Developmental Biology 426 (2017) 1–7

Contents lists available at ScienceDirect

Developmental Biology
journal homepage: www.elsevier.com/locate/developmentalbiology

Development or disease: duality of the mitochondrial permeability MARK

transition pore
⁎
María José Péreza, Rodrigo A. Quintanillaa,b,
a
Laboratory of Neurodegenerative Diseases, Universidad Autónoma de Chile, Santiago, Chile
b
Centro de Investigación y Estudio del Consumo de Alcohol en Adolescentes (CIAA), Santiago, Chile

A R T I C L E I N F O A BS T RAC T

Keywords: Mitochondria is not only a dynamic organelle that produces ATP, but is also an important contributor to cell
Mitochondria functions in both development and cell death processes. These paradoxical functions of mitochondria are
Mitochondrial permeability transition pore partially regulated by the mitochondrial permeability transition pore (mPTP), a high-conductance channel that
Cardiac development can induce loss of mitochondrial membrane potential, impairment of cellular calcium homeostasis, oxidative
Neuronal development
stress, and a decrease in ATP production upon pathological activation. Interestingly, despite their different
Oxidative stress
etiologies, several neurodegenerative diseases and heart ischemic injuries share mitochondrial dysfunction as a
Calcium
common element. Generally, mitochondrial impairment is triggered by calcium deregulation that could lead to
mPTP opening and cell death. Several studies have shown that opening of the mPTP not only induces
mitochondrial damage and cell death, but is also a physiological mechanism involved in different cellular
functions. The mPTP participates in regular calcium-release mechanisms that are required for proper metabolic
regulation; it is hypothesized that the transient opening of this structure could be the principal mediator of
cardiac and brain development. The mPTP also plays a role in protecting against different brain and cardiac
disorders in the elderly population. Therefore, the aim of this work was to discuss different studies that show
this controversial characteristic of the mPTP; although mPTP is normally associated with several pathological
events, new critical findings suggest its importance in mitochondrial function and cell development.

1. Introduction
The mitochondria is a highly dynamic organelle, commonly nick-
named “the powerhouse of the cell” for its ability to produce cellular
energy in a highly efficient manner (Mattson et al., 2008). The main
function of the mitochondria is to convert the energy derived from
nutrients into heat and ATP, but it is also a major contributor to cell
fate determination, as this organelle is able to control both apoptotic
and necrotic cell death (Folmes et al., 2012; Nunnari and Suomalainen,
2012). These diametrically opposed functions of mitochondria are
particularly relevant in the heart and brain, where mitochondria supply
over 90% of the energy demands (Wallace, 2005). In fact, several
studies have demonstrated that, in these specific organs, metabolic
control through mitochondria is not only related to cell fate (Folmes
et al., 2012), but also plays an important role in cell differentiation
(Kasahara and Scorrano, 2014).
Interestingly, it has been shown that all these mitochondrial
functions are at some point related to the opening or activation of
the mitochondrial permeability transition pore (mPTP) (Bernardi et al.,
1999; Hou et al., 2013; Kasahara and Scorrano, 2014; Kwong and
Molkentin, 2015). The mPTP is a high-conductance channel that, upon
opening, generates a sudden increase in inner mitochondrial mem-
brane (IMM) permeability to ions and small solutes (Haworth and
Hunter, 1979; Hunter and Haworth, 1979a, 1979b). These changes
also affect regulation of the mitochondrial membrane potential
(Galluzzi et al., 2009), calcium homeostasis (Elrod et al., 2010),
reactive oxygen species (ROS) production (Hou et al., 2014), ATP
production (Budd and Nicholls, 1996), and cell death (Galluzzi et al.,
2009). Recent advances have determined that the mPTP could also be
the principal mediator of the duality of cell development and cell death.
The mPTP plays a role in cardiac and neurodegenerative pathologies
(Hou et al., 2013; Kwong et al., 2015), but is also involved in heart and
brain development (Hom et al., 2011; Porter et al., 2011; Mattson
et al., 2008), highlighting the importance of the mPTP in health and
disease.

⁎
Corresponding author at: Laboratory of Neurodegenerative Diseases, Universidad Autónoma de Chile, El Llano Subercaseaux 2801, 5to Piso, San Miguel 8910000, Santiago, Chile.
E-mail address: rodrigo.quintanilla@uautonoma.cl (R.A. Quintanilla).

