A icle
Pe u bed Redox Signaling Exace ba es a
Mi ochond ial Myopa hy
G aphical Abs ac
Highligh s
dROS igge mi ochond ial biogenesis in Cox15-
de icien mice
dSa elli e cell ec ui men is ROS dependen in mi ochond ial
myopa hy
dAOX and NAC in e e e wi h ROS signaling
dThe use o an ioxidan s in mi ochond ial diseases should be
ca e ully e alua ed
Au ho s
Suk u Anil Dogan, Ra aele Ce u i,
C is iane Beninca
´, ..., Massimo Ze iani,
Ma en Szibo , Ca lo Viscomi
Co espondence
ma en.szibo @u a. i (M.S.),
c 23@m c-mbu.cam.ac.uk (C.V.)
In B ie
Dogan e al. show ha al e na i e oxidase
a enua es ROS signaling in a COX-
de ec i e mi ochond ial myopa hy model,
hus blun ing ROS-dependen
mi ochond ial biogenesis and sa elli e
cell ec ui men . These indings mus be
conside ed in he ea men o
mi ochond ial myopa hies, sugges ing
he need o ca e ul assessmen o
an ioxidan he apy.
Dogan e al., 2018, Cell Me abolism 28, 764–775
No embe 6, 2018 ª2018 The Au ho (s). Published by Else ie Inc.
h ps://doi.o g/10.1016/j.cme .2018.07.012
Cell Me abolism
A icle
Pe u bed Redox Signaling
Exace ba es a Mi ochond ial Myopa hy
Suk u Anil Dogan,
1
Ra aele Ce u i,
1
C is iane Beninca
´,
1
Glo ia B ea-Cal o,
2
Howa d T e o Jacobs,
3,4
Massimo Ze iani,
1
Ma en Szibo ,
3,4,
*and Ca lo Viscomi
1,5,
*
1
MRC Mi ochond ial Biology Uni , Uni e si y o Camb idge, Wellcome T us /MRC Building Hills Road, Camb idge CB2 0XY, UK
2
Cen o Andaluz de Biologı
´a del Desa ollo and CIBERER, Ins i u o de Salud Ca los III, Uni e sidad Pablo de Ola ide-CSIC-JA, Se illa
41013, Spain
3
Ins i u e o Bio echnology, Uni e si y o Helsinki, Viikinkaa i 5, Helsinki 00790, Finland
4
Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e, A o Ylpo
¨n ka u 34, Tampe e 33520, Finland
5
Lead Con ac
*Co espondence: ma en.szib[email p o ec ed] (M.S.), [email p o ec ed]k (C.V.)
h ps://doi.o g/10.1016/j.cme .2018.07.012
SUMMARY
Al e na i e oxidases (AOXs) bypass espi a o y
complexes III and IV by ans e ing elec ons om
coenzyme Q di ec ly o O
2
. They ha e he e o e
been p oposed as a po en ial he apeu ic ool o
mi ochond ial diseases. We c ossed he se e ely
myopa hic skele al muscle-speci ic COX15 knockou
(KO) mouse wi h an AOX- ansgenic mouse. Su p is-
ingly, he double KO-AOX mu an s had dec eased
li espan and a subs an ial wo sening o he myopa hy
compa ed wi h KO alone. Dec eased ROS p oduc-
ion in KO-AOX e sus KO mice led o impai ed
AMPK/PGC-1asignaling and PAX7/MYOD-depen-
den muscle egene a ion, blun ing compensa o y
esponses. Impo an ly, he an ioxidan N-ace ylcys-
eine had a simila e ec , dec easing he li espan o
KO mice. Ou indings ha e majo implica ions o un-
de s anding pa hogenic mechanisms in mi ochon-
d ial diseases and o he design o he apies, high-
ligh ing he bene i s o ROS signaling and he
po en ial haza ds o an ioxidan ea men .
INTRODUCTION
Oxida i e phospho yla ion (OXPHOS) is he p ocess by which
mi ochond ia con e he ene gy de i ed om nu ien s in o
ATP. Elec ons gene a ed by in e media y me abolism in he
o m o educing equi alen s a e ans e ed along he ou com-
plexes o he mi ochond ial espi a o y chain (complexes I–IV,
cI–cIV) o e en ually combine wi h molecula oxygen o p oduce
wa e . This exe gonic p ocess, e med espi a ion, sus ains he
ex usion o p o ons ac oss he inne mi ochond ial memb ane,
ca ied ou by p o on pumps p esen in cI, cIII, and cIV. P o on
ansloca ion gene a es an elec ochemical g adien , gi ing
ise o a memb ane po en ial, Dj, which is exploi ed by he
ATP syn hase (complex V, cV) o con e ADP and Pi o ATP.
Mu a ions in a as a ay o genes encoded by ei he he nuclea
o mi ochond ial DNA (m DNA) dis up he espi a o y chain and
lead o p ima y mi ochond ial diseases. Se e al in e connec ed
mechanisms accoun o he cellula consequences o OXPHOS
de ec s, including dec eased ATP syn hesis, inc eased p oduc-
ion o eac i e oxygen species (ROS), al e ed ion a icking,
de anged me aboli e le els, and abno mali ies in mi ochon-
d ial- ela ed cell dea h and u no e pa hways such as apop osis
and au ophagy.
In pa icula , ROS a e by-p oduc s o no mal espi a ion, bu
can inc ease d ama ically when he espi a o y chain is impai ed.
ROS a e in ac hough o play a ‘‘ho me ic’’ double ole: in phys-
iological condi ions, low le els o ROS ac as signaling molecules
egula ing homeos a ic pa hways ela ed o mi ochond ial bio-
ene ge ics, whe eas a high le els hey ac as oxic agen s
damaging cellula componen s, including nucleic acids, p o-
eins, and lipids (Yun and Finkel, 2014). Along he espi a o y
chain, ROS a e gene a ed a di e en si es wi h cI, cII, and cIII
playing he main ole (B and, 2016). In pa icula , cI gene a es
ROS h ough e e se elec on ans e (RET), which exploi s
he elec ons lowing back om coenzyme Q (CoQ) when his
is o e - educed by elec ons om cII (Chouchani e al., 2014)
o in he p esence o d ugs o gene ic de ec s ha inhibi cIII
and/o cIV (Gua a
´s e al., 2016). Al hough he de imen al ole
o ROS has ecen ly been challenged (Scialo
`e al., 2016), cells
ha e e ol ed highly e icien ROS sca enging sys ems, which
in mammals a e mainly con olled by an an ioxidan esponse
p og am, based on he Kelch-like ECH-associa ed p o ein 1
(KEAP1) and nuclea ac o e y h oid 2- ela ed ac o 2 (NRF2/
NFE2L2) (Holms o
¨m and Finkel, 2014).
