Dumas, J.F.

Guimaraes, C.

Jarnouen, K.

Lagrave, J.

Loyer, P.

Maillot, F.

Peyta, L.

Pinault, M.

Servais, S.

Elsevier - Publisher Connector 

S2.P10
Uncouplers preserve the respiratory function in the
heteroplasmic Saccharomyces cerevisiae cells carrying
wild type and non-functional mitochondrial DNAs
Dmitry A. Knorre, Julia E. Karavaeva, Ekaterina A. Smirnova,
Svyatoslav S. Sokolov, Fedor F. Severin
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow
State University, Russia
E-mail address: knorre@belozersky.msu.ru
A number of pathologies are associated with the presence of non-
identical mitochondrial DNA (mtDNA) molecules within a single
cell (heteroplasmy). The latter is usually due to the large deletions in a
subset of mtDNAs. The short copies of mtDNA have an advantage
in replication and eventually displace the functional mtDNA. We
proposed that mitochondrial uncouplers can change the competition
conditions in favor of the functional mtDNAs. Here we tested this
by mating of the two Saccharomyces cerevisiae strains: mat a with
the wild type mtDNA (rho+) and mat alpha with hypersuppressive
mutant mtDNA (HS rho–). Then we measured the percentages of the
obtained diploid cells whichwere not able to grow on non-fermentable
carbon source. These percentages reﬂect the efﬁciency of HS rho–
mtDNA in the displacement of wild type rho+mtDNA. It was found that
uncouplers FCCP and pentachlorophenol decrease the frequency of
such displacement. Unlike the uncouplers, the inhibitors of respiratory
chain, an inhibitor of ATP synthase oligomycin or antioxidants (alpha-
tocopherol, N-acetylcysteine) did not show such an effect. Furthermore,
it was found that suppression of DNM1 (a key mediator of mitochon-
drial ﬁssion) signiﬁcantly diminishes the effect of FCCP. We argue that
uncouplers decrease the frequency of mitochondria ﬁssion–fusion
cycles thus preventing the mixing of functional and non-functional
mtDNAs. As a result, the mitochondrial quality control machinery has
more time for the removal of mitochondria with non-functional
variants of mtDNA. We speculate that similar strategy could be applied
to multicellular eukaryotes for preventing clonal expansion of mtDNA
with common deletion.
doi:10.1016/j.bbabio.2014.05.298
S2.P11
Phenotypic cell analysis using automated mitochondrial
morpho-functional ﬁngerprinting and supervisedmachine learning
Dr.Werner J.H. Koopman
Biochemistry RIMLS, Radboud UMC, Nijmegen, The Netherlands
E-mail address: w.koopman@ncmls.ru.nl
Human mitochondrial complex I (CI) dysfunction is often para-
lleled by increased ROS levels and aberrant mitochondrial membrane
potential andmorphology.We generated novel therapeutic molecules
(“KH-compounds”) and studied their multifactorial effects in primary
human skin ﬁbroblasts of Leigh syndrome (LS) patients with isolated
CI deﬁciency. KH-compounds speciﬁcally altered the amount/activity
of fully assembled CI, as well as cellular ROS level. Cluster analysis
highlighted various compound classes with distinct physicochemical
properties and cellular effects. The best performing KH compounds
were analyzed at the single cell level using high-content microscopy,
mitochondrial morpho-functional ﬁngerprinting and machine learning
techniques. Compound KH003 which best counterbalanced mitochon-
drial morpho-functional aberrations in LS cells, was not cytotoxic and
displayed ROS scavenging properties. KH003 is currently tested in
animal models of mitochondrial disease to determine its therapeutic
potential.
References
[1] Koopman et al., OXPHOSmutations and neurodegeneration. EMBO J.
32 (2013) 9–29.
[2] Willems et al., BOLA1 is an aerobic protein that prevents
mitochondrial morphology changes induced by glutathione deple-
tion. Antioxid. Red. Signal. 18 (2013) 129–138.
[3] Koopman et al., Monogenic mitochondrial disorders. N. Eng. J. Med.
366 (2012) 1132–1141.
[4] Distelmaier et al., Trolox-sensitive ROS regulate mitochondrial
morphology, oxidative phosphorylation and cytosolic calcium han-
dling in healthy cells. Antioxid. Red. Signal. 17 (2012) 1657–1669.
[5] Dieteren et al., Solute diffusion is hindered in the mitochondrial
matrix. Proc. Natl. Acad. Sci. USA 108 (2011) 8657–8662.
[6] Koopman et al., Mammalian mitochondrial complex I: biogenesis,
regulation and reactive oxygen species generation. Antioxid. Red.
Signal. 12 (2010) 1431–1470.
[7] Distelmaier et al., Mitochondrial complex I deﬁciency: fromorganelle
dysfunction to clinical disease. Brain 132 (2009) 833–842.
doi:10.1016/j.bbabio.2014.05.299
S2.P12
Reduction of the mitochondrial content in cardiolipins decreases
O2 consumption and increases ATP synthesis efﬁciency in human
hepatocyte-like HepaRG cells
