MOLECULAR AND CELLULAR BIOLOGY, Dec. 2004, p. 10933–10940 Vol. 24, No. 24
0270-7306/04/$08.00⫹0 DOI: 10.1128/MCB.24.24.10933–10940.2004
Copy igh © 2004, Ame ican Socie y o Mic obiology. All Righ s Rese ed.
The Mi ochond ial SDHD Gene Is Requi ed o Ea ly Emb yogenesis,
and I s Pa ial De iciency Resul s in Pe sis en Ca o id Body Glomus
Cell Ac i a ion wi h Full Responsi eness o Hypoxia
Jose´ I. Pi ua ,
1
C. Osca Pin ado,
2
Pa icia O ega-Sa´enz,
1
Ma a Roche,
1
and Jose´Lo´pez-Ba neo
1
*
Labo a o io de In es igaciones Biome´dicas, Hospi al Uni e si a io Vi gen del Rocío,
1
and Cen o de
P oduccio´n y Expe imen acio´n Animal, Uni e sidad de Se illa,
2
Se ille, Spain
Recei ed 19 July 2004/Re u ned o modi ica ion 3 Sep embe 2004/Accep ed 23 Sep embe 2004
The SDHD gene encodes one o he wo memb ane-ancho ing p o eins o he succina e dehyd ogenase
(complex II) o he mi ochond ial elec on anspo chain. This gene has ecen ly been p oposed o be in ol ed
in oxygen sensing because mu a ions ha cause loss o i s unc ion p oduce he edi a y amilia pa aganglioma,
a umo o he ca o id body (CB), he main a e ial chemo ecep o ha senses oxygen le els in he blood. He e,
we epo he gene a ion o a SDHD knockou mouse, which o ou knowledge is he i s mammalian model
lacking a p o ein o he elec on anspo chain. Homozygous SDHD
ⴚ/ⴚ
animals die a ea ly emb yonic s ages.
He e ozygous SDHD
ⴙ/ⴚ
mice show a gene al, noncompensa ed de iciency o succina e dehyd ogenase ac i i y
wi hou al e a ions in body weigh o majo physiological dys unc ion. The esponsi eness o hypoxia o CBs
om SDHD
ⴙ/ⴚ
mice emains in ac , al hough he loss o an SDHD allele esul s in abno mal enhancemen o
es ing CB ac i i y due o a dec ease o K
ⴙ
conduc ance and pe sis en Ca
2ⴙ
in lux in o glomus cells. This CB
o e ac i i y is linked o a sub le glomus cell hype ophy and hype plasia. These obse a ions indica e ha
cons i u i e ac i a ion o SDHD
ⴙ/ⴚ
glomus cells p ecedes CB umo ans o ma ion. They also sugges ha ,
con a y o p e ious belie s, mi ochond ial complex II is no di ec ly in ol ed in CB oxygen sensing.
Mi ochond ial complex II (succina e dehyd ogenase [SDH])
plays majo biological oles in he K ebs cycle and he mi o-
chond ial elec on anspo chain, oxidizing succina e o u-
ma a e and ans e ing elec ons o he ubiquinone pool (9,
12). SDH has also been p oposed o ha e an an ioxidan unc-
ion, p o ec ing agains supe oxide p oduc ion in mi ochon-
d ia, and o pa icipa e in oxygen (O
2
) sensing (3, 7, 22, 27).
De iciencies o any o he ou subuni s o SDH (A, B, C, and
D) a e associa ed wi h a b oad spec um o human diseases,
anging om myo- and encephalopa hies o aging and umo
o ma ion. Mu a ions in SDHA, he ma ix- aced la op o ein,
esul in dys unc ion o he ene gy me abolism, e.g., Leigh
synd ome o exe cise in ole ance, whe eas mu a ions ei he in
he i on-sul u p o ein SDHB o in SDHC and SDHD, he
memb ane-ancho ing p o eins ha con ain one heme and a e
essen ial o ubiquinone binding, a e mo e o en associa ed
wi h pheoch omocy oma and pa aganglioma (1, 22, 23). Spe-
ci ically, mu a ions in SDHD a e he main cause o amilia
he edi a y pa aganglioma (PGL) (3) a mos ly benign, highly
ascula ized umo o he ca o id body (CB). SDHD is he i s
umo supp esso gene iden i ied which encodes a mi ochon-
d ial p o ein.
The CB is he main a e ial chemo ecep o ha senses blood
O
2
concen a ion. I is a highly i iga ed bila e al o gan, lo-
ca ed a he bi u ca ion o he ca o id a e y and de i ed om
he neu al c es . Upon exposu e o acu e hypoxia, neu osec e-
o y CB glomus cells elease ansmi e s which ac i a e a e -
en senso y ibe s connec ed wi h b ain s em cen e s o elici
hype en ila ion and sympa he ic ac i a ion (see e e ences 14
and 15 o e iews). Simila o CBs o indi iduals exposed
ch onically o hypoxia (11, 25), PGL umo s display cellula
hype plasia o anaplasia in he absence o hypoxic s imulus (3).
Mo eo e , he p e alence o pa agangliomas in indi iduals
wi h SDHD mu a ions inc eases in popula ions li ing a high
al i udes (2). Thus, i has been p oposed ha SDHD pa ici-
pa es in O
2
sensing and ha PGL umo s a e induced by
de ec s in he de ec ion o blood O
2
le els (3, 4, 7, 22).
