Integrative Physiology
Determining the Absolute Requirement of G Protein–Coupled
Receptor Kinase 5 for Pathological Cardiac Hypertrophy
Short Communication
Jessica I. Gold, Erhe Gao, Xiying Shang, Richard T. Premont, Walter J. Koch
Rationale: Heart failure (HF) is often the end phase of maladaptive cardiac hypertrophy. A contributing factor is
activation of a hypertrophic gene expression program controlled by decreased class II histone deacetylase (HDAC)
transcriptional repression via HDAC phosphorylation. Cardiac-specific overexpression of G proteinen–coupled
receptor kinase-5 (GRK5) has previously been shown to possess nuclear activity as a HDAC5 kinase, promoting
an intolerance to in vivo ventricular pressure overload; however, its endogenous requirement in adaptive and
maladaptive hypertrophy remains unknown.
Objective: We used mouse models with global or cardiomyocyte-specific GRK5 gene deletion to determine the absolute
requirement of endogenous GRK5 for cardiac hypertrophy and HF development after chronic hypertrophic stimuli.
Methods and Results: Mice with global deletion of GRK5 were subjected to transverse aortic constriction. At 12
weeks, these mice showed attenuated hypertrophy, remodeling, and hypertrophic gene transcription along with
preserved cardiac function. Global GRK5 deletion also diminished hypertrophy and related gene expression due to
chronic phenylephrine infusion. We then generated mice with conditional, cardiac-specific deletion of GRK5 that also
demonstrated similar protection from pathological cardiac hypertrophy and HF after transverse aortic constriction.
Conclusions: These results define myocyte GRK5 as a critical regulator of pathological cardiac growth after
ventricular pressure overload, supporting its role as an endogenous (patho)-physiological HDAC kinase. Further,
these results define GRK5 as a potential therapeutic target to limit HF development after hypertrophic stress.
(Circ Res. 2012;111:1048–1053.)
Key Words: G protein–coupled receptor kinases ◼ cardiac hypertrophy ◼ conditional transgenic mouse
H
eart failure (HF), a leading cause of death in the Western
world, often occurs as an end phase of pathological
myocardial hypertrophy.1 Hypertrophy is initially an adaptive
response to stresses ranging from hypertension, valve disease,
or cardiac injury.2,3 In an attempt to normalize wall stress, cardiomyocytes enlarge, sarcomeres reorganize, fibroblasts proliferate, and hypertrophic genes, including the so-called fetal
gene program, are upregulated. If prolonged, these responses
lead to chamber dilation, myocardial apoptosis, and HF.2,3
In pathological cardiac growth, the molecular pathways affecting transcription lie downstream of the nodal hypertrophic signal
transducer, Gq.1 Among these complex pathways are the class II
histone deacetylases (HDACs). Physiologically opposed to cardiac growth, the HDACs repress expression of key hypertrophic
genes, primarily through inhibition of myocyte enhancer factor
2 (MEF2).3 Genetic deletion of the class II HDAC, HDAC5,
sensitizes mice to cardiac stress, whereas murine deletion of
MEF2 confers cardioprotection, decreasing pathological hypertrophy and attenuating upregulation of the fetal gene program.4
HDAC kinases control nuclear HDAC activity as phosphorylation
of HDAC5 induces its nuclear export and MEF2 derepression.3–5
In This Issue, see p 947
Editorial, see p 957
We recently identified G protein-coupled receptor (GPCR)
kinase-5 (GRK5) as a nuclear HDAC kinase, joining protein kinase D (PKD) and calmodulin-dependent kinase II (CaMKII) as
HDAC-mediated facilitators of cardiac growth after hypertrophic stimuli.5–7 This represents a novel, non-GPCR cardiac role
for this GRK. We showed that mice with cardiac overexpression of GRK5 demonstrate Gq-dependent nuclear translocation of GRK5, where it can phosphorylate HDAC5 and induce
Original received May 8, 2012; revision received July 30, 2012; accepted August 1, 2012. In July 2012, the average time from submission to first decision
for all original research papers submitted to Circulation Research was 11.2 days.
From the Center for Translational Medicine, Thomas Jefferson University, Philadelphia, PA (J.I.G., W.J.K.); Center for Translational Medicine (E.G.,
X.S., W.J.K.) and the Department of Pharmacology (W.J.K.), Temple University School of Medicine, Philadelphia, PA; and the Department of Medicine,
Duke University Medical Center, Durham, NC (R.T.P.).
