Klf5 Regulates Cardiac PPARα and Med13 › idc › groups › ahamah... · CD-14. Sepsis-associated cardiac dysfunction etiology. Cardiac ... SGLT2 inhibition — a novel strategy

Konstantinos Drosatos, PhDAssistant Professor

Laboratory of Metabolic BiologyCenter for Translational Medicine

Department of Pharmacology

Klf5 Regulates Cardiac PPARα and Med13 and affects Fatty Acid Metabolism And Obesity

Name: Konstantinos DrosatosTitle: Klf5 Regulates Cardiac PPARα and Med13 and affects Fatty Acid Metabolism And Obesity

No conflict of interest to be disclosed

Sepsis/endotoxic shock

•Systemic inflammatory response that follows bacterial infection.

•Hypotension and organ failure.

Infection/ Injury

Systemic Inflammatory Response Syndrome

Sepsis

Septic shock

SIRS + Infection

Severe sepsis

Sepsis + Organ dysfunction

Severe sepsis HypotensionVasodilation

*

*SIRS = 2 of the following: Temperature ≥38oC or ≤36oC, HR ≥90 beats/min, Respirations ≥20/min, WBC count ≥12,000/mm3 or ≤4,000/mm3 or >10% immature neutrophils

Sepsis-associated cardiac dysfunction etiology

Cardiac dysfunction

Inflammatory Hypothesis

↑TNFα↑ IL-1↑ ΙL-6

NF-κΒ

Energetic Hypothesis↓Fatty acid

oxidation

↓PPARα

LPSLBP

TLR4CD-14

Endothelial cell

Cardiomyocyte

Lipoprotein lipaseFatty acid

O

OH.

HSPG

Chylomicron

O

OH.

Chyloremnant

O

OH.

VLDL

GPI-HBP1HSPGGPI-HBP1

Albumin

O

OH.

O

OH.

Chyloremnant

VLDLr

VLDL

LRP LRPCD36

O

OH.

O

OH.

O

OH.

O

OH.

O

OH.

O

OH.

VLDLr

O

SCoA.

O

OH. O OH.

O

OH.

O

SCoA.

O

SCoA.

O

SCoA.

ACSO

Ο.

O

Ο.

O

Ο.

- C|

- C|

- C

TriglyceridesO

Ο.

- C|

- C|

- C

O

Ο.

O

Ο.

- C|

- C|

- C

DAG MAG

GPATMGATDGAT

ATGL HSL MGL β-oxidation

Peroxisome Proliferator Activating Receptors PPARs

Nuclear receptors family members

Isoforms Major function Natural ligands Synthetic ligands

PPARα Fatty acid oxidation

Fatty acids Fibrates, WY14643

PPARγ Lipogenesis & Fatty acid oxidation

Fatty acidsProstanoid

Thiazolidinediones (TZD)

PPARδ Fatty acidoxidation

Prostacyclins, FAs GW0742 Wikipedia

Stimulation of cardiac fatty acid oxidation and energy productiontreats septic cardiac dysfunction

Inhibition of JNK prevents inhibition of cardiac PPARα and FAO and protects cardiac function in sepsis

LPS-induced activation of JNK suppressedPPARα gene expression and Fatty AcidOxidation

Inhibition of JNK stimulated PPARα geneexpression, prevented suppression of FAOand improved cardiac function, despiteelevated inflammation

Drosatos K. et. al., Journal of Biological Chemistry, Oct 2011

↓PPARα

AP1 binding site

PPARα promoter

c-junAP1

partner

PPPFatty acid oxidation

Cardiac dysfunction

LPSLBP

CD-14TLR4

c-jun AP1 partnerPPP

JNKP

What is the role of c-Jun in ppara gene regulation?

-800bp

-800bp

-740bp

-740bp

Sense

Anti-sense

Sense

Anti-sense

Does c-Jun bind on the PPARα promoter and inhibit PPARα gene expression?

• Zinc-finger proteins.

• Repressing or activating transcriptional function.

• KLF2, KLF3, KLF7 and KLF15 inhibit PPARγ expression

and adipogenesis

• KLF4, KLF5 and KLF6 induce PPARγ and adipogenesis

• KLF5 promotes cardiac hypertrophy

• KLF5+/- mice have increased FA oxidation in muscle and

are resistant in diet-induced obesity although they

consume more food.

KLFs – Krüppel-like factors

Beth B. Mcconnell & Vincent W. YangPhysiol Rev 90: 1337–1381, 2010

KLF5 and KLF6 show the most prominent increase in LPS-treated C57BL/6 mice

PPARα

William C. ClaycombLSU Health Sciences Center

0

0.5

1

1.5

2

2.5

CTRL LPS CTRL LPS CTRL LPS

PPARα KLF5 KLF6

*

Gen

e ex

pres

sion

(fol

d-ch

ange

)

*

*p

c-junAP1

partner

PPP

KLF5

PPARα promoter

Hypothesis: c-Jun and KLF5 are negative regulators of PPARα

(-) (-)

Gen

e ex

pres

sion

(AU

)

c.a. c-Jun decreases while KLF5 increases PPARα gene expression levels in HL-1 cells

0

1

2

3

4

5

KLF5 PPARα

Gen

e ex

pres

sion

(fol

d-ch

ange

)

AdGFP

AdKLF5 (high conc.)