Ad ance in he s udy o SDH unc ion is, howe e , ham-
pe ed by he lack o mammalian gene ic models o SDH de i-
ciency. To de e mine he possible in ol emen o he mi o-
chond ial complex II in O
2
sensing and he pa hophysiology o
he edi a y CB PGL, we ha e gene a ed a knockou mouse
ca ying a null allele o he SDHD gene. He e we desc ibe he
majo gene al e ec s o SDHD de iciency and he physiological
ea u es o CB glomus cells om SDHD knockou mice.
MATERIALS AND METHODS
Gene a ion o SDHD
ⴙ/ⴚ
knockou mice. A cDNA homolog o he human
succina e dehyd ogenase D gene (accession numbe BF161694) was used as a
p obe o he sc eening o a lambda FIXII ec o -based genomic lib a y o
mouse s ain 129S J by hyb idiza ion o phage-in ec ed bac e ial plaques. Iso-
la ed genomic DNA was used o making a a ge ing cons uc consis ing o a neo
casse e ha eplaced exons 2, 3, and 4 o he SDHD gene, lanked by wo a ms
o homolog DNA (4.0 and 4.4 kb, espec i ely) ha allow o homologous
ecombina ion. The a ge ing cons uc was elec opo a ed in o mouse emb y-
onic s em (ES) cells. Clones esis an o 0.2 mg o Gene icin G418 pe ml we e
selec ed and analyzed o p ope a ge ing a he SDHD locus. Male chime as
we e gene a ed and ma ed wi h 129S J wild- ype emales, which ga e F
1
o -
sp ing wi h he e ozygous SDHD
⫹/⫺
indi iduals. Animal ca e and expe imen a-
ion we e acco ding o he ins i u ional animal ca e commi ee guidelines.
* Co esponding au ho . Mailing add ess: Labo a o io de In es iga-
ciones Biome´dicas, Edi icio de Labo a o ios, 2
a
Plan a, Hospi al Uni-
e si a io Vi gen del Rocío, A enida Manuel Siu o s/n, E-41013,
Se ille, Spain. Phone: (34)-954-617090 o (34)-955-012648. Fax: (34)-
954-617301. E-mail: [email p o ec ed].
10933
Nucleic acid analysis. To al DNA om ES cells, oe ips, and emb yos was
p epa ed by incuba ion in 20 mM T is-HCl (pH 8.5)–5 mM EDTA–1% sodium
dodecyl sul a e–400 mM NaCl–0.1 mg o p o einase K pe ml a 57°C o 2 h. Fo
Sou he n blo ing, 20 ␮g o genomic DNA was diges ed and loaded on o a
T is-ace a e-EDTA–aga ose gel. Fo issue RNA analysis, o gans we e dissec ed
and homogenized in TRIzol eagen acco ding o he manu ac u e ’s ins uc-
ions. Fo No he n blo ing, 10 o 15 ␮g o RNA pe sample was loaded on o a
o maldehyde-aga ose gel. Elec opho esis and ans e o nucleic acids o Hy-
bond-N nylon memb anes we e pe o med by s anda d me hods. Hyb idiza ion
was done a 42°C in Ul a-Hyb hyb idiza ion bu e . Gel-pu i ied DNA p obes
we e adiolabeled wi h [␣-
32
P]dCTP by using he Redip ime ki .
Emb yo analysis. To ob ain SDHD
⫺/⫺
emb yos, he e ozygous SDHD
⫹/⫺
pa -
en s we e ma ed. Females we e sac i iced a di e en days a e copula ook
place o dissec ion o he u e us, a e which ma e nal issue was ca e ully
emo ed, and emb yos we e dissec ed o DNA analysis. In o de o disc imina e
be ween he wild- ype and he mu an allele a h ee-p ime app oach was de-
signed. P ime s 5⬘-TCAGTGACAACGTCGAGCAC-3⬘,5⬘-CAAGGTCGGAA
CCCAGAGAT-3⬘, and 5⬘-ATAGCCAGCCAGGTAGTTCC-3⬘gi e p oduc s o
1.8 kb o he wild ype and 1.25 kb o he mu an SDHD allele.
Isola ion o mi ochond ia. Isola ion o mi ochond ia om mouse issues was
pe o med as epo ed p e iously (6). Tissues we e dissec ed and washed in
ice-cold phospha e-bu e ed saline. The en i e p ocess o mi ochond ion isola-
ion was ca ied ou a 4°C, wi h samples kep on ice. O gans we e cu wi h
scisso s in small agmen s in 2 ml o homogeniza ion medium (320 mM su-
c ose–1 mM EDTA–10 mM T is-HCl [pH 7.4] o li e and kidney and 75 mM
suc ose–225 mM so bi ol–1 mM EGTA–0.1 a y acid- ee bo ine se um albu-
min–10 mM T is-HCl [pH 7.4] o b ain and hea ). Cells we e b oken wi h 10
o 15 s okes in a Dounce homogenize wi h a mo o -d i en pes le. The homog-
ena e was cen i uged a 4,000 pm o 6 min in a mic ocen i uge, and mi o-
chond ion-con aining supe na an was collec ed. Mi ochond ia we e spun down
by cen i uga ion o 10 min a 13,000 pm, washed wice wi h medium B (250
mM suc ose, 2 mM HEPES, 0.1 mM EGTA), and esuspended in a inal olume
o abou 250 ␮l o medium B. The mi ochond ion suspension was aliquo ed (40
␮l), lash ozen in liquid ni ogen, and kep a ⫺80°C un il used. The p o ein
concen a ion was de e mined acco ding o B ad o d’s me hod, wi h samples
dilu ed in 0.05% sodium dodecyl sul a e.