http://dx.doi.org/10.1016/j.ydbio.2017.04.018
Received 7 February 2017; Received in revised form 26 April 2017; Accepted 26 April 2017
Available online 28 April 2017
0012-1606/ © 2017 Elsevier Inc. All rights reserved.
M.J. Pérez, R.A. Quintanilla
2. The mitochondrial permeability transition pore
The mPTP is a non-specific mega-channel that is sensitive to
perturbations in intracellular calcium homeostasis, permitting the
passage of molecules smaller than 1.5 kDa in size (Elrod and
Molkentin, 2013). The opening of the pore results in an increase in
the permeability of the IMM and permits entry of metabolites into the
mitochondrial matrix space, which leads to a reduction in the efficiency
of ATP production by uncoupling the electron transport system from
ATP synthase activity (Elrod and Molkentin, 2013; Mnatsakanyan
et al., 2016). Recent studies suggest that the mPTP is one of the
principal mechanisms that leads to cell death, participating as a
metabolic regulator of cell energy homeostasis, a mitochondrial
calcium transporter, and a superoxide (SO) efflux channel (Izzo et al.,
2016).
The original mPTP model proposed that the channel was formed by
three principal proteins: cyclophilin D (CyPD), located in the mito-
chondrial matrix; the adenine nucleotide translocator (ANT), found in
the inner membrane; the voltage-dependent anion channel (VDAC) in
the outer membrane (Rao et al., 2014); and other interacting mito-
chondrial molecules such as the phosphate carrier, BH3 proteins, and
p53 (Elrod and Molkentin, 2013). However, genetic knock-out studies
showed this model to be invalid, as they demonstrated that only the
deletion of the CyPD gene has a regulatory role in the activation of
mPTP (Baines et al., 2005; Nakagawa et al., 2006). In fact, studies in
mouse models lacking ANT expression showed a functional state of
mPTP that held two-fold more calcium than wild-type mitochondria;
this suggests that ANT has an important regulatory role in the function
of the mPTP, but not in its structure (Kokoszka et al., 2004). On the
other hand, the absence of one or more VDAC isoforms still allowed
opening of the mPTP (Baines et al., 2007), indicating that CyPD is the
major factor responsible for the activation of the mPTP (Elrod and
Molkentin, 2013; Izzo et al., 2016).
Interestingly, in recent years, it has been proposed that ATP
synthase is a major component of the mPTP (Jonas et al., 2015).
This enzyme is a highly conserved molecular machine that catalyzes the
production of ATP from ADP and Pi, powered by an electrochemical
transmembrane gradient in mitochondria (Pogoryelov et al., 2009).
Furthermore, ATP synthase is located in the inner membrane cristae,
made up of a membrane component—known as Fo—composed of nine
polypeptides (a, b, c, d, e, f, g, F6, and F8) and a soluble catalytic core
complex—known as F1—consisting of five different subunits (α, β, γ, δ/
OSCP, and ε) (Pogoryelov et al., 2009). The F1 subcomplex forms a
catalytic head group and a rotating central stalk, and the Fo sub-
complex comprises the membrane region and the peripheral stalk; the
oligomycin sensitivity-conferrin protein (δ/OSCP) subunit connects the
peripheral stalk to the catalytic domain (Pogoryelov et al., 2009).
Regarding ATP synthase and its role in mPTP regulation, important
studies have described that CyPD associates with ATP synthase
specifically through the δ/OSCP subunit; this interaction is inhibited
by cyclosporine A (CsA), a fungus-derived drug that reduces the
probability of mPTP opening through the inhibition of CyPD (Giorgio
et al., 2009, 2013). In fact, this inhibitory drug increased the activity of
both ATP synthesis and hydrolysis through displacement of CyPD from
ATP synthase, suggesting that the interaction of CyPD with ATP
synthase may switch the enzyme to a lower catalytic activity state
(Giorgio et al., 2009). On the other hand, it was suggested that the δ/
OSCP subunit also influences the accessibility of mPTP calcium-
binding sites. It seems that δ/OSCP affects the affinity of the metal
binding sites of ATP synthase, and thus, the ability with which calcium
can interact with the enzyme. Interestingly, when calcium is bound to
the enzyme, ATP synthase activity is not coupled to proton transloca-
tion, suggesting that calcium induces conformational changes in ATP
synthase, which could open the mPTP and stop ATP synthesis (Giorgio
et al., 2013).
