diabetic cardiomyopathy does it exist
TRANSCRIPT
Diabetic cardiomyopathy: does it exist ?New insights with Apolipoprotein O: Upregulated in human diabetic heart and promotes mitochondrial dysfunction and lipotoxicity.
Dr Fatima SMIH
TEAM 7: « Obesity and heart failure: molecular and clinical investigations » I2MC INSERM UMR 1048
1 Avenue Jean Poulhes BP 84225 31432 Toulouse cedex 4. France http://www.i2mc.inserm.fr.
Global Projections for the diabetes epidemic: 2010–2030 => Impact on HEART ?
World2010 = 285 million2030 = 439 millionIncrease 54%
Lei Chen et al.Nature Reviews Endocrinology, 2012
METABOLIC or DIABETIC CARDIOMYOPATHY
Diabetic cardiomyopathy, a consequence of metabolic disorders.
Nutritional/Metabolic disorders
(Obesity, diabetes…)
Left Ventricular HYPERTROPH
Y
ventricularDILATATION FIBROSIS
Metabolic Switch to Fat Consumption
LIPOTOXICITY.
STRUCTURAL CHANGES
Enlarged septum
FONCTIONAL CHANGES
Loss of cardiac cells
DIASTOLIC dysfunction
SYSTOLIC dysfunction
Zhou, Yan-Ting et al. (2000) Proc. Natl. Acad. Sci. USA 97, 1784-1789
Accumulation of lipid droplets in the myocardium of diabetic rat
18 week-old fa/fa diabetic / obese rats’ myocardium
Lipidsdroplets
A hallmark of the diabetic cardiomyopathy ?
Accumulation of lipids occurs also in the diabetic Accumulation of lipids occurs also in the diabetic
failing human heartfailing human heart
Sharma et al., Sharma et al., FASEB JFASEB J. 2004. 2004
HF + Diabetic HF + Obese HF
Analysis of Heart Molecular Adaptation to Obesity: Functional Genomics for Dog Heart Transcriptome
Obese dogsWeight : 14,5 à 17, 5 kg
Control dogsWeight : 12 à 14,5 kg
Os = O - N Ns = N - O
RT-PCR (SMART)
Common
sequencesObese
hypertensiveControl
Suppressive Substractive HybridizationEnrichment for differentially expressed genes
LigationIn plasmid
Transformation
RNA extractionRNA fromObese hypertensive RNA from control heart
Bacterial Amplification
9 weeks HFD
Numerical autoradiography (Storm 860®)Computer analysis X-Dot Reader software (COSE) Microarrays analysis of differential expression
Obese hypertensive control
Organized cDNA Bank
(2400 clones)PCR
Spotting
(4800 spots)
Differential hybridization
Obese target
Differential hybridization
Control Target
PCR
DNA sequencing(~200 differentially expressed sequences
Real-time PCR expression verification
Cloning of the human homolog cDNA
Search for homology in DNA sequence database
cDNA Libray enriched for overexpressed genes
cDNA Libray enriched for downregulated genes
Horm
ones
ENER
GET
IC M
ETA
BO
LISM
Prolifération
ION
IC F
LUX
Rem
odeling
Discovery of 11 genesEncoding for unknownProteins:
1) Apolipoprotein
2) Transcription factor
Philip-Couderc et al.,Hypertension 2003
Discovery of a new apolipoprotein: APOO
obese heart Control heart Cloning of the human ortholog« New » protein:
(Philip-Couderc P., Hypertension, 2003)
Differential expression in the obese heart
9 weeks HFD
cDNA SEQUENCING
11 genes with unknown function
We named : APOO - Secreted protein
- Apolipoproteins’ family
APOO Characterization.
Lamant, J. Biol. Chem, 2006
Ø Associated with lipoproteins. Mainly detected in HDL.
(High Density Lipoproteins)
Can associate with lipids.Can form lipoproteins.
ApoODiscoidal particle
Lipids
In vitro
APOO is overexpressed in human diabetic heart
(Lamant, J. Biol. Chem, 2006)
Ø Up-regulated in diabetic hearts.
What is the role of cardiac APOO overexpression?
