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 polypeptidic sequence is conserved within species.

Lamant, J. Biol. Chem, 2006

APOO (22 kDa)

helix Antibodies

Against two specific

peptides

COOHNH2

APOO Characterization.

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)

APOOCont. shAPOO0

2

4

6

8

10

12**

***

Fatp

4 m

RNA

(A.U

.)

0

5

10

15

20

25

***

******

Triacsin C

***

Palm

itoyl

-CoA

x 1

04

(nm

ol/m

in/m

g pr

otei

n)

APOOCont. shAPOO0

2

4

6

8

10***

***

Cd36

mRN

A (A

.U.)