With respect to the mechanism for channel formation, there are two
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Developmental Biology 426 (2017) 1–7
different working proposals about the structure of the pore itself.
However, both models suggest that ATP synthase forms part of the
functional pore of the mPTP (Alavian et al., 2014; Giorgio et al., 2013).
One hypothesis indicates that the mPTP channel forms at the interface
of two monomers (associated into dimers) of ATP synthase (Giorgio
et al., 2013). Purified dimers of the enzyme were reconstituted into
lipid bilayers and stimulated with regulators of mPTP; it was shown
that dimers of ATP synthase were capable of forming a current that was
electrophysiologically equivalent to that in the mPTP (Giorgio et al.,
2013). Since a current is only seen in dimers, and the genetic ablation
of the subunits that form the dimers did not result in opening of the
mPTP (Carraro et al., 2014), it was suggested that the pore structure of
the mPTP forms at the membrane interface between two adjacent Fo
sectors (Giorgio et al., 2013).
Another proposed model involves the c-subunit ring of the F1
subcomplex (Alavian et al., 2014). Both a- and c-subunits form the
proton channel of ATP synthase, permitting proton flux between the
intermembrane space and the mitochondrial matrix (Pogoryelov et al.,
2009). Interestingly, gene-silencing of isomers of the c-subunit in-
hibited mPTP opening, and the overexpression of these subunits
accelerated the kinetics of the opening response, increasing permeabi-
lization of the outer mitochondrial membrane (OMM) and resulting in
cell death (Azarashvili et al., 2014; Bonora et al., 2013). Furthermore,
in purified ATP synthase extracts, it was found that the ring structure
formed by c-subunits generated a current similar to that of the mPTP.
These currents were inhibited by calcium, CyPD and CsA (Alavian,
2014). Therefore, the properties of the pore formed in the c-subunit
ring suggests that this structure corresponds to the mPTP itself,
indicating a strong relationship between the opening of the mPTP
and ATP synthase activity (Alavian, 2014).
It is still a matter of discussion whether the extraction methods and
protocols used were adequate to reach a clear conclusion about the
components of mPTP (Bernardi et al., 2015a, 2015b; Chinopoulos
et al., 2014; Konig et al., 2016; Maltecca et al., 2015); complementary
evidence has indicated new roles of ATP synthase in mPTP regulation.
Although the mechanism for mPTP formation is an open question,
studies suggest that ATP synthase could not only participate in mPTP
opening by increasing IMM permeability, but could also directly affect
respiratory chain functionality and cell survival (Alavian et al., 2014).
The permeability of the IMM is a critical and decisive point between life
and death in the cell; the mPTP has been suggested as a master
regulator of both metabolism and cell death. With that in mind, a
relative closure or opening of the mPTP could greatly affect cellular
functions in organs and tissues with a high energy demand, such as the
heart and brain (Mnatsakanyan et al., 2016).
3. Pathological implications of mPTP opening
As we previously mentioned, the formation and consequent open-
ing of the mPTP is a key factor in mitochondrial dysfunction and
mitochondria-driven cell death (Bernardi et al., 2015a, 2015b; Bonora
et al., 2013; Jonas et al., 2015; Kroemer et al., 2007). In fact, the
general consensus is that uncontrolled opening of the mPTP leads
irreversibly to necrosis or apoptosis (Bernardi et al., 2015a, 2015b).
Under physiological conditions, when the mitochondria is exposed to
high concentrations of calcium, it can undergo a massive and perma-
nent swelling that leads to an abrupt increase in permeability to small
solutes of the IMM, resulting in the collapse of the chemiosmotic
gradient across the IMM—also known as the mitochondrial perme-
ability transition (Elrod and Molkentin, 2013). The resultant uncou-
pling of oxidative phosphorylation results in a decrease in ATP
generation and a subsequent increase in reactive oxygen species
(ROS) production. In addition, mitochondrial swelling may rupture
the OMM, releasing cytochrome C and initiating apoptosis (Izzo et al.,
2016; Kroemer et al., 2007; Rao et al., 2014).