Diabetic transgenic mice
Overexpressing APOB in heart
Reduced Lipid stores in heartLipid stores in heartReduced Apoptosis
Improved cardiac function Improved cardiac function
Nielsen et al, JBC (2002)
Protection from « Lipoapoptosis »Protection from « Lipoapoptosis »
Physiological Overexpression of APOO in Cardiac Specific Transgenic Mice.
A model of diabetic cardiomyopathy ?
hAPOO cdsMHC promoter
WT
APOO
CALR
APOO-Tg
WT APOO-Tg0
4
8
12 ***
APO
O m
RNA
(A.U
.)
WT APOO-Tg
0
20
40
60 *
PR in
terv
al (m
s)
WT APOO-Tg Depressed heart function
On the contrary to APOB, APOO overexpression in heart is not protective.
High Fat Diet
Turkieh et al. The Journal of Clinical Investigation, 2014
0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8
WT APOO-Tg
0
0.5
1.0
1.5
2.0
**
Cd36
mRN
A (A
.U.)
0
1
2
3
4
Fatp
4 m
RNA
(A.U
.) *
0
2
4
6
Acsl3
mRN
A (A
.U.) *
0
10
20
30
40*
Palm
itoyl
-CoA
x 1
04
(nm
ol/m
in/m
g pr
otei
n)0
2
4
6
8
10 r = 0.7415 P < 0.0001
APOO mRNA (A.U.)
ACSL
3 m
RNA
(A.U
.)
0
3
6
9
12 r = 0.5853 P = 0.0004
FATP
4 m
RNA
(A.U
.)
APOO mRNA (A.U.)0 0.2 0.4 0.6 0.8
0
3
6
9
12 r = 0.5422 P = 0.0024
CD36
mRN
A (A
.U.)
APOO mRNA (A.U.)
Mouse heart
Human heart
APOO induces fatty acid metabolism in mouse and human heart
0
3
6
9
12
15
18
CD36
exp
ress
ion
(A.U
.)
Low APOOexpression
High APOOexpression
***
0
50
100
150
200
FATP
2 ex
pres
sion
(A.U
.)
Low APOOexpression
High APOOexpression
*
Data mining on 107 human heart microarrays from GEO database
APOO induces fatty acids metabolism
TEM of myocardial sections of mouse heart after HFD. WT APOO-Tg
x10,000 x15,000
APOO overexpression induces myocardium disorganization and mitochondrial swelling
-3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.5 1.0 1.5 2.0 2.5 3.0 3.5
-2.0
-1.5
-1.0
-0.5
0.5
1.0
1.5
2.0Oxydative phosphorylation
Mitochondrial dysfunction
P < 0.0001
r = 0.7621 P < 0.0001
r = 0.7757
Mean APOOexpression (A.U.)
Mea
n ge
nes
expr
essio
n (A
.U.)
NDU
FA2
NDU
FA3
NDU
FA6
NDU
FA8
NDU
FAB1
NDU
FB10
NDU
FB11
NDU
FB2
NDU
FB5
NDU
FB8
NDU
FS2
NDU
FS4
NDU
FS7
NDU
FV1
NDU
FV3
SDHA
SDHB
SDHC
SDHD
UQCR
BUQCR
C2UQCR
FS1
UQCR
HCY
C1CY
CSCO
X11
COX1
5CO
X17
COX4
I2CO
X5B
COX6
A2CO
X7A2
COX7
A2L
COX7
CAT
P5C1
ATP5
J0
2
4
6
8
10
12
14
16
18
20
22
C I C II C III C IV C V
Mea
n ex
pres
sion
leve
l (A.
U.)
Low APOOHigh APOO
Data mining on 107 human heart microarrays from GEO database
Cardiac myoblasts cell lines overexpressing APOO
+ cDNA APOO A1/A4/E domain
1981
N-
40 83
-CWT APOO
H9c2 cardiomyoblasts
Expression vectors APOO
Control
Cont. APOO0
5
10
15*
FAM
E x
100
(nm
ol/m
g pr
otei
n)
APOO overexpression promotes lipid overload in cardiac myoblasts
= APOO Cells
CAPOO
Mit.CAPOO
Recom.APOO
APOO
ANT
Cyto.