This manuscript was sent to Elizabeth Murphy, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.112.
273367/-/DC1.
Correspondence to Walter J. Koch, MD, Center for Translational Medicine, Temple University School of Medicine, 3500 N Broad St, MERB 941,
Philadelphia, PA 19140. E-mail Walter.Koch@temple.edu
© 2012 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.112.273367
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Gold et al
Non-standard Abbreviations and Acronyms
AR
CaMKII
%EF
GPCR
GRK
HDAC
HF
HW/BW
KO
LV
LVIDs
LVPWT
MEF2
PBS
PE
PKD
TAC
WT
adrenergic receptor
calmodulin-dependent kinase II
ejection fraction percent
G protein–coupled receptor
G protein–coupled receptor kinase
histone deacetylase
heart failure
heart weight–to–body weight ratio
knockout
left ventricular
systolic left ventricular internal diameter
left ventricular posterior wall thickness
myocyte enhancer factor 2
phospho-buffered saline
phenylephrine
protein kinase D
transverse aortic constriction
wild-type
MEF2 activity.6 Moreover, cardiac GRK5-overexpressing mice
displayed an intolerance to ventricular pressure overload with
potentiated maladaptive hypertrophy and accelerated HF after
transverse aortic constriction (TAC).6 Importantly, these transgenic studies did not address whether endogenous GRK5 plays
a role in the hypertrophic response.
In the present study, we used global and cardiomyocytespecific GRK5 knockout (KO) mice to directly address the
importance of endogenous GRK5 in cardiac hypertrophy. We
found that GRK5 in cardiomyocytes is absolutely required for
hypertrophic responses after stress. Further, our data indicate
that limiting GRK5 expression in the heart can protect against
maladaptive cardiac growth and HF development.
Methods
Generation of GRK5cKO Mice
Detailed mouse protocols are described in the Online Data
Supplement Methods.
Surgical Procedures and Echocardiography
TAC methodology, minipump implantation, and determination of in
vivo cardiac function and morphology are described in the Online
Data Supplement and Methods.
RNA Analysis
Methods for RNA analysis are described in the Online Data Supplement.
Immunoblot Analysis
Detailed methods for heart subfractionation and immunoblotting are
described in the Online Data Supplement.
Results
Global GRK5 Deletion Diminishes In Vivo
Cardiac Hypertrophy
We subjected male global GRK5 gene knockout mice
(GRK5gKO)8 and littermate wild-type (WT) control mice to
GRK5 in Pathological Cardiac Hypertrophy
1049
TAC. Constitutive GRK5 deletion attenuated pressure-overloadinduced cardiac growth seen in WT mice at 12 weeks after TAC
(Figure 1A). Cardiac dimensions were measured serially by
echocardiography to track development of hypertrophy and left
ventricular (LV) dilatation over 12 weeks. WT mice showed a
quick rise in LV posterior wall thickness (LVPWT), with a peak
thickness of 1.99±0.05 mm at 4 weeks after TAC, which then
decreased rapidly, indicative of adverse remodeling (Figure 1B).
Interestingly, global GRK5 deletion significantly delays the initiation of cardiac hypertrophy after TAC, as GRK5gKO mice do
reach a similar LVPWT (1.89±0.09 mm) as WT mice but not
until 12 weeks after TAC (Figure 1B). At the end of 12 weeks,
GRK5gKO mice had significantly less cardiac hypertrophy as
determined by smaller heart weighten-to-enbody weight (HW/
BW) ratios (Figure 1C). Importantly, GRK5gKO mice show no
signs of LV dilatation via echocardiographic measurements of
systolic LV interior diameter (LVIDs), which were significantly
increased in post-TAC WT mice (Figure 1D and Online Figure
I). Further, post-TAC GRK5gKO mice showed preserved cardiac function as determined by ejection fraction percent (%EF)
compared with post-TAC WT mice that had significant LV dysfunction (Figure 1E and Online Figure I).
