Mitochondrie et muscle lisse

Vladimir Veksler

Energy production

Main mitochonrial functions

at maximal stress

0.5 – 1 µmol ATP/min/g

ATP consumption rate in smooth and striated muscle

smooth muscle striated (cardiac) muscle

at rest ≈ 30 µmol/min/g

smooth muscle energetics

Ionic homeostasis

K+, Ca++

Energy production

ATP, PCr

Ca2+

K+

Apoptosis

Synthesis of

steroid hormones

ROS production

H2O2, O2. -

Thermogenesis

UCP

Main mitochonrial functions

Oxygen probe Cell

proliferation

Depolarization of the mitochondrial membrane potential

inhibits IP3-evoked Ca2+ release

MicrocirculationVolume 20, Issue 4, pages 317-329, 10 MAY 2013 DOI: 10.1111/micc.12039http://onlinelibrary.wiley.com/doi/10.1111/micc.12039/full#micc12039-fig-0004

Depolarization of the mitochondrial membrane potential with CCCP inhibits Ca2+ release from an IP3R cluster

MicrocirculationVolume 20, Issue 4, pages 317-329, 10 MAY 2013 DOI: 10.1111/micc.12039http://onlinelibrary.wiley.com/doi/10.1111/micc.12039/full#micc12039-fig-0003

Role of mitochondria in hypoxic pulmonary vasoconstriction

Mitochondrion is an oxygen sensor

MitochondriaROS production

modulation

Vascular smooth

muscle contraction

Hypoxia

Pulmonary arterial hypertension (PAH)

• Severe and progressive vascular disease that frequently leads to right heart failure

and premature death

• Common hallmark: vascular remodeling, due to excessive proliferation and

resistance to apoptosis of pulmonary arterial smooth muscle cells (PASMCs)

Two mitochondrial mechanisms

seem to be involved:

1 - mitochondrial dynamics

2 – mitochondrial biogenesis

(PGC-1αααα)

Mitochondrial fission: DRP1

Mitochondrial fusion: mitofusins

Fusion proteins

TM: transmembrane

HR heptad repeat (coiled coil)

OPA1: protein responsible for autosomal dominant optic atrophy

Possible roles for OPA1.

OPA1 (in pink) anchored to the inner membrane (IM) could controls its dynamic in coordination with the

dynamic outer membrane (OM) by interacting either directly or indirectly with Mfn2 (in grey).

Oligomerized OPA1 could structure the cristae or could control the cristae junction opening, sequestering

cytochrome c (in green) in intra-cristae compartment.

Cyt C

OPA1

Mfn2

Olichon et al., 2006

Excessive fission in human pulmonary arterial hypertension (PAH) pulmonary artery smooth muscle cells

Marsboom et al., 2012

Marsboom et al., 2012

Dynamin-related protein-1 (DRP1) inhibition reduces proliferationof smooth muscle cells

Marsboom et al., 2012

Therapeutic benefit of mitochondrial fission inhibition by Mdivi-1 in the chronic hypoxia model

pulmonary artery

acceleration time tricuspid annular plane systolic excursion

MFN2 Down-regulation Increases Proliferation in Normal PASMC

Ryan et al., 2013

Mitofusin-2 (MFN2) overexpression reduces mitochondrial fragmentation and proliferation in pulmonary arterial smooth muscle

Ryan et al., 2013

MF

N1/C

itra

te s

yn

thase r

ati

o

0,0

0,6

1,2

1,8

2,4

3,0

Control iPAH

OP

A1 (

80)/

Cit

rate

syn

thase r

ati

o

**

0,0

0,4

0,8

1,2

1,6

Control iPAH

*

OP

A1 (

100)/

Cit

rate

syn

thase r

ati

o

Control iPAH

CS

β-actin

OPA1-100 KDa

OPA1- 80 kDa

Expression of mitochondrial fusogenic proteins in human pulmonary artery smooth muscle cells

0,0

0,4

0,8

1,2

1,6

2,0

2,4

Control iPAH

MF

N2/C

itra

te s

yn

thase r

ati

o

0,0

0,5

1,0

1,5

2,0

Control iPAH

*

Ryan et al., 2013

OPA1 downregulation dramatically decreases pulmonary artery smooth muscle cell proliferation and increases apoptosis

C

OP

A1

mR

NA

le

ve

ls(2

-(C

t g

en

e o

f in

tere

st

–C

t r1

8S

)