APOO
Green MitotrackerAPOO/TMR stain Merge
APOO
Recom.APOO
Membranes APOO C
CALR.
What is APOO localization in cardiac myoblasts?APOO Overexpression
APOO:Fluorescentlabelling
Subcellular protein fractionation
Recom.APOO D 1-40 Cyto. Mit.
APOO D 1-40
ANT
ACTIN
APOO D1-40
Green MitotrackerAPOO/TMR stain Merge
APOO localizes to the mitochondria in cardiac cells
APOO D1-40 Overexpression in cardiac myoblasts
APOO D1-40:Fluorescent labelling
A1/A4/E domain
198
N-
40 83
-C ApoO Δ1-40
1
Mutated APOO
Deletion of the mitochondrial adress label
Subcellular protein fractionation
Control
shRNA APOOAPOO D 1-40APOOO 2
Flo
w(p
mol
/s/1
06 cel
ls)
0
25
50
75
100
*
**
Cont.0
1
2
3
4
5
Cyt.
C ox
yd. a
ctiv
ity(µ
mol
/min
/µg
prot
ein) ***
APOO0
500
1,000
1,500
2,000
2,500 ControlApoO
DCFDA (µM)DC
F flu
o/10
6 c
ells
0 1 2 3 4 5 60.0
0.1
0.2
0.3
Cyt.
C ox
yd. a
ctiv
ity(µ
mol
/min
/µg
prot
.)
APOO-TgWT
*
A role for APOO in mitochondrial function?
APOO enhances mitochondrial respiration and oxidative stress in cardiac myoblast.
0
2
4
6
8**
***
Cd36
mRN
A (A
.U.)
0
4
8
12
16
****
Fatp
4 m
RNA
(A.U
.)
0
2
4
6
8
10 *******
Nad
h dh
mRN
A (A
.U.)
0
2
4
6
8 *****
**
Ndu
fa7
mRN
A (A
.U.)
A role for APOO in mitochondrial function?
OxidativePhosphorylationgenes
Fatty acids transportersgenes
Control
shRNA APOOAPOO D 1-40APOO
0
10
20
30
40 ***
Palmitate
**Di
glyc
erid
es(n
mol
/mg
prot
ein)
Mouse heartCardiac myoblasts
APOOControl
APOO D1-40
WT APOO-Tg0
2
4
6
Digl
ycer
ides
(nm
ol/m
g pr
otei
n) **
WT APOO-Tg0
5
10
15
20
Trig
lyce
rides
(nm
ol/m
g pr
otei
n)
Palmitate0
10
20
30
40
Triglycerides
(nmol/mg protein)
0 0.2 0.4 0.60
2
4
6
8 r = 0.6389 P = 0.0002
APOO mRNA (A.U.)
Digl
ycer
ides
(nm
ol/m
g pr
otei
n)
Human heart
0
100
200
300 r = 0.0331 P = 0.8978
APOO mRNA (A.U.)
Trig
lyce
rides
(nm
ol/m
g pr
otei
n)0 0.2 0.4 0.6 0.8
APOO induces lipotoxicity
What happens when lipid uptake exceeds mitochondrial oxidative capacity?
0
2
4
6
8
10
12
****
Bax
mRN
A (A
.U.)
APOOCont. shAPOO
Cardiac myoblasts
shAPOO0
10
20
30**
***
Casp
ase
3 ac
tivity
x10
(RFU
/µg
prot
ein)
APOOCont.2
0 125 250 500 0
100
200
300
400
500
**
******
Casp
ase-
3 ac
tivity
x 1
O2
(RFU
/µg
prot
ein)
*
Palmitate
ControlAPOO
Human heart
0.2 0.4 0.6 0.80
0.1
0.2
0.3 r = 0.7289p < 0.0001
BAX
mRN
A (A
.U.)
APOO mRNA (A.U.)
Mouse heart
0
5
10
15***
Casp
ase
3 ac
tivity
x 1
O R
FU/µ
g pr
otei
n
APOO-TgWT
2
WTBax/
Bcl-2
mRN
A (A
.U.)