Measu emen o ac i i ies o mi ochond ial complexes. Ac i i ies o mi ochon-
d ial complexes we e de e mined in a Beckman DU-640 spec opho ome e , as
desc ibed by Bi ch-Machin and Tu nbull (5) wi h sligh modi ica ions. All e-
agen s we e pu chased om Sigma unless o he wise indica ed. Fo mi ochon-
d ial complex I and II ac i i ies, 30 o 50 ␮g o p o ein was assayed a 30°C.
Samples we e dilu ed 1:4 in he assay eac ion bu e (25 mM KH
2
PO
4
[pH 7.2],
5 mM MgCl
2
, 3 mM po assium cyanide, 2.5 mg o bo ine se um albumin pe ml)
and eeze- hawed h ee imes wi h liquid ni ogen be o e he assay. Fo mi o-
chond ial complex I ac i i y, he o enone-sensi i e NADH dehyd ogenase ac-
i i y was measu ed as he dec ease in abso bance a 340 nm, e e ed o 425 nm,
due o oxida ion o 130 ␮M NADH (Roche) in p esence o 3.6 ␮M an imycin
and 130 ␮M ubiquinone-1. Abso bance was measu ed o 2 min be o e and a e
addi ion o 5 ␮M o enone o he eac ion mix u e. Du ing his pe iod he a e
o dec ease o abso bance was linea . Di e ences be ween a es we e conside ed
o de e mining ac i i y due o mi ochond ial complex I. To e alua e he mi o-
chond ial complex II ac i i y, succina e dehyd ogenase ac i i y was measu ed o
a pe iod o 2 min as he dec ease in he abso bance a 600 nm due o he
educ ion o 50 ␮M 2,6-dichlo ophenol-indophenol (DCPIP) coupled o educ-
ion o 130 ␮M ubiquinone-1. The eac ion was ca ied ou in p esence o 3.6 ␮M
an imycin, 5 ␮M o enone, and 10 mM succina e.
Ca o id body immunohis ochemis y. Bi u ca ions con aining ca o id bodies
we e dissec ed and ixed in o malin (Sigma) a 4°C o 16 h. Tissues we e
dehyd a ed and pa a in embedded, and 10-␮m slices we e ob ained by using an
RM2125 mic o ome (Leica Mic osys ems). Immunohis ochemis y was pe -
o med acco ding o s anda d p ocedu es. Fo de ec ion o glomus cells, issues
we e immunos ained wi h a abbi polyclonal an i y osine hyd oxylase (Pel-
F eez). A e immunode ec ion wi h pe oxidase-conjuga ed seconda y an ibody,
issue samples we e coun e s ained wi h hema oxylin.
Ampe ome ic eco dings in ca o id body slices. Mouse ca o id body slicing
and moni o ing o single-cell sec e ion was done acco ding o he same basic
p ocedu es desc ibed o a s (18, 20). Slices we e con inuously pe used wi h a
solu ion con aining 117 mM NaCl, 4.5 mM KCl, 23 mM NaHCO
3
, 1 mM MgCl
2
,
2.5 mM CaCl
2
, 5 mM glucose, and 5 mM suc ose. The no moxic solu ion was
bubbled wi h a gas mix u e o 5% CO
2
, 20% O
2
, and 75% N
2
(O
2
ension o
⬃150 mm Hg). The hypoxic solu ion was bubbled wi h 5% CO
2
and 95% N
2
o
each an O
2
ension in he chambe o ⬃20 mm o Hg. All expe imen s we e
done a a chambe empe a u e o ⬃36°C. The cumula i e sec e ion signal is he
sum o he ime in eg al o successi e ampe ome ic e en s. The sec e ion a e
(in em ocoulombs pe minu e) was calcula ed as he amoun o cha ge ans-
e ed o he eco ding elec ode du ing a gi en ime pe iod.
Pa ch clamp eco dings in dispe sed CB glomus cells. Mouse ca o id bodies
we e incuba ed o 20 min in an enzyme solu ion (1 ml o phospha e-bu e ed
saline wi h 0.6 mg o collagenase II, 0.3 mg o ypsin, 40 ␮l o elas ase I, 0.3 mg
o bo ine se um albumin, and 10 ␮l o CaCl
2
oma5mMs ock solu ion) a
37°C, and he cells we e mechanically dispe sed by using i e-polished Pas eu
pipe es. Cells we e pla ed on sli e s o glass co e slips ea ed wi h poly-L-lysine
and kep in cul u e medium (Dulbecco’s modi ied Eagle’s medium wi h 10%
e al bo ine se um, 1% an ibio ics, 1% L-glu amine, and 84 ␮U o insulin pe ml).