In spi e o ecen p og ess, no speci ic he apy is cu en ly
a ailable o OXPHOS diso de s. Because o hei huge gene ic
he e ogenei y, an e ec i e he apy should ha e he wides
possible applicabili y o a leas ha e he po en ial o be applied
o mo e han a single disease en i y.
Al e na i e oxidases (AOXs) a e cyanide- esis an , mem-
b ane-bound mi ochond ial enzymes p esen in plan s, lowe eu-
ka yo es, and some speci ic me azoan phyla, consis ing o jus a
single gene p oduc . AOXs main ain elec on low when he es-
pi a o y chain is inhibi ed a he le el o cIII and/o cIV, by di ec ly
ans e ing elec ons om CoQ o O
2
, hus bypassing cIII and
cIV and p e en ing o e - educ ion o he CoQ pool. No ably,
AOX ac i i y is no associa ed wi h p o on pumping ac oss he
inne mi ochond ial memb ane and hus does no con ibu e
764 Cell Me abolism 28, 764–775, No embe 6, 2018 ª2018 The Au ho (s). Published by Else ie Inc.
This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/).
di ec ly o he main enance o Djand ATP syn hesis. Howe e ,
in he p esence o cIII o cIV de ec s, he inc ease in p o on
pumping a cI, due o he e-ac i a ion o elec on low, should
sus ain he elec ochemical g adien and ATP p oduc ion.
Impo an ly, he e-ac i a ion o elec on low by AOXs limi s
he excessi e gene a ion o ROS and main ains edox homeo-
s asis, he eby main aining ica boxylic acid cycle ac i i y (Mills
e al., 2016). This has been exploi ed ex ensi ely o imp o e he
pheno ype o cellula and ly models wi h cIII and cIV de ec s (El-
Khou y e al., 2014). Howe e , i s use in mammalian models has
no been explo ed so a . He e, we epo he in i o e ec s o
AOX exp essed in a skele al muscle-speci ic knockou mouse
o Cox15 (Cox15
sm/sm
, he ea e designa ed KO), encoding he
e minal enzyme o he biosyn he ic pa hway o heme a,an
essen ial p os he ic g oup o cIV (cy och ome coxidase [COX]).
RESULTS
AOX Exp ession Exace ba es he Pheno ype o KO Mice
KO mice de elop a p o ound, muscle- es ic ed COX de iciency
leading o se e e mi ochond ial myopa hy and ea ly dea h (Vis-
comi e al., 2011). A mouse s ain ca ying AOX om he unica e
Ciona in es inalis inse ed in he mu ine Rosa26 locus has
ecen ly been desc ibed (AOX
g
, he ea e designa ed AOX),
and was shown o be pheno ypically indis inguishable om
wild- ype (WT) li e ma es (Szibo e al., 2016).
We c ossed he KO and AOX lines o gene a e KO-AOX double
mu an s, o es whe he AOX could alle ia e he KO pheno ype.
Unexpec edly, KO-AOX mice showed a mo e se e e pheno ype,
Figu e 1. AOX Exp ession Exace ba es he
Physical P ope ies and Li espan o KO Mice
(A) To al mo emen o male 8-week-old WT, AOX,
KO, and KO-AOX mice measu ed by CLAMS and
indica ed as coun s pe nigh (n = 8–10).
(B) T eadmill analysis o mo o pe o mance (n = 4).
(C) Kaplan-Meie su i al cu es (numbe o ani-
mals used a e WT, 17; AOX, 15; KO, 31; KO-AOX,
16; KO-NAC, 8). Mean li espans o KO-AOX and
KO-NAC a e compa ed wi h KO by one-sample
es . N-Ace ylcys eine (NAC) was gi en o KO mice
in he d inking wa e om 3 weeks o age.
Ba s ep esen means ± SEM. As e isks o e he
ba s indica e s a is ical signi icance e sus WT;
o e he b acke s among indica ed g oups. *p %
0.05; **p %0.01; ****p < 0.0001; unpai ed S uden ’s
es .
wi h ea lie onse o symp oms han KO,
cha ac e ized by dec eased body weigh
(Figu e S1A) due o diminished a mass
(Figu e S1B) and dec eased o al spon a-
neous mo emen s (Figu e 1A) as well as
eadmill mo o pe o mance in compa i-
son wi h KO li e ma es (Figu e 1B). The
su i al p obabili y o he KO-AOX mice
was ma kedly lowe as well; i s median
li espan was 60 days compa ed wi h
150 days o KO (log ank, p < 0.0001; Fig-
u e 1C). In ac , all he KO-AOX mice had
o be eu hanized by 90 days o age because o hei poo
condi ion.
Since he COX de ec in KO mice is muscle speci ic, we
easoned ha wo sening o he myopa hy migh be esponsible
o he mo e d as ic pheno ype o KO-AOX mice. We hus
analyzed he skele al muscle om 8-week-old animals o he
ou geno ypes, i.e., be o e KO-AOX mice s a o die. COX/
SDH his ochemical s aining con i med he expec ed p e a-
lence o COX-de icien ibe s in KO e sus WT and AOX
animals. Howe e , he de ec was e en mo e p ominen in
KO-AOX samples (Figu e 2A).Inaddi ion,SDHs ainingwas
inc eased in KO muscles, bu i was simila o he WT in KO-
AOX mice (Figu es S2A and S2B). Quan i a i e spec opho o-
me ic assay o COX-speci ic ac i i y in muscle homogena es
con i med his obse a ion (10.29 ± 1.46 in KO e sus 4.17 ±
0.47 in KO-AOX; p < 0.01; Figu e 2B). Likewise, mo phological
analysis by hema oxylin and eosin (H&E) s aining e ealed
signi ican ly dec eased c oss-sec ional a ea o KO-AOX e sus
KO myo ibe s (Figu e 2C). The numbe o cen alized nuclei, an
index o skele al muscle egene a ion, was ma kedly inc eased
in KO e sus WT and AOX (8.55% ± 0.73% in KO e sus
0.41% ± 0.13% in WT), bu much less so in KO-AOX mice (Fig-
u e 2D). This inding p omp ed us o in es iga e he di e en ia-
ion o sa elli e cells in he skele al muscle o he di e en geno-
ypes. The numbe o nuclei posi i e o PAX7, a ma ke o
esiden myoblas s, and MYOD, a ma ke o he di e en ia ing
sa elli e cells, was signi ican ly inc eased in KO e sus WT
and AOX muscles, bu i was exp essed a no mal le els in
KO-AOX mice (Figu es 3A and 3B).