We have previously shown that elevated GRK5 plays a
critical role in myocytes as an HDAC5 kinase, derepressing
hypertrophy-related transcription.6 Upregulation of generalized
hypertrophy markers, including those of the fetal gene program—
atrial natriuretic factor (ANF), β-myosin heavy chain (βMHC),
and procollagen, type Iα2 (Col1a2)—were significantly attenuated in the GRK5gKO mice compared with WT mice 12 weeks
after TAC (Figure 1F). Loss of GRK5 expression globally also
prevented the post-TAC upregulation of hypertrophic genes directly regulated by MEF29: brain natriuretic peptide (BNP), actin-α1
(Acta-1), and connective tissue growth factor (CTGF) (Figure
1G). Thus, deletion of GRK5 is protective against hypertrophy at
the molecular level, consistent with the in vivo phenotype.
Grk5gKO Mice Are Resistant to
Phenylephrine-Dependent Hypertrophy
Phenylephrine (PE), acting through α-adrenergic receptors
(αARs), induces cardiomyocyte hypertrophy in vitro and in vivo.
Previously, we have found that PE causes GRK5 nuclear translocation and increased MEF2 activity in myocytes.6 Therefore,
we tested whether endogenous GRK5 was necessary for development of PE-induced cardiac hypertrophy. Male mice (WT
and GRK5gKO) were treated with a subpressor dose of PE (35
mg/kg per day) or phospho-buffered saline (PBS) for 14 days via
osmotic minipumps (Figure 2A). This period covers only an initial
hypertrophy stage without decompensation, thereby testing the
requirement of endogenous GRK5 in myocardial αAR-mediated
hypertrophy. PE treatment caused cardiac growth in WT mice,
with a significant 26.9±8% increase in HW/BW ratio at 2 weeks,
whereas GRK5gKO mice subjected to PE had only an 11.2±7%
increase in HW/BW, insignificant compared with PBS-treated
GRK5gKO mice (Figure 2B). Two weeks of PE treatment does
not change cardiac function, which is what we found for %EF
(Figure 2C), although PE significantly altered morphology in WT
mice (Figure 2D and 2E). Importantly, PE-treated GRK5gKO
mice showed no changes in cardiac dimensions (Figure 2D, 2E).
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Figure 1. Attenuated hypertrophy seen in GRK5gKO mice after TAC. A, Hearts from WT and GRKgKO mice subjected to a sham
operation or TAC (top). Histological sections stained are with Masson trichrome for ibrosis (middle; bottom). WT sham, n=6; WT TAC,
n=9; gKO sham, n=6; gKO TAC, n=11. B, Systolic LVPWT measured serially by echocardiogram after sham or TAC operations. *P<0.05.
C, HW/BW ratios were measured 12 weeks after TAC. *P<0.01. LVIDs (D) or %EF (E) as measured by echocardiogram at 12 weeks after
TAC. *P<0.01. RT-PCR was used to measure mRNA expression of known markers of cardiac hypertrophy (F) and genes directly regulated
by MEF2 (G); n=8, *P<0.05.
As with post-TAC, GRK5 deletion significantly decreased
PE-mediated upregulation of hypertrophy markers. Indeed,
whereas PE led to robustly increased expression of our panel
of hypertrophic genes in WT mice, no such upregulation was
seen in GRK5gKO mice (Figure 2F and 2G). Overall, these
data demonstrate that GRK5 plays a key role in cardiac hypertrophy downstream of PE, identical to post-TAC phenotype.
0.91±0.14; WT sham: 1.02±0.17) (Figure 3A and 3B). Similar results were seen in the non-nuclear fraction from WT mice that had
received chronic infusion of PE. These mice showed significantly
greater non-nuclear phosphorylated HDAC5 than PE-infused
GRK5gKO mice and PBS-infused WT mice (Figure 3C), further
reinforcing the role of endogenous GRK5 as an HDAC5 kinase.
Global GRK5 Ablation Decreases Nuclear HDAC5
Export After Hypertrophic Stimulus
The above results show that complete GRK5 ablation attenuates
the cardiac hypertrophic response but does not address the specific role of myocyte GRK5 in maladaptation and post-TAC HF.
