturbing
agents that can either inhibit or promote the PT. In
general, amphipathic anions, such as fatty acids pro-
duced by phospholipase A
2
, favor the PT with an
effect that cannot be explained by depolarization. In
particular, arachidonic acid appears to play a key role
in apoptotic Ca
2+
-dependent apoptotic signalling
through the PTP [104,105]. Conversely, polycations
such as spermine, amphipathic cations such as sphing-
osine and trifluoroperazine, and positively charged
peptides, inhibit pore opening [64]. The putative volt-
age sensor may comprise critical arginine residues, as
suggested by modulation of the PTP voltage depend-
ence by a set of arginine-selective reagents [106,107].
The PTP is regulated by electron flux within respir-
atory chain complex I, with an increased open probab-
ility when flux increases [17]. This finding led to the
discovery that the PT is regulated by quinones, poss-
ibly through a specific binding site, whose occupancy
affects the open–closed transitions depending on the
bound species [108]. Binding of ubiquinone 0 (Ub0) or
decylubiquinone prevents Ca
2+

-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.

With Complex I-linked substrates, PTP opening may
be followed by respiratory inhibition owing to a loss
of matrix PN [16]. With Complex II-linked substrates,
the PT is rather followed by uncoupling. The conse-
quences of a PT on respiration in vivo, and the related
issue of production of reactive oxygen species (ROS)
therefore depend on whether, and to what extent, PN
are lost. Irrespective of whether respiration is inhibited
or stimulated, collapse of the Dp will prevent ATP syn-
thesis as long as the pore is open. ATP hydrolysis by
the mitochondrial ATPase would then worsen ATP
depletion, which, together with altered Ca
2+
homeo-
stasis, is a key factor in various paradigms of cell
death [80].
Persistent PTP opening is followed by equilibration
of ionic gradients and of species that have a molecular
mass of < 1500 Da, which may cause swelling, cristae
unfolding and outer membrane rupture. The occur-
rence of swelling can be prevented by pore-impermeant
solutes, and for short open times solute equilibration
may not occur at all. Thus, assessing whether swelling,
outer membrane rupture, and release of cytochrome c
and other proapoptotic factors follow a PT in situ
needs to be verified in each experimental setting.
An interesting mechanism that links PTP opening to
release of cytochrome c in the absence of outer mem-
brane rupture has recently emerged. Cytochrome c is
compartmentalized within mitochondria in two pools

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
The mitochondrial permeability transition P. Bernardi et al.
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+

-release chan-
nel [117,118], and we refer the reader to previous
reviews that specifically discuss this possibility in some
detail [1,117].
Role of the ANT
The PTP is strikingly modulated by ligands of the
ANT. Atractylate, which inhibits the ANT and stabil-
izes it in the ‘c’ conformation [119], favors PTP open-
ing, while bongkrekate, which also inhibits the ANT
but stabilizes it in the ‘m’ conformation [119], favors
PTP closure. These findings led to the suggestion that
the PTP may be directly formed by the ANT [120]. We
have proposed an alternative explanation that is based
on PTP modulation by the changes of the surface
potential. Indeed, the transition of the translocase
from the ‘m’ to the ‘c’ conformation is accompanied
by a large decrease of the surface potential [121,122],
an observation that may easily explain pore opening
by atractylate and pore closure by bongkrekate within
the framework of PTP modulation by the membrane
potential [1].
The ANT reconstituted in giant liposomes exhibits
high-conductance (but CsA-insensitive) channel activ-
ity that is stimulated by Ca
2+
[123]. The channel dis-
plays a marked voltage dependence with prominent
gating effects that are consistent with the reported
voltage dependence of the pore in intact mitochondria
[123]. In addition, after reconstitution in liposomes,

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

2+
-dependent PT took place [128].
Of note, the PT of ANT null mitochondria was fully
inhibitable by CsA and could be triggered by H
2
O
2
and diamide, indicating that the ANT is neither the
obligatory binding partner of CyP-D nor the site of
action of oxidants [128]. In addition, hepatocytes pre-
pared from control and ANT-deficient livers showed
identical responses to activation of receptor-mediated
apoptotic pathways initiated by tumor necrosis
factor-a (TNF-a) and Fas [128].
It has been argued that a low, undetectable level of
ANT expression could have been present, producing
the PTP observed in ANT-null mitochondria [129]. As
the PTP was insensitive to opening by atractylate and
to closure by ADP, it is very difficult to envisage how
any ANT molecule in mutant mitochondria would not
respond to atractylate and ADP, and yet be able to
promote a CsA-sensitive PT. Another relevant obser-
vation is that mitochondria from the anoxia-tolerant
brine shrimp, Artemia franciscana, do not undergo a
PT, despite a remarkable Ca
2+
-uptake capacity and
the presence of ANT, VDAC and CyP-D [130].
Role of the VDAC
Early studies demonstrated that the PT induced by sul-

