Supplementary Materials for · Fig. S2. Short chronic ISO administration (7 days) promotes ROS...
Transcript of Supplementary Materials for · Fig. S2. Short chronic ISO administration (7 days) promotes ROS...
Supplementary Materials for
Ablation of the stress protease OMA1 protects against heart failure in
mice
Rebeca Acin-Perez,* Ana Victoria Lechuga-Vieco, Maria del Mar Muñoz,
Rocío Nieto-Arellano, Carlos Torroja, Fátima Sánchez-Cabo, Concepción Jiménez,
Andrés González-Guerra, Isabel Carrascoso, Cristiane Benincá, Pedro M. Quiros,
Carlos López-Otín, José María Castellano, Jesús Ruíz-Cabello,
Luis Jesús Jiménez-Borreguero, José Antonio Enríquez*
*Corresponding author. Email: [email protected] (J.A.E.); [email protected] (R.A.-P.)
Published 28 March 2018, Sci. Transl. Med. 10, eaan4935 (2018)
DOI: 10.1126/scitranslmed.aan4935
The PDF file includes:
Fig. S1. Histological analysis of brain, liver, and kidney sections upon work
overload.
Fig. S2. Short chronic ISO administration (7 days) promotes ROS increase and
mitochondrial crista remodeling.
Fig. S3. Modulation of mitochondrial remodeling and calcium homeostasis.
Fig. S4. Analysis of heart performance upon pressure overload.
Fig. S5. Assessment of cardiac function after HFD administration.
Other Supplementary Material for this manuscript includes the following:
(available at
www.sciencetranslationalmedicine.org/cgi/content/full/10/434/eaan4935/DC1)
Table S1 (Microsoft Excel format). Number of samples used in the experiments
shown in Figs. 1 to 7.
www.sciencetranslationalmedicine.org/cgi/content/full/10/434/eaan4935/DC1
Fig. S1. Histological analysis of brain, liver, and kidney sections upon
work overload. H&E staining of wild type brain, liver and kidney sections in
the different conditions.
control ISO
Bra
in (
hyp
oth
ala
mu
s)
Supp. Fig.1. Acin-Perez et al
Liv
er
Kid
ne
y
2.5 mm2.5 mm
1 mm 1 mm
500 µm 500 µm
Fig. S2. Short chronic ISO administration (7 days) promotes ROS increase and mitochondrial
crista remodeling. (A) Rate of ATP synthesis in heart mitochondria driven by glutamate plus malate
(G+M, left panel) or succinate (Succ, right panel) in wild type and OMA1KO in the different conditions
(biological replicates: control, n=4; ISO, n=6. Every biological replicate were measured in duplicate). (B)
OPA1 processing pattern due to the action of both OMA1 and Yme1l proteases. Description of the bands
used for quantification of OPA1 processing. (C) Analysis of OPA1 processing by Western Blot and
quantification. (D) Production of ROS measured by H2O2 released from mitochondria using Amplex Red
(biological replicates: control, n=4; ISO, n=6). (E-F) DHE and DAPI staining in heart sections and
quantification of superoxide levels relative to nuclei staining (biological replicates: WT control, n=3; WT
ISO, n=3; OMA1KO control, n=2, OMA1KO ISO, n=3. For each sample 4 independent fields from two
different slides were analyzed). (G) Determination of mitochondrial SOD (mt-SOD, KCN insensitive)
activity in heart homogenates. (biological replicates: control, n=4; ISO, n=8). Every biological replicate
has been measured in duplicate. (H) Spectrophotometric measurement of ROS sensitive aconitase activity
in heart mitochondria (biological replicates: control, n=4; ISO, n=6). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗∗ P <
0.0001. Gels for Western blots are representative of three independent gels including biological
replicates.
WT OMA1KO0
50
100
150
nmol
ATP
G+M
/min
/mg
vs c
ontro
l
*
WT OMA1KO0
50
100
150
nmol
ATP
Suc
c /m
in/m
g vs
con
trol
****
Control ISO
WT OMA1KO0.0
0.5
1.0
1.5
OP
A1
L /O
PA
1 S
vs
OP
A1
tota
l
****
6
7
8
9
10
11
rate
of R
OS
(Am
plex
Red
) A
U/h
****
WT OMA1KO0
50
100
150
mtS
OD
act
ivity
vs
cont
rol
****
WT OMA1KO0
50
100
150
200
acon
itase
IU/m
g vs
con
trol
**
C ISO
C ISO
Mw
Mw
WT
OMA1KO
OPA1
SDHA
130 -
75 -
Opa1L: sp1l+sp1lOpa1s: sp7so+sp7sy+ sp1so
A B
CD
130 -
75 -
OPA1
SDHA
Control ISO Control ISO
Control ISO
Control ISO
DAPI DHE Merged
OM
A1K
O +
ISO
WT
+ IS
OO
MA
1KO
WT
WT OMA1KO
E
G
H
0.0
0.1
0.2
0.3
DH
E/D
API
Fluo
resc
ence
Inte
nsity 0.4 * *
*Control ISO
WT OMA1KO
F
Fig. S3. Modulation of mitochondrial remodeling and calcium homeostasis. (A) Masson´s trichrome
staining of heart sections of wild type and OMA1KO after ISO+MQ administration. (B-C) Analysis of
OPA1 processing and MCU levels in wild type heart mitochondria when fission was blocked using
Mdivi1 for 3d. (D) TEM analysis of wild type heart mitochondria after 3d in the indicated treatments.
Scale bars correspond to 2M. (E) Analysis of OPA1 processing and MCU levels in wild type and
OMA1KO heart mitochondria after caffeine administration. (F) Analysis of OPA processing and MCU
levels in wild type and OMA1KO heart mitochondria when RyR2 activity was inhibited by Dantrolene
(Dant). In B and C-E-F; SDHA, core2 and Tom20 are used as loading control. Gels for Western blots are
representative of three independent gels including biological replicates.