Therefore, we developed conditional GRK5KO mice in which
GRK5 deletion was cardiac-specific. We bred floxedGRK5
mice with transgenic mice expressing Cre-recombinase under control of the αMHC promoter.10 These conditional GRK5
KO (GRK5cKO) mice had >50% loss of cardiac GRK5 determined by either protein immunoblotting or RT-PCR (Online
Figure II). As above, we stressed GRK5cKO mice and WT
control mice (GRK5floxed) via TAC and studied these groups
alongside sham-operated mice for 12 weeks (Figure 4A). Serial
echocardiography showed significantly attenuated hypertrophy
The diminished upregulation of genetic hypertrophy markers
seen in GRK5gKO mice after TAC suggests that GRK5 can
regulate cardiac gene transcription. This is probably due to the
ability of GRK5 to phosphorylate HDAC5, inducing its export
from the nucleus.6 Hence, we examined location and phosphorylation of HDAC5 after hypertrophic stress in GRK5gKO and
WT mice. Hearts from the above experiments were subjected
to subcellular fractionation. After TAC, WT mice showed a significantly increased amount of phosphorylated HDAC5 in the
non-nuclear subcellular fraction compared with GRK5gKO TAC
mice and WT sham mice (WT TAC: 1.94±0.13;GRK5gKO TAC:
Cardiac-Specific Deletion of GRK5 Attenuates
Hypertrophy After TAC
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Gold et al
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Figure 2. PE induces less hypertrophy in GRK5gKO mice. A, Hearts from WT and GRK5 gKO mice subjected to 2 weeks of chronic
infusion of PBS or subpressor PE (35 mg/kg per day) (top). Histological sections are stained with Masson trichrome for ibrosis (middle;
bottom). WT PBS, n=8; WT PE, n=10; gKO PBS, n=9; gKO PE, n=11. B, HW/BW ratios for WT and GRK5gKO mice were measured after
2 weeks of chronic PBS or PE infusion. *P<0.05. %EF (C), LVIDs (D), and LVPWT (E) as measured by echocardiogram at 2 weeks. *P<0.05.
RT-PCR was used to measure mRNA expression of known markers of cardiac hypertrophy (F) or MEF2-regulated genes (G); n=8, *P<0.05.
in GRK5cKO mice with maximally increased cardiac mass at
12-weeks when WT mice are clearly decompensated after their
peak hypertrophy 4 weeks after TAC (Figure 4B). This delayed
compensatory hypertrophy led to a trend toward lower HW/BW
ratio in GRK5cKO mice at 12 weeks after TAC compared
with WT mice (Figure 4C). This effect is less robust than the
GRK5gKO mice but no doubt due to incomplete GRK5 ablation.
However, from in vivo functional studies, it is clear that the myocyte GRK5 loss protects these hearts from adverse LV remodeling
and HF, as GRK5cKO mice displayed no increased LV dilatation
at the study’s end, compared with significantly increased LVIDs
in WT mice (Figure 4D and Online Figure III). This protection
against HF was also evident in global in vivo cardiac function as
12-week post-TAC WT mice had a significant loss of LV %EF
compared with sham WT mice, while there was absolutely no
drop in %EF in 12-week post-TAC GRK5cKO mice (Figure 4E).
Again, we examined our panel of hypertrophy-related
genes. At 12 weeks after TAC, hearts from GRK5cKO mice
showed significantly diminished upregulation of these common cardiac hypertrophy markers, including those of the fetal
gene program (Figure 4F), and specific MEF2-regulated genes
(Figure 4G). Overall, these data demonstrate that GRK5 expression in cardiomyocytes alone is required for WT molecular, functional, and morphological responses after TAC.
Discussion
Our results indicate an absolute requirement of cardiomyocyte
GRK5 for normal hypertrophic responses and that this kinase
plays a critical pathological role in ventricular decompensation and transition to HF after ventricular pressure overload.
Importantly, merely decreasing cardiomyocyte GRK5 in mice
blunts hypertrophic myocardial growth and prevents HF after
TAC. Importantly, these data demonstrate that endogenous myocyte GRK5 plays a crucial role in adaptive and maladaptive hypertrophy and is required for WT response to stress. Thus, increased
GRK5 in the failing human heart11 has pathological significance,
since lowering GRK5 or inhibiting its activity appears to offer
novel beneficial effects against maladaptive cardiac growth.