Interestingly, despite their different etiologies, several neurodegen-
M.J. Pérez, R.A. Quintanilla
erative diseases and ischemic heart lesions share the common patho-
logic element of cytosolic calcium deregulation, which causes either
overload or uptake defects leading to mPTP opening (Abeti and
Abramov, 2015). Several studies have suggested that in the heart, the
opening of the mPTP during early reperfusion after ischemia is a
harmful event that induces further damage to the myocardium
(Griffiths and Halestrap, 1993). In contrast, in the brain, mPTP is
persistently activated in neurons under pathological conditions, de-
creasing the ability of the cells to regulate calcium homeostasis, which
contributes to neurodegeneration (Friberg and Wieloch, 2002).
Importantly, the genetic and pharmacological blockage of CyPD
prevents mitochondrial dysfunction in both cases (Rao et al., 2014).
3.1. Role of the mPTP in heart injury
Cardiomyopathy is a general term used to describe heart muscle
diseases; these disorders are related to an increase in the size of the
heart or the thickening and stiffening of heart muscle, compromising
the function of the whole organ (Kwong and Molkentin, 2015). One of
the major causes of acute cardiomyopathies is myocardial ischemia
(MI), which is inherited—in a minority of cases—or caused by chronic
diseases such as hypertension and diabetes (Walters et al., 2012).
Regardless of the cause, alterations in mitochondrial bioenergetics
appear to play an important role in the inception and progression of
this disease (Walters et al., 2012), manifesting as an inhibition of
respiratory complex activity, an increase in proton leakage from the
IMM (Ingwall, 2009), increased ROS production (Keith et al., 1998),
mitochondrial calcium overload (Luo and Anderson, 2013), and finally,
opening of the mPTP (Kwong and Molkentin, 2015). During MI
progression, oxygen deprivation alters mitochondrial function and
ATP synthesis, causing an important reduction in cardiac ATP produc-
tion (Jennings et al., 1991). Moreover, because calcium regulation
requires ATP, and the mPTP remains open as ATP pools dissipate,
calcium levels in the ischemic heart are further elevated (Kwong and
Molkentin, 2015). In addition to calcium elevation, ROS production is
also increased in the ischemic heart (Murphy and Steenbergen, 2008).
Thus, the ischemic heart is primed for a prolonged mPTP opening cycle
that ultimately leads to cardiomyocyte death (Murphy and
Steenbergen, 2008).
In support of these findings, CyPD-deficient mice subjected to
prolonged MI displayed reduced mortality with a 50% reduction in the
final infarct size (Lim et al., 2011; Yellon and Hausenloy, 2007),
suggesting that mPTP inhibition could be beneficial for treating this
disease. In fact, additional studies using the CyPD KO mouse model
showed protection not only in ischemia/reperfusion (I/R) models, but
also in mice where the infarction occurred in the brain and kidney
(Elrod et al., 2010). This effect should be considered important as a
potential pharmacological target for cardioprotection (Halestrap,
2009). Drugs that inhibit mPTP activation—such as sanglifehrin
(SfA), CsA, and other non-immunosuppressive analogs of CyPD, such
as [MeAla6]-cyclosporine (Griffiths and Halestrap, 1995), Debio-025
(Gomez et al., 2007), and NIM811 (Argaud et al., 2005)—have the
potential to be of great value in protecting the heart during cardiac
surgery for treatment of coronary thrombosis. More importantly, a
pilot clinical study in patients with MI showed that the administration
of CsA immediately when blood flow was restored reduced infarct size
by 40% compared with the placebo treatment (Piot et al., 2008).
Interestingly, in a subsequent follow-up study with the same group of
patients, reduction of the infarct size upon administration of one dose
of CsA was maintained over 6 months post-treatment, and these
patients displayed improved post-infarction cardiac function
(Mewton et al., 2010). However, these findings were questioned in
another study, where administration of CsA did not show a reduction in
the infarct size (measured by the release of cardiac enzymes) or any
improvement in clinical endpoints and mortality of the patients
(Ghaffari et al., 2013). However, subsequent analysis showed that the
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Developmental Biology 426 (2017) 1–7
formulation of CsA used in the latter study might not have been
effective for preventing myocardial reperfusion injury (Hausenloy and
Yellon, 2015). Thus, although the administration of mPTP inhibitors to
reduce MI damage is still controversial, it remains a promising
alternative treatment.