L. Peytaa, J.F. Dumasb, M. Pinaultb, C. Guimaraesb, K. Jarnouenc,
J. Lagraveb, F. Maillotb, P. Loyerc, S. Servaisb
aTours
bINSERM UMR 1069 “Nutrition, Growth and Cancer”, France
cINSERM UMR 911 “Liver, Metabolisms and Cancer”, France
E-mail address: laure.peyta@etu.univ-tours.fr
Cardiolipins (CL) are major and speciﬁc phospholipids of the
mitochondrial inner membrane known to be essential for mitochon-
drial functions such as the oxidative phosphorylation. However the
implication of CL in mitochondrial bioenergetics is not fully under-
stood, particularly in mammalians. In fact most of the data previously
published have used models of defective yeast mutants lacking
CL or cells derived from patients affected by the Barth syndrome
(tafazzinmutation).We have previously demonstrated an implication
of cardiolipins in cancer cachexia, a paraneoplasic syndrome, charac-
terized by a negative energy balance resulting from variable com-
bination of reduced food intake and hypermetabolismwith a systemic
inﬂammation. We have demonstrated CL involvement in the alter-
ation of rat liver mitochondria bioenergetics in cancer-induced
cachexia. The increase in CL content (+55% p b 0.05) was positively
correlated to the increase in energy wasting observed in liver
mitochondria of cancer cachectic rats (R2 = 0.64, p b 0.05). Energy
wasting was associated to a signiﬁcant decreased in ATP synthesis
efﬁciency. We conﬁrmed in vitro that liver mitochondria enrichment
with CL induced the increase in energy wasting and the reduction
in ATP synthesis efﬁciency. The aim of the present study was to
investigate the impact of the reduction in CL content on hepatic cell
bioenergetics, with a focus on efﬁciency of energy production. For this
purpose, we established recombinant HepaRG cells stably expressing
shRNA triggering the cardiolipin synthase (shCLS) that efﬁciently
reduced CL content (−55% p b 0.05). A signiﬁcant decrease in O2
consumption (−30%, p b 0.05) was observed at phosphorylating, non-
phosphorylating and uncoupling states using different substrates such
as succinate, glutamate +malate, palmitoyl-l-carnitine, palmitoyl CoA
and ascorbate + TMPD. These data demonstrated the impact of CL
reduction on the overall OXPHOS system, independently of nature of
the substrate and respiratory complexes involved. Interestingly, the
Abstractse28
ATP/O ratio, reﬂecting efﬁciency of ATP synthesis, was higher in shCLS
HepaRG cells (+30% p b 0.05) without modiﬁcation of ATP synthesis
rate (using succinate as substrate). Mechanisms involved in these
modiﬁcations are under investigation. In conclusion, our work clearly
demonstrated, for the ﬁrst time, that the reduction of cardiolipin
content regulates directly ATP synthesis in human hepatic cells.
doi:10.1016/j.bbabio.2014.05.300
S2.P13
Effects of reaction probability and occupation area in the electron
transfer kinetics of the mitochondrial chain segment involving
complexes I, II and ubiquinone: A Monte-Carlo simulation study
Jorge Martins
IBB-CBME, DCBB-FCT, Universidade do Algarve, Portugal
E-mail address: jmartin@ualg.pt
The components of mitochondrial chain were conceived to move
freely and interact by random collisions, e.g. ubiquinone moving
more rapidly and shuttling electrons from complexes I and II to
complex III [1]. The behavior of ubiquinone and cytochrome c pools
failed to support the diffusion model of independent complexes
randomly distributed [2]. Given the molecular crowding (and likely
heterogeneous distribution of reactants), lateral diffusion is expected
to be restricted in this membrane. So, the kinetic analysis should
take on theories considering irregular lateral diffusion, reaction-
controlled and domain-limited reactivity [1]. In this work, the effects
of reaction probability varying upon collision and two-dimensional
concentration, involving complexes I, II and ubiquinone, are studied
using Monte-Carlo techniques. The simulations are based on con-
tinuous Pearson-type random walk [3], considering the reactants as
non-overlapping disks, whose diameters mimic the molecular areas
of the lipid and proteins. The probability varied between 1 (the
diffusion-control limit considers the entire perimeter as active),
0.5 (half the perimeter is inactive), 0.25, 0.125 and 0.0625. The
regular lateral diffusion of ubiquinone is equivalent to phospholipids
and proteins are much less mobile (10−10 cm2/s). The stoichiometry
is taken as 1:2:30, for complex I, complex II and ubiquinone,
respectively. The analysis is based on the half-time for the decay
of initial concentration of reactants (within μs time range). For a
disk occupation area of 17.28%, bellow what is expected in vivo,
the outcomes indicate obstructed diffusion effects, comparing with