Cell Me abolism 28, 764–775, No embe 6, 2018 765
Nex , we in es iga ed i AOX de e mined a swi ch in he ibe
ype by immunodeco a ing muscle sec ions wi h an ibodies
agains he di e en myosin iso o ms. Howe e , no di e ences
we e obse ed (Figu es S3A and S3B). Taken oge he , hese
da a clea ly indica e ha he mi ochond ial myopa hy was signi -
ican ly mo e se e e in KO-AOX animals.
Nex , we con i med AOX exp ession and ca aly ic ac i i y in
KO-AOX indi iduals. Wes e n blo immuno isualiza ion showed
obus exp ession o AOX in mos issues excep b ain o adul
mice, as p e iously epo ed (Szibo e al., 2016)(Figu e S4A).
Oxyg aphic analysis o isola ed mi ochond ia in he p esence
o ADP (s a e III) demons a ed subs an ial cyanide- esis an
espi a ion in muscle o bo h AOX and double ecombinan
KO-AOX mice (Figu e S4B). S a e III O
2
consump ion a e was
ma kedly highe in WT and AOX mi ochond ia compa ed wi h
KO and KO-AOX. In con as , oligomycin-sensi i e espi a ion
was ma kedly inc eased in bo h AOX and KO-AOX muscle mi o-
chond ia compa ed wi h he co esponding nai e models, WT
Figu e 2. AOX Exp ession Wo sens he
Biochemical Muscle Pheno ype o KO Mice
(A) His ochemical analyses o cy och ome coxi-
dase (COX), succina e dehyd ogenase (SDH),
double s aining o COX-SDH, and H&E in 8-week-
old WT, AOX, KO, and KO-AOX animals.
(B) Spec opho ome ic speci ic ac i i y o cIV in
skele al muscle o 8-week-old mice (n = 5).
(C) Analysis o he c oss-sec ional a ea o muscle
ibe s (n = 4).
(D) Analysis o he numbe o cen alized nuclei in
muscle ibe s (n = 4).
Ba s ep esen mean ± SEM. As e isks o e he
ba s indica e s a is ical signi icance e sus WT;
o e he b acke s among indica ed g oups. *p %
0.05; **p %0.01; ***p %0.001; ****p < 0.0001;
unpai ed S uden ’s es .
and KO, espec i ely (Figu e S4C). These
esul s indica e ha AOX-dependen
espi a ion is ac i e bu insensi i e o
ei he cyanide o oligomycin inhibi ion.
AOX Exp ession In e e es wi h
Mi ochond ial Biogenesis in
KO-AOX
In addi ion o SDH s aining, CS ac i i y
was also inc eased in KO animals
compa ed wi h con ols and KO-AOX
(Figu e 4A). These da a p omp ed us o
e alua e o he ma ke s o mi ochond ial
biogenesis in skele al muscle. The m DNA
copy numbe (Figu e 4B) and he exp es-
sion le els o mi ochond ial ansc ip ion
ac o A (TFAM) (Figu e 4C) we e
inc eased in he KO e sus WT and AOX
bu we e simila o WT alues in he dou-
ble ecombinan KO-AOX animals. In
addi ion, he amoun o se e al subuni s
o he espi a o y complexes was signi i-
can ly inc eased in KO e sus WT and
AOX, bu no in samples om KO-AOX mice (Figu e 4D). O e all,
hese da a sugges ha AOX exp ession blun s he inc eased
mi ochond ial biogenesis obse ed in he muscle-speci ic
Cox15-de ec i e model.
We hen in es iga ed whe he he main mi ochond iogenic
con ol pa hways we e a ec ed by AOX in KO muscles. AMP-
dependen kinase (AMPK) is an impo an senso o cellula en-
e ge ic s a us and is ac i a ed when he AMP/ATP a io in-
c eases (Lin and Ha die, 2017). Unde such ene gy-de icien
condi ions, AMPK is ac i a ed by phospho yla ion o Th
172
(p-AMPK) by LK1 kinase and, in u n, p-AMPK phospho yla es
a la ge numbe o a ge s, including he ansc ip ional co-ac i-
a o PGC-1a, a mas e egula o o mi ochond ial biogenesis.
As p e iously epo ed (Viscomi e al., 2011), p-AMPK was up e-
gula ed in KO mice, bu i was wi hin he no mal ange in KO-AOX
animals (Figu e 4E). Mo eo e , PGC-1ap o ein amoun was also
inc eased in KO e sus WT and AOX, while i was no mal in KO-
AOX muscle samples (Figu e 4F). Finally, quan i a i e ansc ip
766 Cell Me abolism 28, 764–775, No embe 6, 2018
analysis showed dec eased exp ession o se e al genes ela ed
o he espi a o y chain in KO-AOX e sus KO mice, con i ming
ha mi ochond ial biogenesis is diminished in he p esence o
AOX (Figu e 4G).
AOX Impai s ROS Signaling in KO Mice
Since AOX p e en s excessi e ROS p oduc ion, we easoned
ha i migh in e e e wi h ROS signaling in cIV-de icien muscle.
Thus, we quan i ied ROS p oduc ion in skele al muscle mi o-
chond ia by measu ing hyd ogen pe oxide (H
2
O
2
) p oduc ion.
Succina e-d i en H
2
O
2
p oduc ion was signi ican ly inc eased
in KO compa ed wi h WT and AOX mi ochond ia bu was lowe
in KO-AOX double mu an s han in WT animals (Figu e 5A).
Acco dingly, mi ochond ial aconi ase (ACO2) ac i i y, which is
inhibi ed by H
2
O
2
, was signi ican ly lowe in he KO compa ed
wi h WT and AOX mi ochond ia bu had no mal alues in he
Figu e 3. AOX Impai s he Regene a ion Ca-
paci y o Myo ibe s
(A) Rep esen a i e con ocal 3D z s ack image o
8-week-old muscle ibe s labeled wi h PAX7 ( ed),
MYOD (g een), and DAPI (blue). The image ep e-
sen s a andomly chosen image om ou samples.
Scale ba , 50 mm.
(B) Quan i ica ion o he numbe o posi i e PAX7,
PAX7/MYOD, and MYOD nuclei in muscles o WT,
AOX, KO, and KO-AOX animals (n = 4). Ba s
ep esen means ± SEM. As e isks o e he ba s
indica e s a is ical signi icance e sus WT; o e he
b acke s among indica ed g oups. *p %0.05;
**p %0.01; unpai ed S uden ’s es .
KO-AOX samples (Figu e 5B). Al oge he ,
hese esul s a e consis en wi h AOX be-
ing ac i e only unde s ess condi ions, as
p e iously epo ed (Szibo e al., 2016).