3.2. mPTP and neurodegenerative disorders
In most common neurological disorders, mitochondria have been
suggested as an important factor in the dysfunction and degeneration
of neurons (Mattson et al., 2008). In these diseases, deregulated
calcium homeostasis and increased ROS levels are apparently mediated
by mPTP opening. These disorders include excitotoxicity (Pivovarova
and Andrews, 2010), Huntington's disease (HD) (Quintanilla et al.,
2013, 2016), Alzheimer's disease (AD) (Du et al., 2011), and
Parkinson's disease (PD) (Martin et al., 2014). Under normal condi-
tions, mitochondria can buffer substantial amounts of calcium ions
during neurotransmission; the mitochondrial membrane potential and
several antioxidant mechanisms prevent the opening of the mPTP
(Friberg and Wieloch, 2002). Accumulating evidence indicates that
these abnormalities are rescued by the action of CsA, or by the genetic
ablation of CyPD (Abeti and Abramov, 2015). Indeed, CyPD-deficient
mice displayed resistance to apoptotic and non-apoptotic neuronal cell
death, as well as to mitochondrial dysfunction (Martin et al., 2014).
Different groups have reported increased CyPD expression in AD;
this may facilitate the opening of the mPTP (Du et al., 2010). On the
other hand, several studies have indicated that ROS and calcium
overload are potent inducers of mPTP opening (Du and Yan, 2010).
For example, in AD, the amyloid-beta peptide (Aβ), which accumulates
in high amounts during the development of this disease, induced ROS
formation and calcium stress, which are optimal conditions for mPTP
formation; it is therefore logical to hypothesize that massive mPTP
formation occurs when Aβ interacts with mitochondria (Du and Yan,
2010). Interestingly, the genetic absence of CyPD protects against this
phenomenon and slows cognitive loss in APP/PS1 mice, an animal
model of AD that recapitulates several features of this disease,
including aggregation of Aβ in the brain (Du et al., 2010, 2011; Du
and Yan, 2010). In addition, it was established that neurons that do not
express CyPD show a decrease in Aβ-dependent ROS generation,
increase in the buffering capacity of calcium, improved mitochondrial
respiratory function, and attenuation of abnormalities in synaptic
plasticity present in AD (Du et al., 2011; Guo et al., 2013). Similarly,
in HD, it was shown that mutant huntingtin affects mitochondrial
function by modifying mitochondrial calcium homeostasis, mitochon-
drial morphology, and ROS handling through the opening of mPTP
(Brustovetsky et al., 2003; Choo et al., 2004; Milakovic et al., 2006;
Quintanilla et al., 2013, 2016). These mitochondrial deficiencies in
mutant huntingtin-expressing cells only occurred after calcium over-
load, simulating the pathophysiological conditions that may occur in
HD (Milakovic et al., 2006; Quintanilla et al., 2013). As in AD, CsA
treatment ameliorated these toxic events; treatment also resulted in the
reduction of neuronal loss induced by calcium stress in mutant
huntingtin cells, supporting the role of mPTP in the mitochondrial
dysfunction observed in HD (Quintanilla et al., 2013, 2016).
More interestingly, it seems that opening of the mPTP has effects on
normal ageing (Gauba et al., 2017). For example, analysis of brain
samples of ageing mice showed an increase in CyPD expression and an
increase in its interaction with ATP synthase (Gauba et al., 2017).
These alterations coincided with the presence of mitochondrial un-
coupling, calcium-induced mitochondrial swelling, decreased ATP
production by oxidative phosphorylation, and enhanced SO produc-
tion, indicating that the mPTP could be an important factor that
controls mitochondrial function during ageing. Interestingly, these
mitochondrial alterations were significantly ameliorated by the genetic
ablation of CyPD (Gauba et al., 2017). Those results are in agreement
with studies where the knockout of CyPD presented an improvement in
M.J. Pérez, R.A. Quintanilla
the functional properties of brain mitochondria, and therefore pro-
tected mitochondrial function from calcium-induced stress
(Gainutdinov et al., 2015).
4. Physiological roles of the mPTP
Pioneer studies showed that opening of the mPTP not only leads to
mitochondrial damage and cell death, but is also a physiological
mechanism that mitochondria use to promote cell health (Elrod and
Molkentin, 2013). In fact, evidence of the physiological mechanism of
mPTP opening suggests two possible gating mechanisms: irreversible
full-conductance opening for permanent permeability leading to cell
death, or a second strategy that consists of a transient and flickering
short-term opening of mPTP with smaller and more variable conduc-
tance (Hou et al., 2014; Wang et al., 2008).