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

contain a variety of additional proteins, including
VDAC, the ANT and CyP-D. When reconstituted into
planar lipid bilayers or liposomes, these fractions gave
rise to channels with properties resembling those of the
PTP [140,141]. While these studies establish that the
PTP activity can be reconstituted after detergent
extraction of mitochondria, whether VDAC is essential
in this activity is far less clear (e.g. Fig. 1 in ref. 142).
Indeed, further purification of such extracts by gel fil-
tration chromatography resulted in fractions contain-
ing hexokinase in which the presence of VDAC (and
the ANT) could not be demonstrated, yet remained
capable of forming PTP-like pores on incorporation
into liposomes [140]. Thus, it appears legitimate to
wonder what component(s) of these extracts are, in
fact, responsible for the formation of CsA-inhibitable
pores, an argument that applies to all attempts to
identify the PTP components based on purification of
hexokinase activity [140–142]. More recent biochemical
attempts to place VDAC in the PTP complex have
been based on the characterization of proteins that
co-immunoprecipitate with the ANT, constituting the
‘adenine nucleotide translocase interactome’ [143].
While VDAC, as well as a wide variety of cytosolic,
endoplasmic reticulum, inner membrane and matrix
proteins are present in these immunoprecipitates, their
direct relation to the PTP has yet to be established.
Unfortunately, much of the data used to support the
presence of VDAC in the PTP is based on a tenuous
logical generalization – properties of VDAC in bilayers

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],

while the unconditional elimination of VDAC2 results
in embryonic lethality [151]. Thus, the involvement of
VDAC in PTP function has been difficult to assess in
these animals because it is likely that any VDAC iso-
form may potentially compensate for the absence of
any other isoform, given that all cells appear to
express each isoform.
The idea that VDAC is a component of the PTP has
been considerably reinforced by Cesura et al. who used
a functional assay based on swelling of isolated
mitochondria to screen a chemical library for inhibi-
tors of the PTP [109]. A high-affinity inhibitor was
identified (Ro 68–3400), which was then used to label
mitochondria and identify a protein, of  32 kDa, as
VDAC1 [109]. More recently, we have found that the
PTP in mitochondria prepared from VDAC1-null mice
is fully sensitive to inhibition by Ro 68–3400, and that
the inhibitor labels a 32 kDa protein that is indistin-
guishable from the species labeled in wild-type mito-
chondria. Of note, we have been able to separate the
labeled 32 kDa protein from all VDAC isoforms in
both VDAC1-null and wild-type mitochondria, unam-
biguously proving that the latter are not the targets for
inhibition by Ro 68–3400 (A. Krauskopf et al., unpub-
lished results).
In the end, the involvement of VDAC in the PT
remains reasonable, but convincing data directly impli-
cating this protein in PTP formation in an in vivo, phy-
siological context are absent. Given the redundancy of
VDAC genes in mammals, the application of more

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

testis [157,158]. In these cells, PBR promotes the trans-
port of cholesterol into the mitochondrial matrix, a
rate-limiting step in steroid synthesis [159]. The PBR
also binds some porphyrins, including protoporphyrin
IX, a potent inducer of the PTP [160], and is thought
to be involved in transport of porphyrins into the
mitochondrion [156,161].
Involvement of the PBR in PTP function was ini-
tially suggested following biochemical isolation of the
PBR, which indicated a close association of this pro-
tein with VDAC and the ANT [162]. However, expres-
sion of the 18 kDa protein alone in bacterial cells
demonstrated that binding of high-affinity ligands to
the PBR does not require either of these proteins [163].
Additional evidence that the PBR plays a role in PTP
function was obtained following patch-clamp analysis
of mitoplasts. Treatment with PBR ligands affected
the channel activity of the MMC (i.e. the PTP) [164],
but it is difficult to see how an inner membrane chan-
nel could be affected by the outer membrane PBR
receptor. Indeed, it appears unlikely that the patched
membrane always contains outer membrane fragments.
Experiments directly aimed at testing the activity of
PBR ligands on PTP activity in isolated mitochondria
have demonstrated that the effect of these drugs in
promoting the opening of the PTP, and of apoptosis,
depends on the cell type examined and the concentra-
tions tested [132,155,160,165–167]. Other studies have
shown that PBR ligands inhibit, rather than promote,
the PTP and apoptosis. For example, rat forebrain

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].

