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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