The mPTP appears to act as a normal calcium-release mechanism
that is required for proper metabolic regulation (Elrod and Molkentin,
2013), and it is hypothesized that its transient opening may regulate
cytosolic calcium when a calcium overload occurs in neurons and
myocytes (Bernardi and von Stockum, 2012; Gainutdinov et al., 2015;
Hom et al., 2011). Another possible role of the mPTP is its capacity to
regulate energy production in the cell, because a physical interaction
between CyPD and ATP synthase has been shown; this also suggests
that mPTP has roles as a metabolic regulator in the cell and in the
generation of SO flashes (Beutner et al., 1998; Elrod and Molkentin,
2013; Mnatsakanyan et al., 2016).
In this context, one of the primary functions of mitochondria is the
regulation of redox homeostasis, which could be interrupted by a
constant flow of ROS produced by the respiratory chain (Jensen et al.,
1996). Therefore, mitochondria have the ability to maintain oxidative
equilibrium, mediated by the action of several antioxidant enzymes
such as catalase, thioredoxin, and glutathione peroxidase (Droge,
2002). Even though ROS production by mitochondria could lead to
oxidative damage, recent studies have suggested that ROS could also
contribute to redox signaling in a variety of pathways in differentiation
and organogenesis (Owusu-Ansah and Banerjee, 2009), cell fate
regulation (Maryanovich and Gross, 2013), and the stress response
(Adler et al., 1999). In contrast to basal ROS production, it has been
determined that the generation of SO flashes or “mitoflashes,” which
consist of an intermittent and dynamic liberation of SO in a frequency-
modulated manner, could function in physiological signaling (Wang
et al., 2012, 2014). Further evidence strongly suggests that SO flashes
and oxidative signaling mechanisms are involved in mPTP activation
(Mnatsakanyan et al., 2016). Together with an increase in SO, cells
experience an abrupt dissipation of the mitochondrial membrane
potential, irreversible loss of small solutes from the matrix, and
transient mitochondrial swelling; these events are all characteristic of
mPTP opening (Hou et al., 2014; Wang et al., 2008, 2012). However,
there is still controversy surrounding the exact role of these SO flashes
in development (De Marchi et al., 2014). Recent advances have shown
that mitoflashes not only represent a fundamental step during mito-
chondrial function, but also play an important role in metabolism,
ageing, diabetes, wound healing, and, most importantly, a possible role
in cell differentiation and stemness (Ding et al., 2015; Hom et al.,
2011; Shen et al., 2014; Vega-Naredo et al., 2014). All these functions
are mediated by the opening of the mPTP (Hou et al., 2014).
4.1. The mPTP in cardiac development
Recently, important studies have demonstrated that mitochondria
are essential organelles for the heart and cardiac functions (Hom et al.,
2011; Ingraham et al., 2009). In fact, during cardiac development and
myocyte differentiation, the structures of individual mitochondria and
the cellular mitochondrial network are in constant flux, suggesting
direct participation in developmental mechanisms (Porter et al., 2011).
Recent works have documented that several changes in energetics and
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Developmental Biology 426 (2017) 1–7
mitochondrial biology occur in cardiac stem cells during differentiation
(Chung et al., 2007, 2008; Porter et al., 2011). These changes appear to
mirror what has been observed in the embryonic heart, where the
mPTP is open at early stages of myocyte differentiation (Beutner et al.,
2014; Hom et al., 2011).
Prior to differentiation, cardiac embryonic stem cells have frag-
mented mitochondrial networks with a specific perinuclear distribu-
tion; this event is interestingly related to the fact that the primary
source of energy to the cells is from anaerobic glycolysis, while
mitochondria show minimal participation (Beutner et al., 2014;
Porter et al., 2011). Upon induction of differentiation, mitochondrial
fission decreases and the mitochondrial network spreads throughout
the cell (Porter et al., 2011). There is also an increase in the complexity
of the IMM structure and the utilization of mitochondria and aerobic
respiration as the primary source of energy (Porter et al., 2011).
Interestingly, It was found that mPTP opening in the early heart
primordium is a physiological event in the development of the organ in
which myocytes exhibit low mitochondrial membrane potential, high
levels of ROS, and opening of the mPTP (Hom et al., 2011).
Interestingly, pharmacological closure of the mPTP with CsA led to
dramatic maturation of mitochondrial structure and function, decreas-
ing intracellular ROS levels and increasing mitochondrial membrane
potential, which accelerated myocyte differentiation (Hom et al., 2011).