While PBR-specific drugs have also been used to
examine the role of the PBR in in vivo disease models
[169–171], extension of these results, as well as many
of those outlined above in the context of cultured cells
and mitochondria, to PTP function depends, to some
degree, on the ability of these drugs to specifically tar-
get the PBR. Thus, recent results suggesting that PBR
ligands can generate cellular phenotypes, independently
of the PBR, have further muddled the physiological
role of the PBR and its involvement in PTP activity
[172–174]. Clearly, the examination of mitochondria
prepared from animals in which the expression of PBR
had been eliminated by ‘knockout’ strategies would
greatly help to clarify these issues. Unfortunately, ini-
tial attempts to generate such animals has indicated
that nonconditional elimination of PBR expression
results in embryonic lethality [159]. Thus, although it
cannot be ruled out that the PBR is part of the PTP,
conclusions based on the exclusive use of PBR ligands
should, at this point, be viewed and interpreted with
some caution until genetic tools are generated that will
allow these questions to be addressed directly.
P. Bernardi et al. The mitochondrial permeability transition
FEBS Journal 273 (2006) 2077–2099 ª 2006 The Authors Journal compilation ª 2006 FEBS 2083
Role of CyP-D
Opening of the PTP is inhibited by CsA after binding
to CyP-D, a matrix peptidyl-prolyl cis-trans isomerase
[175]. The relevant binding sites for CsA display a very
high affinity, the estimated K
d

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-

cineurin [44,180,181]. On the other hand, calcineurin
may affect mitochondrial function through the de-
phosphorylation of BAD and the release of apopto-
genic proteins [182].
The most conclusive results on the role of CyP-D in
regulation of the PTP were obtained after inactivation
of the Ppif gene, which encodes CyP-D in the mouse
[46–49]. In three studies, the ablation of CyP-D
approximately doubled the Ca
2+
-retention capacity
(CRC) (i.e. the threshold Ca
2+
load required to open
the PTP), which became identical to that of CsA-trea-
ted, strain-matched wild-type mitochondria, while no
effect of CsA was observed in Ppif
– ⁄ –
mitochondria
[46,47,49]. Nakagawa et al. reported a much higher
increase of the CRC after ablation of CyP-D, which
matched an unusually high CRC after treatment of
control mitochondria with CsA [48]. The basis for this
discrepancy is not clear, but it may depend on the F2
mosaic mice used in this study. Indeed, it has been
noted that a mixed genetic background can probably
also account for many of the discrepancies already des-
cribed for cytochrome c, APAF-1 and caspase 9 null
mice [183].
Taken together, these findings demonstrate that

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-

sion that the PTP participates in cell death pathways
only in response to a restricted set of challenges.
Ppif
– ⁄ –
pups were born at the expected Mendelian
ratio, and were otherwise indistiguishable from wild-
type animals, suggesting that CyP-D is dispensible for
embryonic development and viability of adult mice.
The lack of an overt phenotype could be a result of
adaptive responses bypassing the decreased sensitivity
of the PTP to Ca
2+
(like the increased response to oxi-
dative stress [47], or to effectors that are not detectable
in isolated mitochondria). Furthermore, CyP-D
overexpression desensitizes cells from apoptotic stimuli,
indicating that CyP-D may play an additional role as
a survival-signaling molecule acting on target(s) other
than the PTP [185]. This dual function could lead to a
balance of the pro-apoptotic and anti-apoptic effects
of the protein in animals lacking CyP-D.
Structure of the PTP
As should be clear from the above analysis, to date,
none of the candidate pore components stood rigorous
genetic testing. He & Lemasters have proposed a
model of PTP formation and gating in which the pore
forms by aggregation of misfolded integral membrane
proteins damaged by oxidant and other stresses [186],
which is reminescent of an earlier model suggesting
that the PT is not a consequence of the opening of a