Since AOX equi es a highly educed
CoQ pool o be ac i a ed (D y e al.,
1989), we measu ed he amoun o
educed and oxidized CoQ in ozen skel-
e al muscle o he mice. The ela i e
amoun o educed CoQ was inc eased
in KO e sus WT and AOX mice bu was
compa able wi h WT in he double mu-
an s, con i ming ha AOX e icien ly oxi-
dizes he CoQ pool (Figu e 5C). These e-
sul s suppo he idea ha RET, which is
p omo ed by o e - educ ion o he CoQ
pool, is in ol ed in gene a ing ROS in he
KO model, and ha his phenomenon is
blun ed by AOX.
Inc eased ROS can igge he oxida i e
s ess esponse ia KEAP1/NFE2L2
signaling. Acco dingly, he ansc ip s o
supe oxide dismu ase (Sod2) and glu a-
hione pe oxidase (Gpx1), wo key en-
zymes o he an ioxidan esponse ha
a e NFE2L2 a ge s, we e signi ican ly
inc eased in KO e sus WT and AOX
mice, bu signi ican ly dec eased in KO-
AOX e sus KO animals (Figu e 5D). A simila end was de ec ed
o bo h Ca , encoding ca alase, and Sod1, encoding he cy o-
solic iso o m o supe oxide dismu ase (Figu e 5D). A signi ican
inc ease o he N e2l2 ansc ip was also de ec ed in KO bu
no in KO-AOX samples (Figu e 5D).
Va ious e og ade signals om mi ochond ia can be ac i a ed
unde s ess condi ions associa ed wi h inc eased ROS, ene gy
de iciency, and loss o Dj(Qui o
´s e al., 2016). As low le els o
ATP can ac i a e AMPK, as well as ROS (Rabino i ch e al.,
2017), we assessed he ATP p oduc ion a e. We ound a s ong
bu compa able impai men o ATP syn hesis in bo h KO and
KO-AOX skele al muscle mi ochond ia, using ei he cI- o cII-
linked subs a es (Figu e 5E). In addi ion, he ATP con en was
compa ably lowe han con ols in KO and KO-AOX samples
(Figu e 5F). Likewise, Djwas ma kedly bu compa ably lowe
han con ols in bo h KO and KO-AOX muscle mi ochond ia
Cell Me abolism 28, 764–775, No embe 6, 2018 767

Figu e 4. AOX In e e es wi h Mi ochond ial Biogenesis in KO-AOX Mice
(A) Spec opho ome ic ac i i y o ci a e syn hase (CS)-speci ic ac i i y (n = 5).
(B) m DNA copy numbe by qPCR (n = 8–10).
(C) Wes e n blo and quan i ica ion (n = 5) o TFAM.
(legend con inued on nex page)
768 Cell Me abolism 28, 764–775, No embe 6, 2018
(Figu e 5G). The compa able dec ease o ATP p oduc ion a e,
ATP s eady-s a e le els, and Djin KO and KO-AOX indica es
ha impai ed bioene ge ics is no he main eason o he mo e
se e e pheno ype o KO-AOX mice.
To u he es he hypo hesis ha dec eased ROS p oduc ion
unde lies he agg a a ed pheno ype, we supplemen ed he
d inking wa e o eigh KO mice a e weaning wi h N-ace ylcys-
eine (NAC), a cell-pe meable p ecu so o glu a hione (Viscomi
e al., 2010). The mean su i al was signi ican ly sho e in
ea ed e sus un ea ed animals, and he maximal li espan
was g ossly dec eased in he NAC- ea ed coho , al hough
NAC supplemen a ion also delayed he ea lies dea hs o KO
mice (Figu e 1A).
Au ophagy Is Res o ed in KO-AOX Skele al Muscle
Mi ochond ial biogenesis and au ophagy oge he egula e mi o-
chond ial con en (Pe al a e al., 2016). We measu ed he amoun
o LC3, a ma ke o au ophagosomes, and P62, a ma ke o au-
ophagic ca goes, in skele al muscle samples o he di e en
models. The a io be ween lipida ed, au ophagosome-associ-
a ed LC3 (LC3-II) and non-lipida ed, cy osolic ee LC3 (LC3-I)
was signi ican ly dec eased, whe eas he amoun o P62 was
inc eased, in KO e sus WT and AOX muscles, sugges ing
dec eased au ophagy (Figu e 6A). Con e sely, KO-AOX muscle
showed a ma kedly inc eased LC3-II/LC3-I a io while he le els
o P62 we e compa able wi h WT, indica ing no dec ease in
au ophagy.
Mi ochond ial S ess Ma ke s A e Compa ably
Inc eased in Bo h KO and KO-AOX
A numbe o mi ochond ial pa hways a e ac i a ed in esponse o
mi ochond ial dys unc ion. We measu ed he exp ession a he
p o ein and/o mRNA le el, o se e al componen s o his
in eg a ed s ess esponse, including (1) key playe s o he
mi ochond ial un olded p o ein esponse (UPR
m
)(Shpilka
and Haynes, 2017), such as he bZIP ansc ip ion ac o s Chop
(C/EBP-homologous p o ein), A 4,andA 5 (Fio ese e al.,
2016), and mi ochond ial chape ones HSP60/HSPD1 and
m HSP70/HSPA9 (Dogan e al., 2014); (2) componen s o he
one-ca bon/mi ochond ial ola e cycle, e.g., me hylene e ahy-
d o ola e dehyd ogenase 2 (MTHFD2) (Khan e al., 2017; Kuhl
e al., 2016), and enzymes o p oline biosyn hesis om glu ama e,
e.g., del a-1-py oline-5-ca boxyla e syn hase (ALDH18A1) and
mi ochond ial py oline-5-ca boxyla e educ ase 1 (PYCR1); and
(3) he ib oblas g ow h ac o 21 (Fg 21) and g ow h di e en ia-
ion ac o 15 (Gd 15)mi okines(Leh onen e al., 2016). Inc eased
amoun s o hese ma ke s o hei ansc ip s we e de ec ed in
bo h KO and KO-AOX animals (Figu es 6B–6D). In pa icula ,
Fg 21 mRNA was highly exp essed in KO muscles, and e en
mo e so (260- old) in KO-AOX mice (Figu e 6D). Gd 15 was abou
100- old highe han con ols in KO mice, bu signi ican ly lowe in
KO-AOX, al hough s ill inc eased ela i e o WT animals (Fig-
u e 6D). O he ac o s in ol ed in he in eg a ed s ess esponse,
including A 3 and A 6, ela ed espec i ely o cell dea h and ER-
s ess pa hways, did no change (A 3) o changed o a e y small
ex en (A 6). In e es ingly, he ac i a ion o hese s ess- ela ed
pa hways in ou KO model akes place ia EIF-2alpha and no
ia mTORC1 (Melbe and Haynes, 2018). The le els o mTORC1
a ge euka yo ic ansla ion ini ia ion ac o -binding p o ein 1
(EIF4EBP1), an impo an playe in p o ein syn hesis (Mo i a
e al., 2013) was inc eased in bo h KO and KO-AOX compa ed
wi h WT. Howe e , phospho yla ed e sus unphospho yla ed
EIF4EBP1 a io was dec eased o he same ex en in KO and
KO-AOX e sus WT and AOX mice, indica ing ha mTORC1
signaling, and hus p o ein syn hesis and cell g ow h, was
educed in bo h KO and KO-AOX muscles. In con as , we de-
ec ed inc eased le els o phospho yla ed EIF-2alpha in bo h
KO and KO-AOX mice compa ed wi h bo h WT and AOX li e -
ma es (Figu es S5A–S5C). These indings sugges ha he induc-
ion o majo mi ochond ial s ess esponses co ela es wi h mi o-
chond ial myopa hy and disease p og ession bu AOX did no
signi ican ly modi y hese pa hways, excep inso a as i wo s-
ened he myopa hy, a ec ing he exp ession o ele an endo-
c ine ma ke s, such as Gd 15 (Figu e 6E).
