grk2 inhibition reduces post-myocardial infarction cardiac fibroblast-mediated adverse remodeling...
DESCRIPTION
Post-Infarction Remodeling Increased Interstitial collagen Adverse Remodeling Heart Failure Adapted From: Zouein FA et al. Post-infarct biomaterials, left ventricular remodeling, and heart failure: is good good enough? Congest Heart Fail Sep-Oct;18(5): Konstam MA et al. Left ventricular remodeling in heart failure: current concepts in clinical significance and assessment. JACC Cardiovasc Imaging Jan;4(1):TRANSCRIPT
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 ForumApril 27,2015
Divisions of Cardiothoracic Surgery 1University of Wisconsin & 2University of Chicago
Disclosures
None
Post-Infarction Remodeling
Increased Interstitial collagen
Adverse Remodeling
Heart Failure
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.
Adapted From: Porter KE, Turner NA. Cardiac fibroblasts: at the heart of myocardial remodeling. Pharmacol Ther. 2009 Aug, 123(2):255-78.
β-agonist
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.
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
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.
GPCR uncoupling and endocytosis
GPCR
Phosphorylation and Desensitization
Prolonged Agonist Stimulation
cAMP
Karen M. D'Souza et al. J. Biol. Chem. 2011
0 -7 -6 -50
1000
2000
3000
4000
5000
ControlHF
log [ISO], M[3
H]pr
olin
e in
corp
orat
ion
(cpm
/mg
prot
ein)
* * * *
untreated ISO forskolin0
5
10
15
20
25 ControlHF
cAM
P pr
oduc
tion
(pm
ol/m
L)
#*
GR
K2
Control
Vim
entin
HF
Rhodopsin (38kD)
Control HF0
3
6
9
12
15
18
GRK
2 ac
tivity
(arb
itrar
y u
nits
)
*
Control HF
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
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
Who
le
Hear
tIn
farc
t Ar
eaRe
mot
e Te
rrito
ry
Picrosirius red stain: Collagen stained Red
Con-trol
2 4 8 120%
2%
4%
6%
8%
Weeks Post-MI
Infa
rct F
ibro
tic A
rea
(%)
Control
* * * *
Con-trol
2 4 8 120%
5%
10%
15%
20%
Weeks Post-MIR
emot
e Te
rrito
ry F
ibro
tic A
rea
(%)
*
##
*
2 4 8 12Weeks Post-MI
*p<0.01 vs. Control; n=3-8
*p<0.04 vs. Control, #p<0.01 vs. Control &vs. 2wk Post-MI; n=3-8
Post-MI Ventricular Remodeling
Development of Failing CF Phenotype Post-MI
*
Collagen IGreen
α-SMARed
Control Post-MI
VimentinRed
Nuclei stained Blue with DAPI
Collagen I (134 kDa)
Post-MIControl
Control Post-MI0.0
2.0
4.0
6.0
8.0
10.0
Col
lage
n I/G
APD
H
Expr
essi
on
(rela
tive
units
)
Control Post-MI0.02.04.06.08.0
10.012.014.0
α-SM
A/G
APD
H
Expr
essi
on
(rela
tive
units
) *
*
*p<0.0001 vs. Control; n=4
*p<0.0002 vs. Control; n=3-4
α-SMA (42 kDa)
GAPDH (37 kDa)
*
Increased CF Collagen Synthesis and Uncoupled β–AR signaling 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
*p<0.02 vs. Control; n=3
Control Post-MI0
1000
2000
3000
4000No Drug Iso TGF-β
3[H
]Pro
line
Inco
rpor
atio
n (c
pm/μ
g pr
otei
n)
Control Post-MI 0.0
1.0
2.0
3.0
4.0
5.0
6.0
cAM
P (p
mol
/mL)
*
GRK2 is Upregulated Post-MI
GRK2 (80 kDa)GAPDH (37 kDa)
Control Post-MI
VimentinRed
Nuclei stained Blue with DAPI
GRK2Green
Post-MIControl
Control Post-MI0.00.51.01.52.02.53.0
GR
K2/
GA
PDH
Ex
pres
sion
(rela
tive
units
) *
*p<0.02 vs. Control; n=4
GRK2 (80 kDa)
GAPDH (37kDa)
α-SMA(42 kDa)
GRK2ct (27 kDa)
Control MI+Ad-Null MI+Ad-GRK2ct
GAPDH (37kDa)
Control
MI + Ad-Null
MI + Ad-GRK2ct0.0
0.5
1.0
1.5
2.0
2.5
Fold
Cha
nge
Col
lage
n I
/GA
PDH
Exp
ress
ion
(rel
a-tiv
e un
its)
*
Collagen I (134kDa)GAPDH (37kDa)
Control
MI + Ad-Null
MI + Ad-GRK2ct0.0
0.5
1.0
1.5
2.0
2.5
Fold
Cha
nge
α-SM
A/G
APD
H E
xpre
ssio
n
(rela
tive
units
)
*
Control MI+Ad-Null MI+Ad-GRK2ct
ControlMI+
Ad-NullMI+
Ad-GRK2ct
Inhibition of GRK2 in vivo Decreases Post-MI Activation of Cardiac Fibroblasts
*p<0.02 vs. Control & vs. MI+Ad-GRK2ct, n=5
*p<0.04 vs. Control & vs. MI+Ad-GRK2ct, n=5
*
**
Control MI+Ad-Null MI+Ad-GRK2ct
*
**
Control MI+Ad-Null
MI+Ad-GRK2ct0%
2%
4%
6%
8%
10%
Infa
rct F
ibro
tic
Are
a (%
)Control
MI+Ad-Null MI+Ad-GRK2ct
0%
5%
10%
15%
Rem
ote
Terr
itory
Fib
rotic
Are
a (%
)
Who
le
Hear
tIn
farc
t Ar
eaRe
mot
e Te
rrito
ryInhibition of GRK2 in vivo Decreases
Post-Infarction Ventricular Fibrosis
Picrosirius red stain: Collagen stained Red
*p<0.001 vs. Control, **p<0.002 vs. Ad-Null & vs. Control; n=3-5
*p<0.001 vs. Control, **p<0.002 vs. Ad-Null & vs. Control; n=3-5
***
Control MI+Ad-Null MI+Ad-GRK2ct
Inhibition of GRK2 Preserves Ventricular Function
*p<0.01 vs. Control, **p<0.03 vs. Ad-Null; n=4-5
Control MI+Ad-Null MI+Ad-GRK2ct0%
10%20%30%40%50%60%70%80%90%
Ejec
tion
Frac
tion
(%)
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
