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GRK2 Inhibition Reduces Post-Myocardial
Infarction Cardiac Fibroblast-Mediated
Adverse Remodeling
Jennifer L. Philip1, Xianyao Xu1,Mei Han1, Jinju Li2, Abdur Razzaque1, Shahab A. Akhter1
AATS Lillehei Resident Forum
April 27,2015
Divisions of Cardiothoracic Surgery 1University of Wisconsin & 2University of Chicago
Disclosures
None
Post-Infarction Remodeling
Heart
Failure
Adverse Remodeling
Increased
Interstitial collagen
Adapted From:
Zouein FA et al. Post-infarct biomaterials, left ventricular remodeling, and heart failure: is good good enough? Congest
Heart Fail. 2012 Sep-Oct;18(5):284-90.
Konstam MA et al. Left ventricular remodeling in heart failure: current concepts in clinical significance and assessment.
JACC Cardiovasc Imaging. 2011 Jan;4(1):98-108.
Cardiac Fibroblasts
• Cardiac Fibroblasts (CF) make up 60-70% of cells in the heart
• CF play a critical role in adverse remodeling
• HF phenotype characterized by: differentiation into myofibroblasts and
increased collagen production
β-agonist
Adapted From: Porter KE, Turner NA. Cardiac fibroblasts: at the
heart of myocardial remodeling. Pharmacol Ther. 2009 Aug,
123(2):255-78.
Adapted from: Paul A Insel, Fiona Murray, Utako Yokoyama, Silvia Romano, Hongruo Yun, Loren
Brown, Aaron Snead, David Lu, Nakon Aroonsakool. cAMP and Epac in the regulation of tissue
fibrosis. Br J Pharmacol. 2012 May; 166(2): 447–456.
The Role of β-ARs and GRK2 in CF
• β-adrenergic receptor (β-AR) signaling/cAMP inhibits CF-mediated fibrosis
• G protein-coupled receptor kinase-2 (GRK2) uncouples β-AR signaling
• GRK2 upregulated in human HF
4000
*
*
-7
-6
log [ISO], M
-5
*
*
GRK2
Phosphorylation
and
Desensitization
5000
3000
2000
1000
Control
HF
0
0
Control
Adapted From: Howard A. Rockman, Walter J. Koch & Robert J. Lefkowitz.
Seven-transmembrane-spanning receptors and heart function. Nature. 2002
Jan 10;415(6868):206-12.
25
HF
Rhodopsin
(38kD)
Control
HF
18
20
GRK2 activity
(arbitrary units)
cAMP production (pmol/mL)
cAMP
GPCR
uncoupling
and
endocytosis
HF
Vimentin
GPCR
Prolonged
Agonist
Stimulation
[3H]proline incorporation
(cpm/mg protein)
Control
15
10
5
#
*
15
*
12
9
6
3
0
0
untreated
ISO
forskolin
Control
HF
Karen M. D'Souza et al. J. Biol. Chem. 2011
Hypotheses
• GRK2 is upregulated in cardiac fibroblasts post-MI
and is a primary etiology of maladaptive ventricular
remodeling via uncoupling of β-ARs and decreased
intracellular cAMP production
• Inhibition of GRK2 in vivo can decrease post-MI
ventricular remodeling and cardiac dysfunction by
inhibiting cardiac fibroblast activation and collagen
synthesis
Post-MI Ventricular Remodeling
Weeks Post-MI
2
4
8
12
Whole
Heart
Infarct Fibrotic
Area (%)
Control
10%
8%
*
*
*
*
6%
4%
2%
Infarct
Area
0%
Weeks Post-MI
Picrosirius red stain: Collagen stained Red
Remote Territory
Fibrotic Area (%)
Remote
Territory
*p<0.01 vs. Control; n=3-8
20%
18%
16%
14%
12%
10%
8%
6%
4%
2%
0%
*
#
#
*
Weeks Post-MI
*p<0.04 vs. Control,
#p<0.01 vs. Control &vs. 2wk Post-MI; n=3-8
Development of Failing CF Phenotype Post-MI
Control
Post-MI
Control
α-SMA/GAPDH
Expression
(relative units)
α-SMA (42 kDa)
Collagen I (134 kDa)
GAPDH (37 kDa)