These results in global and cardiac-specific GRK5KO
mice, coupled with our previous results showing that
GPCR-independent nuclear activity of GRK5 can facilitate hypertrophy,6 indicate that nuclear targeting of endogenous GRK5
as a class II HDAC kinase is physiologically significant in normal
and abnormal cardiac growth after stress. Interestingly, loss of
nuclear GRK5 activity can delay HF onset through slower cardiac growth, although its deletion does not completely ameliorate hypertrophy. This may be due to compensatory and discrete
roles of the other known HDAC5 kinases, CAMKII, and PKD,
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Figure 3. Global GRK5 ablation decreases phosphorylated HDAC5 in the cytoplasm. A, Twelve weeks after TAC or a sham operation,
hearts from WT and GRK5gKO mice were fractionated into non-nuclear and nuclear fractions. HDAC5 was immunoprecipitated from the
non-nuclear fraction and immunoblotted for phosphorylated and total amounts (pHDAC5 and tHDAC5, respectively). B, Denistometric
quantiication of pHDAC5 normalized to tHDAC. *P<0.01 versus all groups, n=6. C, Denistometric quantiication of pHDAC5 normalized to
tHDAC5 after immunoprecipitation and immunoblotting for pHDAC5 in WT and GRK5gKO mouse hearts after 2 weeks of chronic PBS or
PE infusion. *P<0.01 versus all groups, n=6.
acting downstream of hypertrophic signaling.5,7 Each kinase has
been shown to cause HDAC5 nuclear export after select receptor
activation such as endothelin-1 receptor for CAMKII and αAR
for PKD.5,12 We now can add GRK5 to the list of physiological
HDAC kinases downstream of TAC and αAR stimulation. These
distinct activators of HDAC kinases appear to underlie a complex network of parallel signaling converging on the same target, HDAC5. Overall, our data demonstrate that targeting only 1
Figure 4. Attenuated hypertrophy seen in GRK5cKO mice after TAC. A, Hearts from WT and GRK5cKO mice subjected to a sham operation
or TAC (top). Histological sections are stained with Masson trichrome for ibrosis (middle; bottom). WT sham, n=9; WT TAC, n=14; cKO sham,
n=8; cKO TAC, n=9. B, Systolic LVPWT measured serially by echocardiogram after sham or TAC operations. *P<0.05. C, HW/BW ratios for WT
and GRK5cKO mice were measured 12 weeks after TAC. LVIDs (D) or %EF (E) as measured by echocardiogram at 12 weeks after TAC. *P<0.01.
RT-PCR was used to measure mRNA expression of known markers of cardiac hypertrophy (F) and MEF2-regulated genes (G); n=8, *P<0.05.
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Gold et al
HDAC kinase can delay HF and may be advantageous in therapeutic intervention by allowing some signaling.
Because GRK5 plays a dual role in the cardiomyocyte—
membrane-associated GPCR desensitizing kinase and nuclear
kinase facilitating transcription—the most critical activities for
cardiac signaling and function are uncertain. Classic GRK5 activity toward β-adrenergic receptors (βARs) has been shown to
induce transactivation of the cardioprotective epidermal growth
factor receptor.13 A human polymorphism of GRK5 (Q41L) appears to increase βAR desensitization, protecting some HF patients chronically.14 Additionally, transgenic overexpression of
Gαq caused a slight cardiac dilatation in GRK5gKO mice compared with WT controls.15 Therefore, there is some question as
to whether the increased GRK5 is protective or injurious.
In the present study, simply decreasing myocyte GRK5,
either completely in GRK5gKO mice or significantly in
GRK5cKO mice, attenuated cardiac hypertrophy and prevented pathogenesis of HF. We show that GRK5 ablation does
not completely prevent hypertrophy but significantly delays it.
Therefore, despite potential beneficial activities at the plasma
membrane, removing cardiomyocyte GRK5 has a profound
positive effect on outcomes after pressure overload and chronic
α-adrenergic stress. Clearly, our data now indicate that GRK5,
and, most likely, its nuclear HDAC kinase activity, represent
a novel target to prevent maladaptive cardiac hypertrophy and
protect against ventricular decompensation and HF.
Acknowledgments
We thank Zuping Qiu for excellent technical support.
Sources of Funding
This research was supported in part by National Institutes of Health
grants P01 HL091799 (to W.J.K.) and P01 HL07544 Project 2 (to
W.J.K.) and a Pre-Doctoral Fellowship Award from the American
Heart Association Great Rivers Affiliate (to J.I.G.).
Disclosures
None.
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KS, Martin JL, Bossuyt S, Robia SL, Bers DM. Spatiotemporally distinct
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Novelty and Significance
What Is Known?