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

comment is in order on the popular ideas that the pore
(a) forms at ‘contact sites’ between the inner and outer
mitochondrial membranes and (b) that it spans both
membranes [191]. As discussed in the paragraphs on
the ANT and VDAC, the idea that the PTP forms at
contact sites is based on a set of assumptions rather
than on established facts, and should be considered
with great caution also because the very existence of
points of fusion between the outer and inner mem-
branes has been questioned by tomography of unfixed
mitochondria [192]. The second point does not take
into account that the permeability pathway resulting
from a pore spanning both membranes would directly
connect the matrix with the cytosol, resulting in the
release of matrix solutes, but not of cytochrome c and
of other intermembrane pro-apoptotic proteins.
We would like to stress that the PT is primarily an
inner membrane event that may cause secondary outer
membrane changes, and that in an in vivo setting, the
outer membrane can affect the probability of pore
opening through protein–protein interactions, as exem-
plified in the scheme of Fig. 1. Interaction of an outer
membrane protein (e.g. VDAC or the PBR) with the
PTP might depend on a specific conformation, which
could be conferred by cytosolic regulator(s) after modi-
fication by upstream signaling pathway(s). Binding
would be followed by a conformational change that
allows interaction with the PTP and its stabilization in
the closed conformation (panel 1; note that the ligand-
dependent change could instead favor the open confor-

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

studies is so large that we must refer the reader to
reviews covering both general and specific aspects of
the problem [81–95]. We have already discussed the
major sources of artifacts, particularly those arising
from the use of fluorescent potentiometric probes [78].
Largely through the use of CsA, however, and more
recently through the study of Ppif
– ⁄ –
mice, key
sro
t
aluger cil
o
sotyC
)
s(
rotpada enarb
mem
re
t
u
O
P
TP
deso
l
C
.m.
o
.m.i

.
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-

mia-reperfusion. As already mentioned, the role of
mitochondria in cell death extends beyond the shortage
in ATP supply and involves the generation of ROS,
the impairment of ion homeostasis, the accumulation
and release of potentially harmful metabolites, and the
release of pro-apoptotic proteins. These derangements
are prominent in the heart where, paradoxically, mit-
ochondrial function is also required for the sudden
onset of cell death that occurs when coronary flow is
re-established after a prolonged ischemic episode (i.e.
postischemic reperfusion) [82,192,193]. Under these
conditions, the partial recovery of mitochondrial func-
tion generates an amount of ATP that is sufficient for
contraction, but not for relaxation, resulting in hyper-
contracture and sarcolemma rupture [4–6]. This dra-
matic sequence of events is prevented by respiratory
chain inhibitors or mitochondrial uncouplers [194,195],
as well as by inhibition of ATP utilization by myosin
ATPase [196]. Obviously, these findings are not
directly amenable to clinical application, yet they indi-
cate that the ischemic heart could be protected by pre-
venting mitochondrial dysfunction.
As first proposed at the end of the 1980s [197,198],
and now documented by numerous reports [199,200],
the PTP represents an ideal target for cardioprotection.
While ischemia per se does not appear to cause PTP
opening, probably because of the protective effects of
intracellular acidosis [201,202], it creates the conditions
for PTP opening at reperfusion. Indeed, recovery of
respiration in the presence of increased intracellular

[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-

membrane and the cytosolic spaces, where they
become the substrate of a wide array of NAD
+
util-
izing enzymes [206]. The release of NAD
+
is not only
a tool for PTP detection. Formation of cyclic nucleo-
tides (such as cADP ribose) from NAD
+
may further
increase the probability of PTP opening through the
release of Ca
2+
from sarcoplasmic reticulum, and the
released NAD
+
can be utilized by poly(ADP-ribose)
polymerase (PARP) for DNA repair [207].
It has been proposed that uncontrolled PARP acti-
vation might deplete intracellular NAD
+
and ATP,
resulting in mitochondrial depolarization and eventu-
ally cell death [208,209]. As the major fraction of PN
is compartmentalized within the mitochondrial matrix,
we think that pore opening must precede rather than
follow PARP activation. Consistent with our view,
compounds and procedures that cause DNA damage
and PARP activation are also powerful PTP agonists.

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
The mitochondrial permeability transition P. Bernardi et al.
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-

ting that calcineurin is not involved in cardioprotection
[213,214]. Definite support for a role of the PTP
in myocardial pathophysiology is provided by the
reduced susceptibility to ischemic injury observed in
mice lacking CyP-D [46,48] (see also the paragraph on
the role of CyP-D).
PTP inhibition may also be involved in ischemic pre-
conditioning (i.e. the ability to confer protection
against a prolonged and otherwise lethal ischemia by
repeated, short ischemic episodes; [215] reviewed in ref.
[200]; and in ischemic postconditioning (i.e. the ability
to confer protection by a brief ischemic episode
applied at the onset of reperfusion [211]). Both issues
are controversial [92,216], and represent good examples
of the complexity of the problem and of the limits of
current approaches largely based on the effects of
CsA.
Recent efforts have been devoted to identifying the
signaling pathways linking myocardial protection to
PTP inhibition, which may involve protein kinase Ce
[217] and mitogen-activated protein kinase kinase 6
[218]. An integrated model has recently been proposed
where various protective pathways would impinge on
glycogen synthase kinase-3b causing its inhibition,
which would, in turn, result in a decreased probability
of PTP opening [219]. Further studies will be necessary
to identify the relevant signalling molecules and to
clarify how cytosolic kinases can eventually modify the
activity of an inner membrane channel.
Liver diseases