Supp. Fig. 3. Acin-Perez et al
AOMA1KOWT
C
D
E
F WT OMA1KO
Control ISO Dant
Control ISO Dant
Control ISO Dant
Control ISO Dant
OPA1
SDHA
SDHA
core2
MCU
Tom20
Control ISO+Mdivi1
Control
OPA1
MCU
SDHA
core2
WT
WT OMA1KO
Control Caffeine
Control Caffeine
Caffeine
Caffeine
OPA1
SDHA
SDHA
core2
MCU
Tom20
SDHA
ISO ISO+Mdivi1
Con
Con
ISO+Dant
ISO+Dant
ISO+Dant
ISO+Dant
ISO
100
5
MCU Core 2
Control ISO ISO + Mdivi1
SDHA
ControlCaffeine
MCU
Tom 20
0
4
35
2 µm
2 µm
2 µm 2 µm
2 µm
2 µm 2 µm
ISO
+M
div
i1M
div
i1IS
OD
MS
O
B
ISO+MQ ISO+MQ
1 mm1 mm
MCU
Tom 20
2 µm
Fig. S4. Analysis of heart performance upon pressure overload. (A) Echocardiography analysis of
%EF and heart rate in mice subjected to the indicated treatments (biological replicates: WT control, n=22;
WT AngII, n=10; WT AngII+MQ, n=8; OMA1KO control, n=22; OMA1KO AngII, n=11; OMA1KO
AngII+MQ, n=6). (B) Cardiac hypertrophy evaluated by heart weight vs body weight (HW/BW)
treatments (biological replicates: WT control, n=10; WT AngII, n=12; WT AngII+MQ, n=4; OMA1KO
control, n=15; OMA1KO AngII, n=13; OMA1KO AngII+MQ, n=4). (C) H&E staining of wild type brain,
liver and kidney sections in the indicated situations conditions. (D) Serum creatinine in control and AngII
wild type and OMA1KO treated mice (biological replicates, n=4). (E) Serum urea in control and AngII
wild type and OMA1KO treated mice (biological replicates: WT control, n=2; WT AngII, n=5; OMA1KO
control, n=3; OMA1KO AngII, n=3). (F) Masson’s trichrome staining of transverse aortic sections under
the indicated treatments. (G) Collagen quantification of the aortic sections in F. (H) Media to lumen ratio
in aortic sections. In G and H biological replicates: WT control, n=3; WT AngII, n=5; OMA1KO control,
n=3; OMA1KO AngII, n=3. * P < 0.05; ∗∗ P < 0.01
OMA1KO0
20
40
60
80
100
% E
F
300
400
500
600
700
HR
3
4
5
6
7
8
HW
/BW
(mg/
g)
**
control AngII AngII+MQA B controlAngIIAngII+MQ
Brain Liver Kidney
Con
trol
AngI
I
C
D
OMA1KO0.0
0.1
0.2
0.3
0.4
0.5
seru
m c
reat
inin
e (m
g/dl
)
ControlAngII
*
20
25
30
35
40
45
seru
m u
rea
(mg/
dl) *
ControlAngII
E
2.5 mm 100 µm
2.5 mm 100 µm 250 µm
250 µm
2.5 mm
2.5 mm
Con
trol
AngI
I
WT
OMA1KO
F
500 µm
500 µm
500 µm
500 µm
500 µm
500 µm
500 µm
500 µm
Con
trol
AngI
I
G
0
100
200
300
AngIIControl
**
WT OMA1KO
Col
lage
n/pe
rimet
er
0.0
0.2
0.4
21
AngIIControl****
WT OMA1KO
Tuni
ca Media/Lumen
H
WT OMA1KOWT OMA1KOWT
WT
OMA1KOWT
Fig. S5. Assessment of cardiac function after HFD administration. (A) Weekly weight
gain profile in mice fed with HFD. GTT (B) and ITT (C) analysis in mice after being in
HFD for 8-10 weeks. For A-C, n=7 biological replicates. Echocardiography determination
of heart mass (V mass corrected, D) and cardiac output (CO, E) in mice after 8-10 weeks in
HFD (biological replicates: WT SD, n=5; WT HFD, n=6; OMA1KO SD, n=4; OMA1KO
HFD, n=4). (F) Assessment of ATP synthesis driven by glutamate+malate (G+M) or
succinate (Succ) in heart mitochondria isolated from mice subjected to HFD. (biological
replicates: WT SD, n=4; WT HFD, n=6; OMA1KO SD, n=4; OMA1KO HFD, n=4. HFD
biological replicates were measured in duplicate). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗∗ P < 0.0001.
0 2 4 6 80
5
10
15
20
time (weeks)
weig
ht gain
vs t0
C57:HFD
OMA1KO:HFD
C57:SD
OMA1KO:SD
****
WT OMA1KO
0
10
20
30
40
CO
(m
l/m
in)
SDHFD
0 50 1000
200
400
600
time (mins)
Glu
cose (
mg/d
l) **** **** ******** ****
**
WT OMA1KO
0
50
100
150
LV
mass c
orr
ecte
d
** **
SD
HFD
0
50
100
nm
olA
TP
/m
in/m
g v
s S
D G+M Succ
WT OMA1KO WT OMA1KO
SD HFD SD HFD SD HFD SD HFD
0 20 40 600.0
0.5
1.0
1.5
time (mins)
ITT
FI glu
cose (
mg/d
l) v
s t0
****
A B C
D E F
Supp Fig 5.- Acín-Pérez et al.