• G protein-ncoupled receptor kinase 5 (GRK5) is upregulated in numerous models of heart failure (HF), as well as in the failing human heart.
• GRK5 enters the nucleus and acts as a histone deacetylase 5 (HDAC5)
kinase, increasing transcription of cardiac hypertrophy genes.
• Increased nuclear GRK5 is pathological in the setting of chronic
pressure overload.
What New Information Does This Article Contribute?
• Ablation of GRK5 significantly delays maladaptive cardiac remodeling and HF
after chronic pressure overload or α-adrenergic receptor (αAR) stimulation.
• Removing nuclear GRK5 by global ablation decreases HDAC5 export.
• Deletion of GRK5 in cardiomyocytes alone significantly delays the onset of HF.
Pathological cardiac hypertrophy, a process commonly ending
in HF, occurs through activation of nodal signal transducer, Gq.
Downstream of Gq, class II HDACs represses hypertrophic gene
transcription. GRK5, a recently identified HDAC kinase, has
been shown to be upregulated in human HF, although the is unknown. Here, we investigated the role of endogenous GRK5 in
maladaptive cardiac remodeling. Our results show that global
and cardiomyocyte-specific ablation of GRK5 significantly attenuates pathological cardiac hypertrophy, delaying HF onset.
This cardioprotection may be attributed to decreased nuclear
HDAC5 export after GRK5 deletion. Overall, this study suggests
that endogenous nuclear GRK5 plays an injurious role in maladaptive hypertrophy and may represent a novel therapeutic
target.
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Determining the Absolute Requirement of G Protein−Coupled Receptor Kinase 5 for
Pathological Cardiac Hypertrophy: Short Communication
Jessica I. Gold, Erhe Gao, Xiying Shang, Richard T. Premont and Walter J. Koch
Circ Res. 2012;111:1048-1053; originally published online August 2, 2012;
doi: 10.1161/CIRCRESAHA.112.273367
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2012 American Heart Association, Inc. All rights reserved.
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Supporting Information Materials and Methods
Generation of Conditional GRK5 knockout mice. . All animal procedures and
experiments were performed in accordance with the guidelines of the Institutional Animal
Care and Use Committee of Thomas Jefferson University. The GRK5/flox line was
created as described in Gainetdinov et al, 19991, except that a Cre transfected, GRK5targeted ES cell line was selected for deletion of the TK-NEO marker cassette but
maintenance of the exon 7/8 region of the GRK5 gene flanked by loxP sites. These
GRK5/flox cells were microinjected into day 3.5 blastocysts (c57BL/6J) and implanted
into pseudopregnant mice to generate founder chimeric pups. After germline
transmission was verified, GRK5/flox mice were backcrossed with c57BL/6J mice for
>12 generations. An initial cross between GRK5flox/flox and αMHC-Cre mice led to a
cardiac-specific GRK5 deletion. This conditional deletion was maintained by continued
breeding of the GRK5flox/flox with GRK5flox/flox/αMHC-Cre+/-. Only 8 week-old males from
the above cross were utilized in our experiments. Genotyping was performed using
specific PCR primers designed for the loxP site.
TAC. Transverse aortic constriction was performed as described previously2. Briefly, 8
week-old mice were sedated in an isoflurane sedation box (induction 3% and
maintenance 5%) and anesthetized to a surgical plane with an i.p. dose of ketamine
(50mg/kg) and xylazine (2.5mg/kg). Anesthetized mice were intubated using a blunt 20gauge needed that was connected to a volume-cycle rodent ventilator on supplemental
oxygen at a rate of 1 L/min and respiratory rate of 140 bpm/min. A midline cervical
incision was made to expose the trachea, carotid arteries and rib cage. Aortic
constriction was performed by tying a 7.0 nylon suture ligature against a 27-gauge
needle that was promptly removed to yield a 0.4 mm constriction. Pressure gradients
were determined by in vivo echocardiography of the transverse aorta and mice with
gradients greater than 30 mmHg were used.
Echocardiography. Echocardiography was performed as previously described3. To
measure global cardiac function, echocardiography was performed at 8 weeks of age
prior to TAC and at 1, 2, 4, 8, and 12 weeks post-TAC by use of the VisualSonics VeVo
770 imagingsystem with a 707 scan head in anesthetized animals (1.5% isoflurane,
vol/vol). The internal diameter of the left ventricle was measure in the short-axis view
from M-mode recordings in end diastole and end systole. At 1-week post-TAC, pressure
gradients were determined by PW Doppler mode.