DISCUSSION
While he exp ession o an AOX xenogene on a mu ine WT
backg ound had ha dly any consequences, we he e showed
ha AOX en ains a d ama ic wo sening o he clinical and
biochemical pheno ype in a mouse model o COX-de ec i e
mi ochond ial myopa hy. Unde s ess condi ions, including de-
ec s o cIII o cIV, AOX di ec ly oxidizes CoQ, main aining elec-
on low om NADH and FADH
2
bu abolishing he con ibu ion
o cIII and cIV o he o ma ion o he Dj. The unexpec ed
ou come obse ed in he KO-AOX double mu an model sug-
ges ed wo possible mechanisms: a di ec bioene ge ic ailu e
caused by he exclusion o he p o on pumping ac i i y o cIII
and cIV, o an indi ec e ec consequen o dec eased ROS
signaling and blun ing o mi ochond ial biogenesis. We
excluded he i s possibili y, since no di e ence was de ec ed
in he ATP p oduc ion a e, ATP le els, o Djbe ween KO and
KO-AOX muscle samples, as expec ed since AOX does no
ansloca e p o ons ac oss he mi ochond ial inne memb ane.
Con e sely, ROS p oduc ion was signi ican ly dec eased in
KO-AOX muscle as we e se e al indica o s o mi ochond ial
biogenesis.
Redox signaling con ols a la ge numbe o ansc ip ional
pa hways (Holms o
¨m and Finkel, 2014). Howe e , i s impo -
ance in i o has no been adequa ely in es iga ed. While ou
esul s a gue agains he gene al applicabili y o AOX as a he a-
peu ic ool in mi ochond ial diso de s, hey p o ide solid gene ic
(D) Wes e n blo and quan i ica ion (n = 5) o MRC complexes. Indi idual subuni s and he complexes a e indica ed on he le .
(E) Rep esen a i e wes e n blo o phospho yla ed and o al AMPK.
(F) Wes e n blo and quan i ica ion (n = 5) o PGC-1a.
(G) Rela i e exp ession le els o espi a o y chain ansc ip s (n = 6). Resul s ep esen old inc ease no malized agains WT.
All expe imen s we e pe o med on 8-week-old mice wi h wes e n blo s using skele al muscle homogena es o 8-week-old mice and GAPDH as loading con ol.
Ba s ep esen means ± SEM. As e isks o e he ba s indica e s a is ical signi icance e sus WT; o e he b acke s among indica ed g oups. *p %0.05; **p %
0.01; ***p %0.001; unpai ed S uden ’s es .
Cell Me abolism 28, 764–775, No embe 6, 2018 769
Figu e 5. AOX Impai s ROS Signaling in KO Mice
(A) H
2
O
2
p oduc ion a e caused by RET in isola ed skele al muscle mi ochond ia (n = 4).
(B) Aconi ase2 ac i i y in ozen skele al muscle samples (n = 5).
(C) Pe cen age o educed CoQ in ozen muscle samples.
(D) Rela i e exp ession le els o an ioxidan esponse ansc ip s (n = 6). Resul s ep esen old inc ease no malized agains WT.
(legend con inued on nex page)
770 Cell Me abolism 28, 764–775, No embe 6, 2018
e idence in a mammalian mi ochond ial disease model o he
c ucial pa hophysiological ole o ROS-dependen pa hways as
compensa o y esponses o mi ochond ial dys unc ion, a leas
in skele al muscle, as p e iously sugges ed (Ho n e al., 2017;
Reczek and Chandel, 2015). This has ob ious implica ions in
unde s anding some o he pa hogenic mechanisms o mi o-
chond ial diseases and can po en ially be exploi ed o de elop
e ec i e he apies. Fo ins ance, an ROS-based mechanism
can unde pin he o ma ion o agged- ed ibe s (RRFs), a
mo phological hallma k o mi ochond ial myopa hy. RRFs a e
de e mined by he segmen al accumula ion o dys unc ional
mi ochond ia along he muscle syncy ium, especially in he
sub-sa colemmal egion. Howe e , he mechanism leading o
hei o ma ion is poo ly unde s ood. We p opose ha ROS
can be a majo signal inducing he local p oli e a ion o mi o-
chond ia, in an a emp o compensa e hei unc ional de ec
h ough he ac i a ion o a mi ochond iogenic p og am by he
su ounding nuclei. Fu he mo e, ou indings suppo he idea
ha he induc ion o mi ochond ial biogenesis, o ins ance by
ac i a ing he SIRT1-and/o AMPK-dependen PGC-1aaxis, o
o he mi ochond iogenic pa hways, could be a a ional and
po en ially e ec i e app oach in he he apy o mi ochond ial
diseases. In e es ingly, he e is inc easing e idence ha AMPK
is di ec ly egula ed by ROS ei he h ough oxida ion and S-glu-
a hionyla ion o cys eines 299 and 304 in he asubuni , leading
o he ac i a ion o he enzyme, o oxida ion o cys eines 130 and
174 in he asubuni , esul ing in i s inac i a ion (Shao e al.,
2014). An addi ional mechanism po en ially con ibu ing o he
dec ease o mi ochond ial con en is he main enance o au o-
phagic lux in KO-AOX, as sugges ed by an inc eased LC3-II/
LC3-I a io and no maliza ion o he P62 le el, which is impai ed
in KO muscle. Fu he in es iga ion will be needed o unde s and
whe he his e ec is di ec ly ela ed o dec eased ROS p oduc-
ion o is an indi ec consequence o he ac i a ion o s ess e-
sponses. The in eg a ed s ess esponse is indeed ac i a ed in
bo h KO and KO-AOX mice. This seems o be con olled by
he ac i a ion o he ansla ion ini ia ion ac o EIF-2alpha by
phospho yla ion ope a ed by GCN2 o o he kinases unde mi o-
chond ial s ess condi ions (Melbe and Haynes, 2018) and as
obse ed in o he mouse models o mi ochond ial dys unc ion
(Sei e ling e al., 2016). In con as , mTORC1 signaling, which
was shown o egula e mi ochond ial in eg a ed s ess esponse
in a model o impai ed m DNA eplica ion (Khan e al., 2017), is
inhibi ed in bo h KO and KO-AOX mice as indica ed by educed
le els o phospho yla ed EIF4EBP1. In e es ingly, we ound ha
wo mi okines ecen ly in oduced as bioma ke s o mi ochon-
d ial myopa hies (Leh onen e al., 2016) we e bo h highly
inc eased in KO and KO-AOX mice. Howe e , while Fg 21 was
much highe in KO-AOX han in KO, Gd 15 was educed in
KO-AOX compa ed wi h KO, implying a s ess-dependen
modula ion o he la e , as sugges ed by p e ious wo k (Chung
e al., 2017).