**

***

*p

Potential KLF and c-Jun binding sites on PPARa promoter

0

1

2

3

4

5

6

(cJunAb)

(IgGAb)

(cJunAb)

(IgGAb)

Ad-GFP Ad-cJun-Asp

cJun

enr

ichm

ent

**

01

2

345

6

78

Ad-GFP(KLF5Ab)

Ad-GFP(IgG Ab)

Ad-KLF5(KLF5Ab)

Ad-KLF5(IgG Ab)

KLF5

enr

ichm

ent

**

-792/-772 bp (PPARa promoter)

00.5

11.5

22.5

33.5

44.5

5

(cJunAb)

(IgGAb)

(cJunAb)

(IgGAb)

Ad-GFP Ad-cJun-Asp

cJun

enr

ichm

ent

**

0

1

2

3

4

5

6

(KLF5Ab)

(IgGAb)

(KLF5Ab)

(IgGAb)

Ad-GFP Ad-KLF5

KLF5

enr

ichm

ent

-719/- 698 bp (PPARa promoter)

c-jun AP1 partnerPPP

KLF5

PPARα promoter

c-Jun and KLF5 compete for binding on PPARα promoter

(-) (+)

LPS-induced binding of c-Jun on PPARa promoter prohibits binding of KLF5

0

0.5

1

1.5

2

2.5

3

CTRL (cJun Ab) LPS (cJun Ab) CTRL (KLF5 Ab) LPS (KLF5 Ab)

-792/-772 bp (PPARa promoter)

*

NS

*p

XKLF5loxP/loxPαMHC-Cre-KLF5+/+

αMHC-Cre-KLF5+/loxP KLF5loxP/loxP

X

αMHC-KLF5-/-

Provided by Prof. Jeffrey WhitsettCincinnati Children’s Hospital Medical Center

Generation of cardiomyocyte-specific KLF5-/- mouse model

0

10

20

30

40

50

60

floxed αMHC-KLF5-/-

FS (%)

0

0.5

1

1.5

2

2.5


LVIDS

0

0.5

1

1.5

2

2.5

3

3.5

4


LVIDd

αMHC-KLF5-/- have normal cardiac function

*

KLF5

0

0.2

0.4

0.6

0.8

1

1.2

1.4

CTRL aMHC-KLF5-/-

PPARα

**

*p

Cardiomyocyte-specific ablation of KLF5 reduces cardiac PPARα and FAO-related gene expression

Reduced cardiac PPARα is associated with cardiac lipotoxicity

Is diabetes associated with changes in cardiac KLF5 and PPARα?

C57BL/6

CTRL (saline) Diabetes (STZ)

Streptozotocin-induced diabetes induces mild cardiac dysfunction in C57BL/6 mice

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

wt

Frac

tiona

l Sho

rten

ing

(%)

CTRL STZ

*

Diabetes reduces cardiac KLF5 and PPARα gene expression

0

0.2

0.4

0.6

0.8

1

1.2

1.4

KLF5 PPARα

Gen

e ex

pres

sion

(fol

d-ch

ange

)

fl/fl

fl/fl + STZ

αMHC-KLF5-/- + STZ

*p

Glucose elevation drives KLF5 and PPARα downregulation

HL-1 cells

CTRL Glucose (1 mg/ml, 24h)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

KLF5 PPARα

CTRL

Glucose

***

***

n = 5-6

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Glut1 Glut4 PDK4 AOX Cpt1b LCAD VLCAD

CTRL

Glucose*

*

*** ***

n = 5-6

*********

Hyperglycemic ob/ob mice have reduced cardiac KLF5 and PPARα

**

0

0.2

0.4

0.6

0.8

1

1.2

1.4

KLF5 PPARα

wt

ob/ob

***

**

n ≥ 4

Type-II diabetes model

Glucose is reabsorbed prior to excretion through kidneys

Nature Reviews Drug Discovery (July 2010)SGLT2 inhibition — a novel strategy for diabetes treatment

Edward C. Chao & Robert R. Henry

Correction of hyperglycemia with SGLT2 inhibition restored KLF5 and PPARα levels in C57BL/6 mice

SGLT2Pharmacological inhibitorDapagliflozin Anti-sense-SGLT2 oligo

Floxed

HF diet

20

22

24

26

28

30

32

Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6

WTaMHC-KLF5-/- * *

*

*p

αMHC-KLF5-/-Floxed αMHC-KLF5-/-Floxed

*

0

0.2

0.4

0.6

0.8

1

1.2

1.4

05

10152025303540

*

Wei

ght g

ain

(%)

WAT

wei

ght (

g)

αMHC-KLF5-/- mice gain more weight in HF diet

CTRL

αMHC-KLF5-/-

High fat-fed αMHC-KLF5-/- mice have larger adipocytes in WAT and higher lipid accumulation in BAT

CTRL

αMHC-KLF5-/-

HFD-fed αMHC-KLF5-/- mice have increased expression of lipid metabolism markers in WAT

0

2

4

6

8

10

12

PPARγ2 PPARγ1 LPL CD36 DGAT2 GLUT4

Gen

e ex

pres

sion

(fol

d-ch

ange

)

fl/fl

αMHC-KLF5-/-

**

*********

***

n = 4

***

αMHC-KLF5-/-

HF diet

More profound body weight

increase

A change in cardiomyocytes promotes DIO!