0

0,2

0,4

0,6

0,8

1

Scramble-Seq OPA1-SiRNA

controls

IPAH

**

§§§§§

0

500

1000

1500

2000

2500

3000

Scrambl-Seq OPA1-SiRNA Scrambl-Seq OPA1-SiRNA

Basal FCS (5%)

[3H

]th

ym

idin

e i

nc

orp

ora

tio

n(c

pm

)

**

*

§§

§

Ap

op

toti

cc

ell

s(%

)

H2O2

0

20

40

60

80

Basal scrambl-seq OPA1-siRNA Basal scrambl-seq OPA1-siRNA

Control

iPAH

Basal

§§

§§

*

§§§

§§§

Control iPAH

OPA1-deficient mice have neither hypoxia-induced pulmonary artery hypertension nor right ventricle hypertrophy

0

10

20

30

40

OPA1 +/+ +/- +/+ +/-

Normoxia Hypoxia

0

10

20

30

OPA1 +/+ +/- +/+ +/-

Normoxia Hypoxia

RV

SP

(m

mH

g)

0

20

40

60

80

100

NM PM FM

Normoxic OPA1 (+/+) mice

Normoxic OPA1 (+/-) mice

Hypoxic OPA1 (+/+) mice

Hypoxic OPA1 (+/-) mice

Pu

lmo

na

ry V

es

sels

(%

)NS

*** ******

NS

NS*** ***

***

NS

RV/(LV

+S), %

NM – non muscularPM – partially muscularFM – fully muscular

vehicle PDGF

PGC1α

tubulin

0

0,2

0,4

0,6

0,8

1

1,2

1,4

PGC-1alpha

Re

lati

ve

ba

nd

de

ns

ity

vehiclePDGF

*

Proliferating smooth muscle cells have downregulated PGC-1αααα

Mitochondrial biogenesis

Metabolic fitness

Energy depletion

Redox alterations

Phosphorylation Deacetylation

interplay

scramble siSIRT1

BrdU incorporation assay

*

β actin

SIRT1

scramble siSIRT1

CNTR PASMCs

rd 1 0,16

Effects of SIRT1 downregulation on human PASMC proliferation

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

CNTR PASMCs

Re

lati

ve

ab

so

rba

nc

e (

A4

50

nm

)

SIRT1 downregulation favours human PASMC proliferation.

Effects of STAC-3 on human PAH PASMC proliferation

SIRT1 activator 3 (stac-3)

SIRT1 activation by 10 µM stac-3 strongly inhibits human PAH PASMC proliferation.

WST-1 assay BrdU assay

0

0,2

0,4

0,6

0,8

1

1,2

1,4

PAH PASMC

Re

leti

ve

ab

so

rba

nc

e (

A4

50

nm

)vehicle stac-3 10µM

*

0

0,2

0,4

0,6

0,8

1

1,2

1,4

PAH PASMCs

Re

lati

ve

ab

so

rba

nc

e

(A4

40

nm

-A6

90

nm

)

*

Sirtuin1

activation

PGC-1αααα

deacetylation

(activation)

↑↑↑↑ mitochondrial

antioxidant activity

↑↑↑↑ mitochondrial

biogenesis

↓↓↓↓ mitochondrial

fragmentation

?

inhibition of smooth muscle

cell hyperproliferation

Putative mechanisms of sirtuin1-dependent inhibition of smooth muscle proliferation

Take home message

• Abnormal pulmonary artery vascular smooth muscle proliferation is associated with alterations in expression of proteins involved in mitochondrial dynamics and apoptosis (DRP-1, mitofusin, OPA-1, PGC-1α).

• Correction of these proteins (DRP-1 and OPA1 down-regulation; mitofusin and PGC-1α up-regulation) could be a therapeutic target in pulmonary artery hypertension.

U-769 INSERM Châtenay-Malabry, France

Giada ZURLOFrédéric JOUBERT Christophe LEMAIRE Matthieu RUIZ Anne GARNIER Renée VENTURA-CLAPIER

Participants

Tartu University EstoniaAllen KAASIK

Johannes-Gutenberg Universität Mainz GermanyMarcel ALAVI

U-999 INSERM, Hospital Marie-Lannelongue, Le Plessis-Robinson, France

Mohamed IZIKKISaadia EDDAHIBI