An in iguing inding o ou s udy is he dec ease in he numbe
o cen alized nuclei in KO-AOX e sus KO muscle ibe s.
Cen alized nuclei a e a p ima y sign o muscle egene a ion
(Yin e al., 2013). Du ing muscle egene a ion, sa elli e cells,
which cons i u e he esiden pool o s em cells in skele al mus-
cle, unde go a p ocess o ac i a ion, cha ac e ized by p oli e a-
ion and mig a ion o he si e o damage, whe e hey di e en ia e
in o myocy es and e en ually use wi h he exis ing myo ibe s.
Speci ic ansc ip ion ac o s a e exp essed du ing his p ocess,
including Pax7,MyoD, and Myogenin. Ou da a sugges ha
ROS in luence his pa hway, simila o wha is obse ed in o he
popula ions o s em cells. Myogenic di e en ia ion is associa ed
wi h high ROS le els, mainly due o he concomi an induc ion o
mi ochond ial biogenesis (L’hono e
´e al., 2014). An ioxidan de-
enses a e ac i a ed a he same ime o p e en cellula damage
(La ella and Pu i, 2014). The dec eased numbe o cen alized
nuclei and educed PAX7- and MYOD-posi i e nuclei in KO-
AOX muscles indica e ha sa elli e cells a e p esen bu canno
di e en ia e in o myo ubes, hus impai ing he capaci y o
epai / egene a ion o he myo ibe s. This e ec is likely o
ha e an impo an ole in he obse ed wo sening o he pheno-
ype in KO-AOX mice.
The majo i y o ongoing clinical ials o mi ochond ial dis-
eases a e based on he use o an ioxidan s, s emming om he
assump ion ha excessi e ROS p oduc ion causes oxida i e
damage o cellula componen s. Howe e , he e has hi he o
been only a minimal in es iga ion o he ex en o ROS p oduc-
ion and oxida i e damage in mi ochond ial diseases in i o,
and he a ailable da a a e con adic o y. Fo ins ance, in he mu-
a o mouse (T i uno ic e al., 2004), no signs o oxida i e dam-
age ha e been de ec ed in pos -mi o ic issues (T i uno ic
e al., 2005), while ex ensi e ROS damage has been shown in
eplica ing cells (Hamalainen e al., 2015). Ou da a using bo h
AOX and NAC highligh a po en ial isk associa ed wi h an ioxi-
dan use, o in e e ing wi h compensa o y ROS signaling, a
leas in mi ochond ial myopa hies. This obse a ion is in ag ee-
men wi h p e ious epo s showing ha an ioxidan s can ha e
dele e ious e ec s on plu ipo en s em cells wi h impai ed mi o-
chond ial unc ion (Hamalainen e al., 2015). Howe e , i should
be no ed ha NAC and AOX ac on edox homeos asis h ough
di e en mechanisms, as AOX p e en s excess ROS p oduc ion
by inc easing elec on low along he espi a o y chain and is ex-
p essed ansgenically h oughou emb yonic de elopmen ,
while NAC, a glu a hione p ecu so , was adminis e ed sho ly a -
e weaning, hus explaining he obse ed di e ences in hei
su i al cu es.
In ou model, COX de iciency leads o accumula ion o he
educed o m o CoQ, and hus o excess p oduc ion o supe ox-
ide anion ia RET. By e-ac i a ing he elec on low along he
espi a o y chain, AOX no malizes he CoQ pool and abolishes
he inc eases in RET and ROS p oduc ion. Ou da a indica e
ha his e ec , in u n, p ecludes he ac i a ion o a numbe o
(E) ATP syn hesis lux in skele al muscle mi ochond ia in he p esence o cI-linked subs a es (py u a e, mala e, glu ama e) + ADP o cII-linked subs a e
(succina e, o enone) + ADP (n = 4).
(F) ATP con en in ozen skele al muscle samples (n = 6–8).
(G) Analysis o mi ochond ial memb ane po en ial using sa anin in isola ed skele al muscle mi ochond ia (n = 4).
All expe imen s we e pe o med on 8-week-old mice. Ba s ep esen means ± SEM. As e isks o e he ba s indica e s a is ical signi icance e sus WT; o e he
b acke s among indica ed g oups. *p %0.05; **p %0.01; ***p %0.001; ****p < 0.0001; unpai ed S uden ’s es .
Cell Me abolism 28, 764–775, No embe 6, 2018 771
METHOD DETAILS
His ochemis y, Immuno luo escence and Imaging
Fo his ochemical analysis, skele al muscles we e ozen in isopen ane p e-cooled wi h liquid ni ogen. 8 mm hick sec ions we e
s ained o COX and SDH ac i i y. Fo SDH analysis six images pe sample we e acqui ed andomly in places o mixed in ensi y ibe s
keeping he same ligh in ensi y and exposu e se ings using an Axio Obse e Z1 wi h ApoTome 2 (Ca l Zeiss L d.), composed by a
Zeiss 10x ApoPlan objec i e and an AxioCam ICc1 came a. Images we e acqui ed using Zen P o so wa e and analysed wi h Fiji
(% o o al a ea) (Schindelin e al., 2012) keeping he same h eshold o all samples.