Mac oscopic ionic cu en s we e eco ded by using he whole-cell con igu a ion
o he pa ch clamp echnique as adap ed o ou labo a o y (20, 24). The s anda d
ex acellula solu ion con ained 117 mM NaCl, 4.5 mM KCl, 23 mM NaHCO
3
,
1 mM MgCl
2
, 2.5 mM CaCl
2
, 5 mM glucose, and 5 mM suc ose, and he pH was
adjus ed by bubbling wi h 5% CO
2
. The s anda d in e nal solu ion (inside he
pipe e and he cell) con ained 80 mM K-glu ama e, 30 mM KCl, 20 mM KF, 1
mM EGTA, and 4 mM Mg-ATP (pH 7.3) adjus ed wi h KOH. Cell capaci y was
es ima ed om he ime in eg al o capaci a i e cu en ansien s eco ded by
applica ion o 2-ms depola izing pulses o 20 mV om he holding po en ial o
⫺80 mV. To calcula e cell size, we assumed a cons an memb ane-speci ic ca-
paci y o 1 ␮F/cm
2
. Conduc ance- ol age cu es we e ob ained om he no mal-
ized ampli ude o inwa d K
⫹
ail cu en s eco ded a he end o depola izing
pulses. Fo hese expe imen s, we used an ex e nal solu ion wi h 30 mM KCl
(NaCl was educed o 87 mM o main ain cons an he osmolali y). All o he
elec ophysiological expe imen s we e done a oom empe a u e (⬃22°C).
S a is ical analysis. Unless o he wise speci ied, da a a e exp essed as means ⫾
s anda d e o s o he means, wi h he numbe o expe imen s (n) indica ed.
S a is ical analysis was pe o med wi h he unpai ed S uden es . A P alue o
⬍0.05 was conside ed s a is ically signi ican .
RESULTS
Gene a ion o he SDHD
ⴙ/ⴚ
knockou mouse. In o de o
gene a e a mu an allele o he SDHD gene, we i s pe o med
a sc eening o a lib a y o mouse genomic DNA, based on
lambda phage, wi h a cDNA p obe con aining he en i e
SDHD coding sequence. Two agmen s o app oxima ely 16
and 19 kb we e isola ed. Res ic ion analysis and sequencing
con i med, by compa ison wi h he Ensembl Genome B owse
(www.ensembl.o g), ha hey bo h con ained he en i e geno-
mic DNA co esponding o he SDHD gene. These DNA ag-
men s we e used o making he a ge ing cons uc . The gen-
e a ion o he mu an SDHD allele is shown schema ically in
Fig. 1A. The a ge ing cons uc con ained he p oka yo ic
neomycin esis ance gene (neo) lanked by genomic DNA ho-
mologous o he SDHD locus. ES cells we e elec opo a ed
wi h his a ge ing cons uc and selec ed o Gene icin esis-
ance. ES cell clones we e isola ed and analyzed by Sou he n
blo ing o p ope in eg a ion o he non unc ional allele (Fig.
1B). Homologous ecombina ion eplaced he wild- ype allele
by he mu an allele lacking exons 2, 3, and 4. The selec ed
clones we e used o blas ocys injec ion o gene a e chime as
om which we ob ained he e ozygous F
1
o sp ing, which we e
also iden i ied by Sou he n blo ing (Fig. 1C).
Emb yo le hali y o SDHD
ⴚ/ⴚ
mice. To da e, we ha e geno-
yped 152 animals bo n om ma ing be ween he e ozygous
pa en s, o which 50 (33%) we e SDHD
⫹/⫹
and 102 (66%)
we e SDHD
⫹/⫺
, wi h no SDHD
⫺/⫺
animals de ec ed. These
a e he ela i e equencies expec ed o a Mendelian- ype
inhe i ance wi h le hali y o homozygous mu an emb yos.
The analysis o emb yos om he e ozygous p egnan emales
ma ed wi h he e ozygous males indica es ha SDHD
⫺/⫺
ani-
mals die a ea ly s ages o o ganogenesis. Figu e 2 shows ha
a 7.5 days pos concep ion (dpc) all de eloping emb yos om
10934 PIRUAT ET AL. MOL.CELL.BIOL.
a ma ing had he same appea ance (Fig. 2A), whe eas a 9.5
dpc app oxima ely one- ou h o emb yos we e s alled a a
p e ious s age (Fig. 2B). To u he analyze he emb yonic
de elopmen o hese mice, we dissec ed he emb yos om he
ma e nal decidua a 7.5 dpc, among which some we e clea ly
a es ed se e al hou s be o e (Fig. 2C and D). The dissec ed
emb yos we e analyzed by PCR (Fig. 2E). The use o speci ic
p ime s o bo h he wild- ype and mu an alleles showed ha
hose emb yos wi h no mal appea ance (Fig. 2C) co espond-
ed o ei he he wild- ype (SDHD
⫹/⫹
; one 1.8-kb band) o he
he e ozygous (SDHD
⫹/⫺
; wo bands o 1.8 and 1.25 kb) mice,
whe eas he s alled emb yos (Fig. 2D) co esponded o he
homozygous SDHD
⫺/⫺
(1.25-kb band) mice. These obse a ions
indica e ha SDHD
⫺/⫺
mu an s die be o e 7.5 dpc in he emb yo
de elopmen , coinciding wi h he ea ly s age o o ganogenesis.