Closure of the mPTP using CsA and antioxidant treatments also led to
further myocyte differentiation; the latter treatment was effective
regardless of the state of the mPTP (Hom et al., 2011). These studies
indicate that mPTP function regulates myocyte differentiation via
redox-signaling pathways and that changes in mPTP function, mito-
chondrial maturation, and ROS levels contribute to a novel mechanism
that protects the embryonic heart during a period of increasing energy
demand and low oxygen supply (Hom et al., 2011). These reports
highlight the mitochondria, and more specifically the mPTP, as a gating
mechanism underlying differentiation in the developing heart, impli-
cating a cross-talk between genetic and metabolic signaling (Folmes
et al., 2012; Hom et al., 2011).
In the same context, other studies have related ANT, a regulator of
mPTP, with the development and repair of cardiac tissue (Kokoszka
et al., 2016). Fetal and adult heart tissues express two ANT isoforms:
ANT1, which is highly expressed in the heart and brain; and ANT2,
which is expressed in the whole body (Bauer et al., 1999; Teixeira et al.,
2015). Mouse fetal and adult hearts express both ANT isoforms
(Haraguchi et al., 1993; Neckelmann et al., 1987). Interestingly,
genetic knockdown of ANT1 does not impair fetal development, but
does generate hypertrophic cardiomyopathy and induce apoptosis in
postnatal mice (Chevrollier et al., 2011). This effect is mediated
through increased association with the IκBα–NFκB complex, which is
later sequestered within the mitochondrial intermembrane space,
impeding migration to the nucleus (Bauer et al., 1999). In addition,
overexpression of ANT1 inhibited expression of the anti-apoptotic
proteins Bcl-XL, c-IAP2, and Sod2, favoring the opening of the mPTP
and eventually activating the intrinsic apoptosis pathway (Bauer et al.,
1999). Further studies generated a heterozygous ANT2 null mouse
model, in which fetuses presented with embryonic death at E14.5
(Kokoszka et al., 2016). ANT2 null mice showed significant cardiac
developmental failure with immature cardiomyocytes and a high
degree of hyperproliferation (Kokoszka et al., 2016). Interestingly,
these mice suffered from cardiac failure due to hypertrabeculation and,
more importantly, showed a high number of swollen mitochondria,
indicating that this mutation has an important effect on mitochondrial
function (Kokoszka et al., 2016). ANTs have two main functions; they
function as mitochondrial–cytosol ATP/ADP exchangers, and in mod-
ulating the opening of the mPTP (Kokoszka et al., 2004). Previous
studies suggest that ANT2 stimulates the closing of the mtPTP
(Chevrollier et al., 2011), while ANT1 enhances its opening (Bauer
et al., 1999). Therefore, the developmental toxicity shown in ANT2-null
mice may be the result of the mPTP being constantly opened in the
M.J. Pérez, R.A. Quintanilla Developmental Biology 426 (2017) 1–7

Fig. 1. Dual roles of the mitochondrial permeability transition pore (mPTP) in health and disease. (A). During myocyte stemness, which is defined as the ability to self-renew and
differentiate into different cell types, the mPTP is open; this results in a decrease in levels of ATP and mitochondrial membrane potential, as well as an increase in levels of cytosolic
calcium and reactive oxygen species (ROS). On the contrary, in neuronal stemness it seems that the mPTP remains closed, maintaining decreased levels of ROS and appropriate
production of ATP. (B). In cardiomyocytes, closure of the mPTP led to dramatic maturation of mitochondrial structure and function, decreasing intracellular ROS levels and increasing
mitochondrial membrane potential, which ultimately accelerated cell differentiation. However, neuronal development seems to be mediated by the opening of the mPTP, specifically by
the controlled increase in ROS levels in the form of superoxide mitoflashes leading to differentiation. (C). Despite their different etiologies, several neurodegenerative diseases and
cardiomyopathies share mitochondrial dysfunction as a common element, specifically the impairment of cytosolic calcium regulation; this may cause either calcium overload or uptake
defects in calcium homeostasis, leading to mPTP opening. In the heart, the opening of the mPTP during early reperfusion after ischemia is a harmful event that induces further damage
to the myocardium. In contrast, in the brain, mPTP is persistently activated in neurons exposed to pathological conditions, impairing their ability to regulate calcium. Only at this point is
the opening of the mPTP considered a pathological event.