APOO-Tg
Is APOO inducing lipoapoptosis?
APOO induces apoptosis
• PPAR is a transcription factor involved in lipid uptake.
• Ppara overexpression in mice led to cardiac dysfunction.
• The expression and activity of PPAR increase in diabetic hearts.
• The increase in intracellular lipid content in APOO models, should be associated with an induction of the expression of PPAR
Are APOO models mimicking diabetic cardiomyopathy?
Finck BN, et al. J Clin Invest. 2002
APOO associates with an increase in Ppar expression.
Cont.APOO0
2
4
6
8
10
12
Ppar
a m
RNA
(A.U
.) *
P-AMPK
CALR
AMPK
Cont. APOO
Mouse heart
5 10 15 20 250
2
4
6
8 r = 0.81 P < 0.0001
Apoo mRNA (A.U.)
Ppar
a m
RNA
(A.U
.)
0 0.2 0.4 0.6 0.80
0.1
0.2
0.3
0.4
0.5 r= 0.48
PPAR
A m
RNA
(A.U
.) P = 0.0006
APOO mRNA (A.U.)0
Human heart
Cardiac myoblasts
• PGC-1 , a master regulator of mitochondrial biogenesis, is upregulated in diabetic heart
• Ultrastructural analyses have revealed mitochondrial damage in mouse models of the metabolic syndrome and in models of type 1 and type 2 diabetes.
Are APOO models mimicking diabetic cardiomyopathy?
Handschin C. et al. Endocr. Rev. 2006
Mitochondrial degradation in APOO cells
0 5 10 15 20 250
2
4
6
8r = 0.81 P < 0.0001
Apoo mRNA (A.U.)
Pgc1
a m
RNA
(A.U
.)
Mouse heart
Human heart
0 0.2 0.4 0.6 0.82
4
6
8
10
12 r= 0.63 P = 0.0002
mRN
A (A
.U.)
PGC-
1A
APOO mRNA (A.U.)
nM
nM
nMN
Control +100µM palmitate
aM
mf
mFN
APOO + 100µM palmitate
mf
mF
0.0
0.5
1.0
1.5
2.0
2.5
***
ATP/
AMP
ratio
Cont. APOO
PPARPPAR
Multilamellar bodies
ROSFATPs
LCFAs
NADHFADH2
Metabolic sink
b-Oxid.
Plasma membrane
1
2
4
3
Lipotoxic byproducts
5
DNA
PGC1PPAR
Nucleus
6
OxidativePhospho.
Cytoc.C
Cell death
BAX
Apoptosome
7
IMM
OMM
APOO
Conclusion: The pathological overexpression of APOO induces a mitochondrial metabolic sink
APOO overexpression drives the cell into a vicious cycle consisting of a cascade of maladaptive events that ends in apoptosis and cell death
mimicking the diabetic cardiomyopathy.
Turkieh et al. The Journal of Clinical Investigation, 2014
INSERM UMR 1048 Team 7
Toulouse University Hospital
Dr Philippe Rouet, PhDProf. Galinier, MD-PhDProf. C. Dambrin, MD-PhDDr P. Massabuau, MDDr M.Berry, MDM. BarutautM. F. EvaristiDr C. Caubère, Dr A. Turkieh,
Dr. V. Le Berre, PhDDr S. Sokol, PhDL. Trouilh
Toulouse Genopole and Genotoul Network
Cardiology Federation
Acknowledgments
05
1015202530 ***
***
Palm. 100 µM- + -+ +-
Oleate 10 µM -
Digl
ycer
ides
(nm
ol/m
g pr
otei
ns)
+
***
0
0.25
0.50
0.75
1.00
1.25
Palm. 100 µM-
+-++
-Oleate 10 µM - +
******
***
DG/T
G
0
25
50
75
100
Palm. 100 µM-
- + +-
--
-+
++
---
-+
+-Oleate 10 µM
Oleate 1 µM
******
Casp
ase-
3 ac
tivity
x 1
02 (R
FU/µ
g pr
otei
ns)