Func ional analysis o he SDHD gene in he e ozygous an-
imals. We analyzed he e ec o he emo al o one copy o he
SDHD gene on bo h he exp ession o he emaining allele and
he ac i i y o mi ochond ial complex II. In SDHD
⫹/⫺
animals
a gene al dec ease o he s eady-s a e le el o he SDHD
mRNA was obse ed in hea , b ain, kidney, and li e (Fig.
3A). Consis en ly, in mi ochond ia isola ed om he same is-
sues, succina e dehyd ogenase ac i i y was ⬃50% lowe han
no mal, whe eas mi ochond ial complex I ac i i y emained
unchanged (Fig. 3B). Hence, he de ici o mi ochond ial com-
plex II ac i i y co ela es wi h he absence o one o he unc-
ional alleles o he SDHD gene, indica ing ha a compensa-
o y up- egula ion o he emaining wild- ype allele does no
ake place. Despi e he gene al dec ease o complex II ac i i y,
SDHD
⫹/⫺
animals did no show signs o g oss physiologic al-
e a ions. A 6 mon hs, he animal weigh o males was 30.8 ⫾
0.7g(n⫽13) o SDHD
⫹/⫹
animals e sus 31 ⫾0.6g(n⫽22)
o SDHD
⫹/⫺
animals; ha o emales was 23.4 ⫾1.1g(n⫽
11) o SDHD
⫹/⫹
animals e sus 22.7 ⫾0.5g(n⫽13) o
SDHD
⫹/⫺
animals.
CB glomus cell sec e o y ac i i y in esponse o hypoxia.
The mos equen mani es a ion o SDHD de iciency in hu-
mans is he he edi a y CB PGL (3, 4). PGL umo s display
cellula hype plasia and CB hype ophy, simila o wha oc-
cu s in indi iduals exposed o ch onic hypoxia (11, 25). The e-
o e, i has been p oposed ha SDHD pa icipa es in O
2
sens-
ing and ha he ul ima e cause o PGL umo s is a de ec in
sensing en i onmen al O
2
le els (3, 4, 7, 22). We ha e es ed in
young adul mice whe he a pa ial SDHD de ici and he e-
duc ion o mi ochond ial complex II ac i i y al e CB unc ion
by moni o ing ca echolamine sec e ion om single glomus
cells in slices o he whole o gan. Simila o he case o a CB
slices (18, 20), he esponse o hypoxia o glomus cells in mouse
CB slices was cha ac e ized by a sha p and e e sible bu s o
sec e o y e en s (Fig. 4A). This esponse was main ained, o
FIG. 1. (A) Gene a ion o a null allele o he SDHD gene. Gene a ge ing eplaced he wild- ype allele by a non unc ional allele lacking exons
2, 3, and 4. The wo homologous agmen s o genomic DNA (4.0 and 4.4 kb) lanking he p oka yo ic neomycin esis ance (neo) gene allow o
homologous ecombina ion. B, BamHI; H, HindIII. (B) Selec ed ES cell clones we e analyzed o co ec gene a ge ing a he SDHD locus by
Sou he n blo ing. Diges ed DNA was hyb idized wi h he indica ed p obes, gi ing addi ional bands o he indica ed sizes in he he e ozygous
(⫹/⫺) ES clone wi h espec o he homozygous wild ype (⫹/⫹). (C). Sou he n blo geno yping o SDHD knockou mice.
VOL. 24, 2004 SDHD KNOCKOUT MOUSE 10935
e en augmen ed, in SDHD
⫹/⫺
mice (Fig. 4B and C), indica ing
ha a pa ial de iciency o complex II ac i i y does no al e
glomus cell esponsi eness o hypoxia. Howe e , spon aneous
CB ac i i y unde no moxic condi ions was inc eased by ⬃2.4-
old (P⬍0.05) in SDHD
⫹/⫺
animals compa ed wi h wild- ype
li e ma es (Fig. 4D). The highe es ing exci abili y o CB
cells in SDHD
⫹/⫺
animals also explained he sligh inc ease o
he hypoxic esponse. The spon aneous sec e o y ac i i y o
SDHD
⫹/⫺
glomus cells was e e sibly abolished by blockade o
Ca
2⫹
channels wi h Cd
2⫹
(Fig. 4E), hus sugges ing ha i was
due o pe sis en ex acellula Ca
2⫹
in lux h ough memb ane
channels (18, 20, 24).
Elec ophysiological and mo phological cha ac e iza ion o
CB glomus cells in SDHD
ⴙ/ⴚ
mice. To analyze in u he de ail
he molecula changes unde lying CB ac i a ion in SDHD
⫹/⫺
mice, we s udied he K
⫹
cu en s in pa ch-clamped glomus
cells, as i is known ha in his p epa a ion K
⫹
channels eg-
ula e memb ane po en ial and cellula exci abili y (see e e -
ence 20 and e e ences he ein). Rep esen a i e amilies o K
⫹
cu en s om SDHD
⫹/⫹
and SDHD
⫹/⫺
glomus cells a e illus-
a ed in Fig. 5A and B. The a e age K
⫹
cu en densi y- and
conduc ance- ol age ela ionships a e shown in Fig. 5C and D,
espec i ely. In wild- ype glomus cells he inc ease in K
⫹
cu -
en ampli ude induced by memb ane depola iza ion showed a
cha ac e is ic sa u a ion a posi i e memb ane po en ials (Fig.