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

weight. The inhibitory effect returned to the basal level
within 24 h [223]. By using proper times of treatment
we achieved full protection from the otherwise lethal
effects of lipopolysaccharide plus d-galactosamine,
which act through liver sensitization to the proapop-
totic effects of TNF-a [223]. It is worth mentioning
that encouraging results were already obtained in 13
patients with fulminant viral hepatitis by treatment
with an initial dose of 3 mg CsAÆkg
)1
by continuous
drip infusion for 2 days, followed by a maintenance
dose of 1 mgÆkg
)1
[234].
The PTP is also involved in a rodent model of
hepatocarcinogenesis. Feeding rats with the arylamine
2-acetylaminofluorene (AAF) causes onset of liver
tumors within 30–50 weeks [235]. Onset of tumors is
preceded by a sequence of alterations that closely
resembles the clinical course of chronic hepatitis, with
hepatocyte damage and fibrosis gradually evolving into
cirrhosis [235]. Hepatic metabolism of AAF generates
a large variety of compounds that are responsible for
the complex toxic and mutagenic effects of AAF.
These include 2-nitrosofluorene, a redox cycling com-
pound that drains electrons from the respiratory chain
[236] and causes opening of the PTP in isolated mito-
chondria [237]. Our studies have shown that very early
into AAF feeding an adaptative response takes place,

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

that the anti-apoptotic properties of CsA may contrib-
ute to the long-term appearance of cancers [243]. As
the immunosuppressant, FK506, does not affect the
PTP [177], CsA may be the drug of choice for organ
storage only.
Neurological diseases
Many neurological disorders are caused or worsened
by mitochondrial dysfunction that is not necessarily a
result of PTP opening, and coverage of this large area
is beyond the scope of this review. On the other hand,
a convincing set of results linking the PTP to neuropa-
thology has been obtained in experimental models of
neuronal injury. Based on the protective effects of
CsA, the PTP has been implied in brain damage as a
result of hyperglycemia [244,245], hypoglycemia
[246,247], ischemia [248–251], trauma [252–256] and
injection of 3-nitropropionic acid [257], in cell death
following facial motoneuron axotomy in neonatal
rodents [258] and in photoreceptor apoptosis [259].
Furthermore, mice with genetic inactivation of the
Ppif
– ⁄ –
gene encoding CyP-D displayed a striking
decrease of the emispheric damage that follows middle
cerebral artery occlusion [49]. These latter experiments
are also important because they firmly establish that
the neuroprotective effects cannot be caused by the
inhibition of calcineurin. Lack of involvement of cal-
cineurin had already been suggested by the findings (a)
that the immunosuppressant FK506 (which inhibits

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-

ital muscular dystrophy [269]. Collagen VI-deficient
(Col6a1
– ⁄ –
) mice display a muscle phenotype strongly
resembling Bethlem myopathy, with loss of contractile
strength associated with major ultrastructural altera-
tions of sarcoplasmic reticulum and mitochondria, and
spontaneous apoptosis [270]. These defects are caused
by inappropriate PTP opening, and could be normal-
ized by treatment with CsA, which rescued the muscle
ultrastructural defects and dramatically decreased the
number of apoptotic nuclei in vivo in what represents
the first successful pharmacological treatment of an
animal model of a genetic muscle disease [270]. We
have recently found that the same defect can be dem-
onstrated in myoblast cultures from patients with
Ullrich congenital muscular dystrophy (A. Angelin
et al., unpublished results), a finding that has paved
the way to a pilot clinical trial. Taken together, these
findings suggest that mitochondrial dysfunction could
be a component, if not a determinant cause, in other
muscle diseases with altered Ca
2+
homeostasis, such as
Duchenne muscular dystrophy.
Conclusions
Although the molecular nature of the PTP remains
unsolved, we conclude that the PT is an effector
mechanism that can explain the involvement of mito-
chondria in many pathological conditions and

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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