simulations with overlapping particles. The results emphasize the
importance of reaction kinetics and molecular crowding in models
of mitochondrial electron transport. This work was supported by
national Portuguese funding through FCT — Fundação para a Ciência
e a Tecnologia, project ref. PEst-OE/EQB/LA0023/2013.
References
[1] E.Melo, J. Martins, Kinetics of bimolecular reactions inmodel bilayers
and biological membranes. A critical review, Biophys. Chem. 123
(2006) 77–94.
[2] H. Boumans, L.A. Grivell, J.A. Berden, The respiratory chain in yeast
behaves as a single functionalunit, J. Biol. Chem.273 (1998)4872–4877
[3] J. Martins, K. Razi Naqvi, E. Melo, Kinetics of two-dimensional
diffusion-controlled reactions: a Monte Carlo simulation of hard-
disk reactants undergoing a Pearson-type random walk, J. Phys.
Chem. B 104 (2000) 4986–4991.
doi:10.1016/j.bbabio.2014.05.301
S2.P14
Targetingmitochondria throughmodulation of eIF5A hypusination
protects from anoxia-induced cell death
Nicolas Melis
LP2M — CNRS-UMR 7370, France
E-mail address: nmelis@unice.fr
eIF5A, for eukaryotic initiation factor 5 A, is a highly conserved
protein throughout evolution. Firstly described as a translation
initiation factor, it became more noteworthy for its unique post-
translational activation through hypusination. This modiﬁcation, a
4-aminobutyl moiety from spermidine transferred onto a lysyl residue,
is sequentially catalyzed by two enzymatic steps involving respectively
the deoxyhypusine synthase (DHPS) and the deoxyhypusine hydrox-
ylase (DOHH). We took advantage of this unique characteristic and
use the highly speciﬁc DHPS competitive inhibitor GC7 (N-guanyl-1,7-
diaminoheptane) to demonstrate an unsuspected link between eIF5A
hypusination, mitochondria activity and the cellular resistance to
anoxia. To investigate the involvement of this pathway in the resistance
to low oxygen level, renal proximal tubular cells (PCT) were exposed to
anoxia (b0.1% O2, 24 h). GC7 pre-treatment (30 μM) or RNA silencing-
mediated inhibition of DHPS or DOHH was largely protected from
the anoxia-induced cell death. This tolerance to anoxia is paralleled
by a marked increase in glucose consumption and lactate production
reﬂecting a reversible metabolic shift from aerobic OXPHOS to
anaerobic glycolysis, preserving the cellular energetic status. We also
studied the effect of GC7 on mitochondrial status and showed that
GC7 induced a reversible “mitochondrial silencing” characterized by a
decrease inmitochondrial potential (ΔΨm) and a drasticmitochondrial
structure remodeling associated to a down-regulation of respiratory
chain complex expression. The resulting effect is a decrease in O2
requirements associated with a reduction of the deleterious reactive
oxygen species produced during anoxia. Thus, targeting mitochondria
through modulation of eIF5A hypusination pathway may offer an
innovative therapeutic strategy for ischaemic human diseases—e.g.,
stroke or myocardial infarction—or organ transplantation.
doi:10.1016/j.bbabio.2014.05.302
S2.P15
Mitochondrial nucleoid visualization in isolated pancreatic β-cells
Tomas Olejara, Lukas Alanb, Monika Cahovac, Zuzana Berkovac,
Frantisek Saudekc, Petr Ježekd
aDepartment of Membrane Transport Biophysics, Institute of Physiology,
Academy of Sciences of the Czech Republic, Czech Republic
bMembrane Transport Biophysics, Institute of Physiology, AS CR, v.v.i.,
Czech Republic
cInstitute of Clinical and Experimental Medicine, Czech Republic
dDepartment of Membrane Transport Biophysics, Institute of Physiology,
ASCR, Czech Republic
E-mail address: olejar@biomed.cas.cz
Quality of mitochondrial (mt) network and mtDNA is widely
discussed in relationship to reactive oxygen species (ROS) production
and development of the type 2 diabetes mellitus. Photoactivatable
ﬂuorescence proteins like Green ﬂuorescence protein (GFP) and Eos
tagged to different lentiviral protein vectors are utilized to study
respective targets in vivo. As an approach to study mt morphology and
mt nucleoid distribution in isolated pancreatic β-cells, we implemented
lentiviral transfection of matrix-addressed GFP (Mito-GFP) to visualize
mt network and Eos-tagged Single-strand DNA binding protein-1
(SSBP1-Eos) to image nucleoids. Diabetic Goto-Kakizaki (GK) rats,
Abstracts e29


Reduction of the mitochondrial content in cardiolipins decreases O2 consumption and increases ATP synthesis efficiency in human hepatocyte-like HepaRG cells 

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Reduction of the mitochondrial content in cardiolipins decreases O2 consumption and increases ATP synthesis efficiency in human hepatocyte-like HepaRG cells

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