Grueter CE et al.; A cardiac microRNA governs systemicenergy homeostasis by regulation of MED13. Cell. 2012

αMHC-MED13-/- mice gain more weight in HF diet

Gen

e ex

pres

sion

(fol

d-ch

ange

)

0

0.2

0.4

0.6

0.8

1

1.2

MED13

*** ***

***

αMHC-KLF5-/- mice have reduced cardiac MED13 expression without changes in miR-208

0.0

0.2

0.4

0.6

0.8

1.0

1.2

miR-208am

iR-2

08a

expr

essi

on (f

old-

chan

ge)

fl/fl male

αMHC-KLF5-/- male

fl/fl female

αMHC-KLF5-/- female

0

0.5

1

1.5

2

2.5

AdGFP AdKLF5 AdGFP AdKLF5

KLF5 MED13

Adenovirus-mediated overexpression of KLF5 increases MED13 gene expression in HL-1 cells

**

Ad-KLF5 provided by Ceshi Chen, Ph.D.Center for Cell Biology and Cancer Research

Albany Medical College

KLF5 binds the promoter of mMED13 gene

0.00.51.01.52.02.53.03.54.04.55.0

Ad-GFP(KLF5 Ab)

Ad-GFP(IgG Ab)

Ad-KLF5(KLF5 Ab)

Ad-KLF5(IgG Ab)

KLF5

enr

ichm

ent

-730 bp

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Ad-GFP(KLF5Ab)

Ad-GFP(IgG Ab)

Ad-KLF5(KLF5Ab)

Ad-KLF5(IgG Ab)

-142 bp

KLF5

enr

ichm

ent

*

↑MED13

KLF binding site

MED13promoter

KLF5

Increased systemic metabolic

rate

KLF5

↓MED13

KLF binding site

MED13promoter

Obesity

KLF5X Reduced systemic metabolic

rate

Proposed model

Summary

1.KLF5 and cJun compete for binding on PPARα gene promoter insepsis.

2.KLF5 is a positive regulator of PPARα gene expression that isdriven by hyperglycemia.

3.KLF5 is a positive regulator of MED13 gene expression.

4.Cardiomyocyte-specific KLF5 deletion leads to downregulationof MED13 and accelerates diet-induced obesity.

Long-term plan

KLF5

Activation

↑Cardiac PPARα ↑Cardiac MED13

Improved cardiac fatty acid oxidationand alleviation of

cardiac lipotoxicity

Improved systemic metabolism and

inhibition of obesity

Acknowledgements

Ira J. Goldberg

Leslie Anderson

P. Christian Schulze

Yunying Hu

Chad Trent

K99/R00 award, 2012-2017

Nina Pollak

Iannis AifantisHHMI & NYU

Panos Ntziachristos

Temple University

Mesele Valenti Walker Lyons

New York UniversityUniv of Graz, Austria

Columbia University

Florian Willecke

Laboratory of Metabolic BiologyCenter for Translational Medicine

Department of PharmacologyTemple University School of Medicine

www.Drosatos.com

Slide Number 1Slide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Stimulation of cardiac fatty acid oxidation and energy production�treats septic cardiac dysfunctionInhibition of JNK prevents inhibition of cardiac PPARα and FAO and protects cardiac function in sepsisSlide Number 10Slide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23Reduced cardiac PPARα is associated with cardiac lipotoxicitySlide Number 25Diabetes reduces cardiac KLF5 and PPARα gene expressionGlucose elevation drives KLF5 and PPARα downregulationHyperglycemic ob/ob mice have reduced cardiac KLF5 and PPARαSlide Number 29Correction of hyperglycemia with SGLT2 inhibition �restored KLF5 and PPARα levels in C57BL/6 miceSlide Number 31Slide Number 32Slide Number 33HFD-fed αMHC-KLF5-/- mice have increased expression of lipid metabolism markers in WATSlide Number 35Slide Number 36Slide Number 37Slide Number 38Slide Number 39Slide Number 40Slide Number 41Slide Number 42Slide Number 43Slide Number 44

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Klf5 Regulates Cardiac PPARα and Med13 › idc › groups › ahamah... · CD-14. Sepsis-associated cardiac dysfunction etiology. Cardiac ... SGLT2 inhibition — a novel strategy