5A and C). This e lec s a K
⫹
cu en componen media ed by
ol age- and Ca
2⫹
-ac i a ed K
⫹
channels whose p obabili y o
being open dec eases a memb ane ol ages nea he appa en
Ca
2⫹
equilib ium po en ial (16, 21). In e es ingly, unde he
expe imen al condi ions o ou s udy, his cu en componen
almos disappea ed in glomus cells om SDHD
⫹/⫺
mice (Fig.
5B). In SDHD
⫹/⫺
glomus cells, he K
⫹
cu en - ol age ela-
ionship was almos linea (Fig. 5C), and he o al K
⫹
cu en
densi y dec eased (3.55 ⫾0.63 pA/␮m
2
[n⫽11] o SDHD
⫹/⫹
mice and 2.96 ⫾0.11 pA/␮m
2
[n⫽8] o SDHD
⫹/⫺
mice a
⫹20 mV). In addi ion, close inspec ion o he mac oscopic K
⫹
cu en eco dings indica ed ha hei ac i a ion h eshold
inc eased in SDHD
⫹/⫺
glomus cells (compa e he cu en
eco ds ob ained a ⫺30, ⫺20, and ⫺10 mV in Fig. 5A and B).
This was con i med by he analysis o he K
⫹
conduc ance-
ol age ela ionships, which demons a ed a ⬃10-mV displace-
men o posi i e memb ane po en ials o he ac i a ion h esh-
old o K
⫹
channels in SDHD
⫹/⫺
glomus cells (Fig. 5D).
Al hough a comple e biophysical analysis o ion channels in
SDHD
⫹/⫺
glomus cells is ou side he scope o he p esen
s udy, he da a sugges ha he sensi i i y o Ca
2⫹
o K
⫹
channels could be dec eased in SDHD-de icien animals. I is
known ha Ca
2⫹
-ac i a ed K
⫹
channels con ibu e o he es -
ing po en ial o oden glomus cells (20, 26) and ha in mos
issues hese channels ac as coun e egula o y de ices ha
p e en excessi e cell depola iza ion. The e o e, he biophys-
ical changes obse ed in pa ch-clamped SDHD
⫹/⫺
glomus cells
could accoun o he pe sis en sec e o y ac i i y de ec ed by
ampe ome y in CB slices.
The cons i u i e unc ional ac i a ion o CB glomus cells
obse ed in SDHD-de icien mice occu ed wi hou ob ious
FIG. 2. (A and B) Emb yos dissec ed a (A) 7.5 dpc and (B) 9.5 dpc om SDHD
⫹/⫺
emales ma ed wi h SDHD
⫹/⫺
males. No e di e ences
be ween emb yos in panel B, which a e no obse able in panel A. (C and D) Emb yos dissec ed om ma e nal decidua a 7.5 dpc. (E) PCR
analysis o emb yos dissec ed a 7.5 dpc. S alled emb yos show only he band co esponding o he mu an SDHD allele (⫺/⫺; 1.25 kb), whe eas
no mal emb yos show he pa e n expec ed o ei he wild- ype (⫹/⫹; 1.8 kb) o he e ozygous (⫹/⫺; bo h bands) indi iduals.
10936 PIRUAT ET AL. MOL.CELL.BIOL.
o gan enla gemen o g oss his ological modi ica ions. How-
e e , he glomus cell memb ane su ace (es ima ed om he
alues o cell capaci ance measu ed in pa ch-clamped cells)
inc eased om 254 ⫾57 ␮m
2
(n⫽25) in wild- ype o 297 ⫾
17 ␮m
2
(n⫽17) in SDHD
⫹/⫺
glomus cells (P⬍0.05). We ha e
also obse ed a sligh , al hough signi ican , inc ease in he
pe cen age o glomus cells, iden i ied by hei immuno eac i -
i y o y osine hyd oxylase (TH), ha exis in CBs o emale
SDHD
⫹/⫺
(44.3 ⫾1.3%; n⫽4) compa ed wi h SDHD
⫹/⫹
(37.8 ⫾1.5%; n⫽3) animals (Fig. 6). The sub le glomus cell
hype plasia and o gan hype ophy p obably p ecede umo
ans o ma ion, al hough we so a ha e no obse ed CB u-
mo s in SDHD
⫹/⫺
mice. In humans, loss o he e ozygosi y
(LOH) o he wild- ype allele is equi ed o PGL o occu (3,
4). The absence o umo s in he SDHD
⫹/⫺
mice sugges s ha
he induc ion o LOH in CB glomus cells depends on di e en
ac o s in humans and oden s. Howe e , since sys ema ic oc-
cu ence o pa hologies associa ed wi h complex II de iciency
depends on age (23), he possibili y ha CB pa agangliomas
could appea in aged SDHD
⫹/⫺
mice canno be uled ou .
DISCUSSION
We epo he e a mammalian gene ic model o SDH de i-
ciency. To ou knowledge, his is he i s knockou mouse o
a componen o he mi ochond ial elec on anspo chain.