cardiomyocytes, thus impairing cardiac cell maturation and resulting in
cardiomyocyte hyperproliferation (Kokoszka et al., 2016).
4.2. Role of the mPTP in nervous system development
During development of the nervous system, neural stem cells
proliferate and differentiate into neurons in the process of neurogen-
esis (Mattson et al., 2008). The newly formed neurons then grow an
axon and dendrites and eventually form synapses; during this process,
many newly generated neurons undergo constant changes in metabo-
lism and energy sources (Mattson et al., 2008). It was reported that the
proliferation and differentiation of cultured embryonic mouse cortical
neural progenitor cells (NPCs) was affected by the concentration of
oxygen in the culture; lower oxygen levels promoted proliferation,
while higher oxygen levels suppressed this process (Morrison et al.,
2000; Smith et al., 2000; Tsatmali et al., 2005).
Interestingly, Hou et al. described that in NPCs, neuronal differ-
entiation may be regulated by mitochondrial SO flashes; specific, focal,
and temporally-constrained bursts of mitochondrial SO negatively
regulate NPC proliferation independently of global changes in cytosolic
ROS levels (Hou et al., 2012). Indeed, when the authors limited the
mitoflash frequency using ROS scavengers, NPC proliferation in-
creased; conversely, increasing mitochondrial SO flashes promoted
neuronal differentiation (Hou et al., 2012). On the other hand, it has
been shown that during neurogenesis of NPCs there is a switch in
cellular energy production from anaerobic glycolysis to aerobic mito-
chondrial oxidative phosphorylation in differentiated neurons
(Candelario et al., 2013). Interestingly, increasing bursts of mitochon-
drial SO production are associated with this switch mechanism in
cellular energy metabolism (Hou et al., 2012).
With respect to the participation of the mPTP in neural develop-
ment, it was reported that NPCs exhibit intermittent spontaneous
bursts of mitochondrial SO generation that require a transient opening
of mPTP (Hou et al., 2012). Therefore, it has been shown that both
mitochondrial ROS scavengers and mPTP inhibitors—such as CsA—
reduced the frequency of mitoflashes and enhanced NPC proliferation,
whereas prolonged mPTP opening and SO generation increased the
incidence of mitoflashes and promoted neuronal differentiation of
NPCs (Hou et al., 2012). This minimal and transient ROS production
negatively regulates the self-renewal of NPCs and suggests that
dynamic mitochondrial ROS generation in the form of mPTP-depen-
dent SO flashes serves as a signaling mechanism that controls the fate
of NPCs (Hou et al., 2012). Interestingly, similar to these findings, CsA
inhibited the differentiation of hematopoietic progenitor cells and
vascular progenitor cells (Arnold et al., 2000; Davies et al., 2005),
but enhanced the differentiation of natural killer cells from hemato-
poietic progenitors and cardiac cells from induced pluripotent stem
cells (Fujiwara et al., 2011; Kosugi and Shearer, 1991). These findings
reveal that there is great complexity and specificity in SO generation
and opening of the mPTP. These events are dependent on the specific
cell type, the subcellular localization of mitochondria, and the temporal
occurrence within the developmental stage (Hou et al., 2013, 2012).
5. Conclusions
The opening of the mPTP was normally associated with pathologi-
cal events such as neurodegenerative diseases or ischemic injury in
brain and heart, which all share a mitochondrial dysfunction condition
directly related to the activation of mPTP (Fig. 1). However, several
novel physiological functions of mPTP in development have also been
discovered, as mPTP participates in the physiological calcium-release
mechanisms that are required for proper metabolic regulation of the
cell; this structure could be an important player in the regulation of
heart and brain development (Fig. 1). Furthermore, it seems that the
conversion of a physiological opening event into a pathological channel
opening may be related to the energetic context and the metabolic

5
M.J. Pérez, R.A. Quintanilla
status of the cell; nevertheless, the factors that regulate the transition
from physiological to pathological mPTP opening in the mitochondria
are not completely understood.
It is relevant to emphasize that these findings could help us to
understand the mechanisms of cell differentiation and the importance
of mitochondria in health and disease, but they also highlight the
significance of research on drugs that may modulate the activation of
mPTP in the search for possible therapies in degenerative diseases
associated with either ageing or development.
Acknowledgments
This work has been supported by FONDECYT # 1140968 and
1170441 (RAQ), as well as CONICYT PIA Anillo ACT1411 (RAQ).
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