Analysis o c oss sec ional a ea and cen alized nuclei was pe o med on hema oxylin and eosin (H & E)-s ained sec ions using Fiji
on ou samples/geno ype (600 ibe s/sample).
Fo immuno luo escence, slides we e ixed in 4% PFA o 10 min, washed in PBS and hen incuba ed in 0.2% i on in PBS o
15 min. A e washing in PBS, sec ions we e incuba ed in blocking solu ion (5% no mal goa se um, 2% BSA, 1:40 M.O.M. blocking
eagen in PBS) o 1 h a RT and hen incuba ed wi h p ima y an ibodies dilu ed in DAKO An ibody diluen wi h backg ound educing
componen s o 1 h a RT. Slides we e washed in PBS o 15 min and incuba ed o 1 h a RT wi h Alexa Fluo seconda y an ibodies
(1:300 in DAKO An ibody diluen wi h backg ound educing componen s). Sec ions we e washed in PBS and moun ed wi h P olong
Diamond An i ade wi h DAPI (In i ogen).
Fo PAX7/MYOD analysis i e images we e acqui ed andomly o each slide using a D agon ly Spinning Disk imaging sys em
(Ando Technologies L d.), composed by a Nikon Ti-E mic oscope, Nikon 20x ApoPlan objec i e and an Ando Ixon EMCCD
came a. The z-s acks we e acqui ed using Fusion so wa e (Ando Technologies) and he 3D images analysed using Ima is so -
wa e (Bi plane) c ea ing spo s su aces o each channel (MYOD and PAX7) keeping he same condi ions o all he samples. A
su ace was c ea ed using he au o luo escence o he issue o measu e he a ea and he numbe o posi i e nuclei was no mal-
ized pe a ea.
Fibe yping analysis was calcula ed om en i e muscle sec ions using a Tile image acqui ed using he D agon ly Spinning Disk as
p e iously desc ibed o MYOD/PAX7. The mo phome ic analysis was pe o med using Ima is so wa e c ea ing su aces o each
channel.
No blinding was used o any o he his ological/immuno luo escence analysis. No samples we e excluded om analysis.
Analysis o Mi ochond ial Enzyme Ac i i ies
Skele al muscle samples we e snap- ozen in liquid ni ogen and homogenized in 10 mM phospha e bu e (pH 7.4). The spec opho-
ome ic ac i i ies o cIV and ci a e syn hase (CS) we e measu ed as desc ibed (Bugiani e al., 2004). In b ie , complex IV ac i i y was
measu ed by ollowing educ ion o cy och ome c a 550 nm a 37C. 10 mg mi ochond ia we e incuba ed in a mix con aining 10 mM
KH
2
PO
4
, pH 7.2 and be ween 5 and 50 mM cy och ome c.
Ci a e syn hase ac i i y was measu ed a 30C by incuba ing 10 mg mi ochond ia in a eac ion mix u e con aining 125 mM T is–
HCl, 100 mM DTNB (5,50-di hiobis(2-ni obenzoic acid)) and 300 mM ace yl coenzyme A. The eac ion was ini ia ed by he addi ion o
500 mM oxaloace a e, and DTNB educ ion a 412 nm measu ed o 2 min.
Analysis o Body Composi ion
Nuclea magne ic esonance (NMR) was employed o de e mine he body a con en o li e animals using he NMR Analyze Min-
ispec mq7.5 (B uke Op ik, E lingen, Ge many).
Real-Time Quan i a i e Polyme ase Chain Reac ion
RNA was isola ed om skele al muscles using TRIzol eagen (In i ogen, 15596026) acco ding o manu ac u e ’s ins uc ions,
ea ed wi h DNAse (DNA- ee Ki , Ambion) and subsequen ly e e sely ansc ibed wi h he Omnisc ip e e se ansc ip ion ki
(Qiagen, 205113). Fg 21 (Mm00840165_g1), Gd 15 (Mm00442228_m1), Gapdh (Mm99999915_g1) and Hp (Mm00446968_m1)
p obes we e ob ained om TaqMan Assay-on-Demand ki s (Applied Biosys ems).
All he o he ansc ip s, including m DNA con en , we e analyzed by SYBR G een eal- ime PCR. Samples we e adjus ed o o al
RNA con en by Hp ,Gapdh o in he case o m DNA con en , RNaseP. Rela i e exp ession o mRNAs was de e mined using a
compa a i e me hod (2
DDCT
). P ime sequences a e p o ided in Table S1.
Wes e n Blo Analysis
P o eins, ex ac ed om homogenized skele al muscle samples, we e sepa a ed by dena u ing NuPAGE 4%–12% Bis-T is gels.
The p ima y an ibodies we e used in dilu ions ecommended by he supplie s. The lis o an ibodies is included in he Key Resou ces
Table.
Mi ochond ia Isola ion and H
2
O
2
P oduc ion
Mi ochond ia we e isola ed om skele al muscle as desc ibed (F ezza e al., 2007). B ie ly, small pieces o skele al muscles we e
incuba ed in ice-cold PBS/10 mM EDTA and 0.05% ypsin o 30 minu es on ice. A e cen i uga ion a 200 g o 5 min a 4C,
e3 Cell Me abolism 28, 764–775.e1–e5, No embe 6, 2018

he pelle was esuspended in IBM1 bu e (67 mM Suc ose, 50 mM KCl, 50 mM T is-HCl pH 8, 10 mM EDTA, 0,2% BSA ee om
a y acids, pH 7.4). The pieces we e homogenized by hand, cen i uged a 700 g o 10 minu es a 4C. The supe na an was u he
cen i uged a 8000 g o 10 minu es a 4C o pelle mi ochond ia, which was esuspended in IBM2 bu e (250 mM suc ose, 0.3 mM
EGTA-T is, 10 mM T is-HCl, pH 7.4).
H
2
O
2
p oduc ion a e was measu ed a 37C using 130 mg o mi ochond ia dilu ed in 2 ml o mi ochond ial espi a ion bu e
(120 mM suc ose, 50 mM KCl, 20 mM T is–HCl, 4 mM KH
2
PO
4
, 2 mM MgCl
2
, 1 mM EGTA, 1 mg/ml a y-acid- ee BSA, pH 7.2)
in an Oxyg aph-2k using O2k-Fluo LED2-Module (OROBOROS INSTRUMENTS, Innsb uck, Aus ia). The a ionale behind he
me hod is moni o ing he oxida ion o he luo ogenic indica o Amplex Red Reagen (Li e Technologies, A12222) in he p esence
o ho se adish pe oxidase (Sigma-Ald ich, P8250) and supe oxide dismu ase (Sigma-Ald ich, S8409) (K umschnabel e al., 2015).