We show ha he comple e lack o he SDHD subuni is le hal
du ing emb yonic de elopmen and ha pa ial SDHD de i-
ciency p oduces a ⬃50% gene alized dec ease o he mi o-
chond ial complex II ac i i y, as de e mined by enzyma ic assay
o succina e dehyd ogenase ac i i y in isola ed mi ochond ia. I
has been shown ha mu a ion o he SDHC homolog in Cae-
no habdi is elegans esul s in ⬎80% educ ion o complex II
ac i i y (10). The null C. elegans mu an is o some ex en
iable due o a small emaining SDH ac i i y. In e es ingly, his
is no he case o he SDHD knockou mouse model, whe e
he comple e absence o he gene p e en s emb yo de elop-
men . Le hali y occu s in he ea ly s ages o o ganogenesis,
mos likely be ween 6.5 and 7.5 dpc, possibly due o he high
ene ge ic demands o emb yos and/o o he gene al me abolic
modi ica ions esul ing om he accumula ion o succina e.
We ha e no obse ed in he he e ozygous SDHD
⫹/⫺
mouse a
gene dosage compensa ion by o e exp ession o he wild- ype
allele. I is no ewo hy ha his unc ional de ici was simila in
FIG. 3. (A) SDHD mRNA exp ession in he indica ed issues. To-
al RNA was hyb idized wi h an SDHD cDNA-con aining p obe. The
Ppia gene, encoding cyclophilin, was used as loading con ol. (B) Mi o-
chond ial succina e-dehyd ogenase (MCII) and NADH-dehyd oge-
nase (MCI) ac i i ies in mouse issues. Da a a e he a e ages om ou
animals pe issue and geno ype. E o ba s indica e he s anda d e o . In
all issues MCII ac i i ies in SDHD
⫹/⫹
and SDHD
⫹/⫺
mice we e signi i-
can ly di e en (P⬍0.05). Numbe s in pa en heses indica e he el-
a i e ac i i y (in pe cen ) in SDHD
⫹/⫺
wi h espec o SDHD
⫹/⫹
mice.
FIG. 4. Sec e o y ac i i y and esponsi eness o hypoxia o CB
cells. (A and B) Sec e o y esponse o hypoxia o CB cells o SDHD
⫹/⫹
(A) and SDHD
⫹/⫺
(B) mice. Spike-like quan al e en s co espond o
ca echolamine elease om indi idual esicles. Cumula i e sec e ion
(in em ocoulombs) o each expe imen is shown. (C) Quan i ica ion
o he sec e o y esponse o low O
2
ension (in em ocoulombs/las
minu e o hypoxia) in SDHD-de icien (5,105 ⫾810 C/min [mean ⫾
s anda d e o ]; n⫽16) and wild- ype (3,539 ⫾603 C/min; n⫽18)
mice (P⫽0.12 by analysis o a iance). (D) Quan i ica ion o he
spon aneous sec e o y ac i i y in CB slices om SDHD
⫹/⫹
(462 ⫾94
C/min [mean ⫾s anda d e o ]; n⫽16) and wild- ype (198 ⫾35
C/min; n⫽18) mice (P⫽0.01 by he K uskal-Wallis es ). (E) Spon-
aneous sec e o y ac i i y in a SDHD
⫹/⫺
CB cells and e e sible block-
ade by applica ion o 0.3 mM ex acellula cadmium.
VOL. 24, 2004 SDHD KNOCKOUT MOUSE 10937

issues wi h ela i ely high (hea o kidney) and low (li e and
b ain) le els o SDH ac i i y, poin ing o a gene alized de ici
o succina e dehyd ogenase ac i i y. S ikingly, his SDH de i-
ciency did no p oduce any g oss mo phological al e a ion o
changes in body weigh in SDHD
⫹/⫺
animals. Whe he he
exis ing mi ochond ial SDH ac i i y is su icien o mee he
me abolic demands o he issues in i o o whe he he e is a
sys emic compensa ion o SDH ac i i y ha is no de ec able in
FIG. 5. Mac oscopic K
⫹
cu en s in pa ch-clamped dispe sed glomus cells o wild- ype and pa ially SDHD-de icien mice. (A and B) Families
o ep esen a i e ou wa d K
⫹
cu en s in SDHD
⫹/⫹
and SDHD
⫹/⫺
glomus cells eco ded du ing 100-ms depola izing pulses eaching memb ane
po en ials be ween ⫺30 and ⫹60 mV in s eps o 10 mV. (C) K
⫹
cu en densi y (o dina e)- e sus- ol age (abscissa) ela ionship in wild- ype and
SDHD
⫹/⫺
glomus cells. Each poin is he a e age om a leas eigh di e en expe imen s. (D) No malized K
⫹
conduc ance (o dina e)- e sus-
ol age (abscissa) ela ionship in wild- ype and SDHD
⫹/⫺
glomus cells. Each poin is he a e age om a leas h ee di e en expe imen s. Da a
poin s we e i ed by an equa ion o he o m G⫽1/[1 ⫹exp(V
1/2
⫺V
m
)/k]. The hal ac i a ion (V
1/2
)(⫺11.7 and ⫺10.6 mV o SDHD
⫹/⫹
and
SDHD
⫹/⫺
, espec i ely) and he slope ac o (k) (14.6 and 12.6 mV o SDHD
⫹/⫹
and SDHD
⫹/⫺
, espec i ely) we e simila o he wo cu es.
Howe e , he ac i a ion h eshold was clea ly highe in SDHD
⫹/⫺
glomus cells (inse in panel D). In panels C and D, e o ba s a e omi ed.
