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: jaenriquez@cnic.es (J.A.E.); rebeca.acin@cnic.es (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.