*
15.0
Post-MI
α-SMA
Red
10.0
5.0
Collagen I
Green
0.0
Control
Post-MI
*p<0.0001 vs. Control; n=4
Collagen I/GAPDH
Expression
(relative units)
10.0
*
Vimentin
Red
8.0
*
6.0
4.0
Nuclei stained Blue with DAPI
2.0
0.0
Control
Post-MI
*p<0.0002 vs. Control; n=3-4
Increased CF Collagen Synthesis and
Uncoupled β–AR signaling Post-MI
Iso
TGF-β
#
5000
6.0
**
4000
cAMP (pmol/mL)
3[H]Proline
Incorporation
(cpm/μg protein)
No Drug
*
3000
2000
1000
*
4.0
*
3.0
2.0
1.0
0
0.0
Control
Post-MI
*p<0.02 vs. Control + No Drug, **p<0.005 vs. Control + Iso,
#p<0.01 vs. Control + TGF-β; n=7-9
*
5.0
Control
Post-MI
*p<0.02 vs. Control; n=3
GRK2 is Upregulated Post-MI
Control
Control
Post-MI
GRK2/GAPDH
Expression
(relative units)
GRK2
Green
Vimentin
Red
*
Post-MI
GRK2 (80 kDa)
GAPDH (37 kDa)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
*
Control
Post-MI
*p<0.02 vs. Control; n=4
Nuclei stained Blue with DAPI
Control
MI+Ad-Null MI+Ad-GRK2ct
α-SMA
(42 kDa)
GAPDH
(37kDa)
Control
Fold Change α-SMA
/GAPDH Expression
(relative units)
Inhibition of GRK2 in vivo Decreases Post-MI
Activation of Cardiac Fibroblasts
2.5
*
2.0
1.5
1.0
0.5
0.0
MI+Ad-Null MI+Ad-GRK2ct
Collagen I
(134kDa)
GAPDH
(37kDa)
Control
MI+
MI+
Ad-Null Ad-GRK2ct
GRK2 (80 kDa)
Fold Change Collagen I
/GAPDH Expression
(relative units)
*p<0.02 vs. Control & vs. MI+Ad-GRK2ct, n=5
3.0
2.5
*
2.0
1.5
1.0
0.5
0.0
GRK2ct (27 kDa)
GAPDH (37kDa)
*p<0.04 vs. Control & vs. MI+Ad-GRK2ct, n=5
Inhibition of GRK2 in vivo Decreases
Post-Infarction Ventricular Fibrosis
Control
MI+Ad-Null
MI+Ad-GRK2ct
Infarct
Area
Whole
Heart
Infarct Fibrotic
Area (%)
10%
*
8%
6%
**
4%
2%
0%
Remote
Territory
Remote Territory
Fibrotic Area (%)
*p<0.001 vs. Control,
**p<0.002 vs. Ad-Null & vs. Control; n=3-5
16%
14%
12%
10%
8%
6%
4%
2%
0%
*
**
Picrosirius red stain: Collagen stained Red
*p<0.001 vs. Control, **p<0.002 vs. Ad-Null & vs.
Control; n=3-5
Inhibition of GRK2 Preserves
Ventricular Function
Control
MI+Ad-Null
MI+Ad-GRK2ct
Ejection Fraction (%)
100%
80%
**
*
60%
40%
20%
0%
Control
MI+Ad-Null
MI+Ad-GRK2ct
*p<0.01 vs. Control, **p<0.03 vs. Ad-Null; n=4-5
Conclusions
• Post-MI LV remodeling is characterized by
significant remote territory fibrosis and CF
differentiation and activation
• Uncoupling of β-AR signaling via increased
GRK2 appears to be an important mechanism
of CF-mediated myocardial fibrosis
• Inhibition of GRK2 decreases cardiac fibroblast
transformation and collagen synthesis
resulting in decreased maladaptive ventricular
remodeling
Future Directions
• Investigation of the potential role of GRK2 in
MMP and TIMP biology
• Studying additional regulators of GPCR
signaling including β-arrestin in CF biology
• Creation of a fibroblast-specific transgenic
mouse model for inhibition of GRK2 to further
explore CF-specific inhibition of GRK2 as a
potential therapeutic strategy to prevent
maladaptive ventricular remodeling
Acknowledgements
• PI
– Shahab Akhter, MD
• Lab members
–
–
–
–
–
Xianyao Xu
Mei Han
Abdur M. Razzaque, PhD
Tiju Theccanat
Jinju Li, PhD
• University of Wisconsin Department of Surgery
• University of Chicago Department of Surgery
• Funding:
– Howard Hughes Medical Institute
– National Institutes of Health