FIG. 6. (A and B) Immunohis ochemis y o SDHD
⫹/⫹
(A) and SDHD
⫹/⫺
(B) ca o id bodies. Tissues we e incuba ed wi h an an ibody o TH
a e which hey we e coun e s ained wi h hema oxylin o coun ing o nuclei. No e he clus e o ganiza ion o glomus cells. (C) Pe cen age o
TH-posi i e cells in he CBs o emale mice (n⫽3 o SDHD
⫹/⫹
;n⫽6 o SDHD
⫹/⫺
). The as e isk indica es s a is ical signi icance (P⬍0.05).
10938 PIRUAT ET AL. MOL.CELL.BIOL.
he mi ochond ial assay a e ques ions ha emain o be in es-
iga ed.
De ailed analysis o CB unc ion in SDHD
⫹/⫺
animals has
allowed us o de ec a pe sis en CB o e s imula ion ha e-
sembles he Ca
2⫹
-dependen CB ac i a ion induced by pha -
macological inhibi ion o he mi ochond ial elec on anspo
chain (18). Elec ophysiological s udies on glomus cells sugges
ha a sub le, bu measu able, dec ease in he densi y and
ac i a ion h eshold o Ca
2⫹
-dependen K
⫹
channels in
SDHD
⫹/⫺
indi iduals could accoun o he pe sis en sec e-
o y ac i i y o CB cells. Howe e , u u e expe imen s should
add ess how de ec s in SDHD ac i i y lead o K
⫹
channel
dys unc ion. In his espec , i is emp ing o specula e ha
o ma ion o eac i e oxygen species a complex II migh play
a ole, as has been p oposed o hypoxic pulmona y ascula-
u e (19), bu his has ye o be shown in SDHD
⫹/⫺
mice.
The unc ional changes obse ed in SDHD
⫹/⫺
CB cells a e
no accompanied by majo his ological modi ica ions. We ha e
de ec ed an inc ease in he SDHD
⫹/⫺
glomus cell memb ane
su ace, which could e eal some deg ee o cell hype ophy.
Immunohis ochemical s udies ha e also shown a small bu
signi ican inc ease in he pe cen age o TH-posi i e cells in
he CBs o emale SDHD
⫹/⫺
mice wi h espec o wild- ype
animals. Whe he his is due o an inc ease in he exp ession o
TH o o p oli e a ion o glomus cells is unknown. Ne e he-
less, inc eases ei he in cell olume and numbe o in TH
exp ession a e hallma ks o hypoxia-induced CB hype ophy
(25). The cons i u i e CB o e s imula ion, which implies a
cons an Ca
2⫹
in lux and he ac i a ion o Ca
2⫹
-dependen
biochemical e en s in CB cells, is p obably one o he mecha-
nisms ha induce i s cellula hype ophy and hype plasia
and subsequen ly umo ans o ma ion in SDHD
⫹/⫺
pa ien s.
The ac ha he edi a y PGL sha es his ological cha ac e is ics
wi h CBs exposed o ch onic hypoxia (3, 4) and he highe e-
quency o SDHD mu an alleles in popula ions li ing a low al i-
udes (2) ha e led o he sugges ion ha SDHD pa icipa es in
CB esponses o lowe ing O
2
ension. We ha e shown ha CB
esponsi eness o hypoxia was no impai ed in he mu an
SDHD
⫹/⫺
mice despi e a 50% gene alized dec ease in SDH ac-
i i y, and pe sis en CB o e s imula ion was obse ed. Al hough
i has no ye been possible o es he esponsi eness o hypoxia
o SDHD
⫺/⫺
glomus cells, he da a a ailable so a sugges ha
SDHD is no di ec ly esponsible o CB acu e O
2
sensing.
Tumo o ma ion in he edi a y CB PGL appea s o equi e
he LOH o he SDHD wild- ype allele. To da e, we ha e no
obse ed an inc eased suscep ibili y o umo igenesis in ou
mouse model. Thus, i is likely ha he biological de e minan s
making he human SDHD egion a ho spo o LOH do no
exis in mouse. On he o he hand, he a e o PGL umo
onse , like ha o mos o he pa hologies associa ed wi h
mi ochond ial complex II de iciencies, depends on age (1, 22,
23). Since he a e age age o he mice in ou colony does no
each 1 yea , we canno exclude he possibili y ha umo s will
appea in aged mice. As in he CB, soma ic SDHD de iciency
could also p oduce cons i u i e subclinical al e a ions in o he
o gans, especially in hose con aining pa aneu al issues, whe e
he dec ease o complex II ac i i y p ecedes he appea ance o
diseases (8, 13, 17). The SDHD knockou mouse model could
help o p o ide p ognos ic insigh s o indi iduals ca ying
mu a ions. In addi ion, i could be a aluable ool o he s udy
o he pa hophysiological mechanisms ha induce LOH-de-
penden umo igenesis and o he pa hologies associa ed wi h
SDHD de iciency in humans.
ACKNOWLEDGMENTS
We hank J. H. Hoeijmake s and R. Fe na´ndez-Chaco´n o he
supply o mouse ES lines and J. A. En íquez o echnical ad ice.
J. Lo´pez-Ba neo ecei ed he Ayuda a la In es igacio´n 2000 o he
Juan Ma ch Founda ion. Resea ch was also suppo ed by The Lilly
Founda ion and by g an s om he Spanish Minis ies o Heal h and o
Science and Technology.
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