The inal concen a ions o Amplex Red, ho se adish pe oxidase and supe oxide dismu ase in he incuba ion medium we e
10 mM, 4 U/ml and 5 U/ml, espec i ely. H
2
O
2
p oduc ion a e measu emen s we e ini ia ed by succina e ( inal concen a ion
10 mM) addi ion as a subs a e o cII. In a sepa a e expe imen , a s anda d cu e was ob ained by adding known amoun s o
H
2
O
2
o he assay medium in he p esence o he eac an s. The H
2
O
2
p oduc ion a e was de e mined om he slope o a plo o
he luo ogenic indica o e sus ime.
Fo (cyanide- esis an ) espi a ion, a e addi ion o cI-linked o cII-linked subs a es, including 1 mM ADP, 1 mM KCN o 2.5 mg/ml
oligomycin was added in o he Oxyg aph-2k chambe s.
Mi ochond ial Aconi ase Ac i i y
Mi ochond ial aconi ase 2 ac i i y was measu ed by Aconi ase Ac i i y Assay Ki (Sigma-Ald ich, MAK051) acco ding o he manu-
ac u e ’s p o ocol.
QH
2
/Q Measu emen s
In o de o measu e he p opo ion o bo h educed and oxidized CoQ, a apid lipid ex ac ion was pe o med. B ie ly, 10 o
15 mg o muscle issue was homogenized wi h a manual mic opes le in 20 mM po assium phospha e bu e pH 7.5, and deb is
we e disca ded by maximum speed cen i uga ion, a 4C. 100 ml o sample was mixed by o ex wi h 330 mlo n-p opanolin
hep esenceo 0.5mMb-me cap oe hanol. Sample deb is we e disca ded a e spin a maximum speed and clean lipid
ex ac s we e immedia ely injec ed in o a e e se-phase Beckmann 166 HPLC sys em, equipped wi h a C18 column (5 mm,
150 x 4.6 mm) and column o en se up a 40C. Mobile phase, low a e and g adien se ings we e as desc ibed p e iously
(Rod ı
´guez-Aguile a e al., 2017). CoQ de ec ion was pe o med by a Coulochem III ESA elec ochemical de ec o linked o he
HPLC sys em and condi ioning cell was se up be o e injec ion al e in o de o main ain he oxida ion s a e o he sample
analy es.
Measu emen o ATP Syn hesis Flux and ATP Con en
ATP p oduc ion a e was assessed as desc ibed (Mou ie e al., 2015). B ie ly, isola ed skele al muscle mi ochond ia (65 mg/ml) we e
suspended in he mi ochond ial espi a ion bu e (see ‘‘Measu emen o H
2
O
2
p oduc ion’’). A e addi ion o ADP (1 mM), succina e
(2 mM) and o enone (10 nM) o addi ion o ADP (1 mM), py u a e (10 mM), glu ama e (5 mM) and mala e (5 mM), aliquo s we e
collec ed e e y 20 seconds and p ecipi a ed in 7% HClO
4
/25 mM EDTA, cen i uged a 16,000 g
max
o 10 minu es, and hen neu al-
ized wi h 2 M KOH and 0.3 M MOPS. The ATP con en in hese samples was de e mined wi h ATP de e mina ion ki (Molecula
P obes, A22066) acco ding o he manu ac u e ’s ins uc ions. In a pa allel expe imen , oligomycin (2.5 mg/ml p o ein) was added
o he mi ochond ial suspension o de e mine he nonoxida i e ATP syn hesis a e.
ATP con en in ozen skele al muscle samples was measu ed as desc ibed (Fe nandez-Ma cos e al., 2012) using he same ki as
abo e. B ie ly, issues we e weighed and lysed in 600 ml o 6% ( / ) pe chlo ic acid, and cen i uged a 4,000 g
max
o 10 minu es a
4C. 500 ml o he supe na an we e neu alized wi h 200 ml o 2.5 M KOH, and p ecipi a e was emo ed by cen i uga ion a 4,000 g
max
o 5 minu es a 4C. Luci e ase ac i i y was measu ed wi h a lag ime o 1 second and an in eg a ion ime o 3 seconds using a
GloMax 96 Mic opla e Luminome e (P omega).
Mi ochond ial Memb ane Po en ial Measu emen
Mi ochond ial memb ane po en ial was assessed as desc ibed (Chowdhu y e al., 2015). In b ie , Oxyg aph-2k was used wi h O2k-
Fluo escence LED2 module equipped wi h il e se s o sa anin (exci a ion a 495 nm and emission a 587 nm). Sa anin (Sigma-
Ald ich, S2255) dissol ed in dis illed wa e up o a inal concen a ion o 2.5 mM. Isola ed skele al muscle mi ochond ia (65 mg/ml)
we e suspended in he mi ochond ial espi a ion bu e (see ‘‘Measu emen o H
2
O
2
p oduc ion’’) including 2.5 mM sa anin. The
memb ane po en ial was acked li e wi h he addi ion o ADP (2.5 mM), py u a e (5 mM), glu ama e (10 mM) and mala e (5 mM)
o ADP (2.5 mM), succina e (10 mM) and o enone (0.5 mM). In a sepa a e expe imen , a s anda d cu e was ob ained by adding
known amoun s o sa anin o he assay medium in he p esence o mi ochond ia. The signal was hen no malized (0–1) o de ec
changes a e addi ion o subs a es (2.5 mM sa anin se as 1).
Cell Me abolism 28, 764–775.e1–e5, No embe 6, 2018 e4
QUANTIFICATION AND STATISTICAL ANALYSIS
Fiji so wa e (Schindelin e al., 2012) was used o quan i ica ion. S a is ical analyses we e pe o med using G aphPad P ism 7 o Mac
OS X (G aphPad So wa e; La Jolla, CA, USA).
Unpai ed S uden ’s - es was used o pai wise compa isons o independen expe imen al g oups. Kaplan–Meie dis ibu ion and
one sample - es we e used o su i al analysis. No pa icula me hod was used o de e mine whe he he da a me assump ions o
he s a is ical app oach. G ubb’s es was used o de e mine ou lie s. E o ba s ep esen s anda d e o o he mean (S.E.M.).
e5 Cell Me abolism 28, 764–775.e1–e5, No embe 6, 2018