Mini-osmotic pumps. Chronic infusion of phenylephrine (Sigma) was achieved using
Alzet 2 week osmotic minipumps (model 2002, DURECT Corporation). Pumps were
filled following the manufacturer’s specifications with sterile PBS, or PE (35µM/kg/day).
Briefly, Mice were anesthetized with isofluorane (2.5% vol/vol) and pumps were
implanted subcutaneously through a sub-scapular incision, which was then closed using
4.0 silk suture (Ethicon). The contents of the pumps were delivered at a rate of
0.5µl/hour for 2 weeks. Mice were followed by echocardiogram prior to pump
implantation and then weekly until the end of the 2 week period when mice were
euthanized.
RNA Isolation and Semiquantitative PCR. RNA isolation and analysis was performed
as previously described2. Total RNA isolation was performed using the Ultraspec RNA
Isolation System (Biotecx), according to the manufacturer’s protocol and as previously
1
described2. After RNA isolation, cDNA was synthesized from 200ng of total RNA using
the iScript cDNA Synthesis Kit (Bio-Rad Laboratories). Semiquantitative PCR was
carried out on cDNA using iQ SYBR Green Supermix (Bio-Rad Laboratories) and 100
nM of the following gene-specific oligonucleotides—18S, ANF, βMHC, BNP, Col1a2,
CTGF, and Acta-1. Quantitation was established by comparing 18s rRNA, which was
similar between groups, for normalization. For each run, saturation of amplification
cycles was controlled by the use of MyiQ software (version 1.0); subsequently, a melting
curve was generated by heating the product to 95°C, cooling to and maintaining at 55°C
for 20 seconds, and then slowly (0.5°C/s) heating to 95°C to determine the specificity of
PCR products, which was then confirmed by gel electrophoresis.
Immunoblot Analysis. Isolated cardiac tissue was first homogenized using a Dounce
homogenizer in a buffer containing: 4mM Hepes, 320mM sucrose, 10mM KCL, 5mM
EDTA, 2mg NaF, 8mg MgCl2, .1% Triton x-10, 1.094g DTT, and protease inhibitors. The
homogenate was filtered through a 70µm cell filter. Total cell lysate was taken at this
point. Then the lysate was subjected the same protocol as previously described for
NRVM2. Following subcellular fractionation, 500 µg of non-nuclear protein was
immunoprecipitated with protein A/G and anti-HDAC5 (sc-133106, Santa Cruz). Proteins
were subjected to SDS-PAGE, and immunoblotted with antiphospho-HDAC5 (PA114187, Thermo Scientific), and anti-HDAC5 (sc-133106, Santa Cruz). Visualization of
Western blot signals was performed using secondary antibodies coupled to Alexa Fluor
680 or 800 (Molecular Probes) on a LI-COR infrared imager (Odyssey). Pictures were
processed by Odyssey version 1.2 infrared imaging software. All densitometry scans
were carried out in the linear range of detection.
Statistics. All the values in the text and figures are presented as mean ± SEM from
given n sizes. Statistical significance of multiple treatments was determined by one-way
ANOVA followed by the Bonferroni’s post hoc test when appropriate. Statistical
significance between two groups was determined using the two-tailed Student’s t test. P
values of <0.05 were considered significant.
Supplemental References
1. Gainetdinov RR, Bohn LM, Walker JK, Laporte SA, Macrae AD, Caron MG, Lefkowitz
RJ, Premont RT. 1999. Muscarinic supersensitivity and impaired receptor
desensitization in G protein-coupled receptor kinase 5-deficient mice. Neuron.
24:1029-36
2. Martini JS, Raake P, Vinge LE, DeGeorge BR, Jr., Chuprun JK, Harris DM, Gao E,
Eckhart AD, Pitcher JA, Koch WJ. 2008. Uncovering G protein-coupled receptor
kinase-5 as a histone deacetylase kinase in the nucleus of cardiomyocytes.
Proceedings of the National Academy of Sciences of the United States of
America. 105:12457-62
3. Brinks H, Boucher M, Gao E, Chuprun JK, Pesant S, Raake PW, Huang ZM, Wang
X, Qiu G, Gumpert A, Harris DM, Eckhart AD, Most P, Koch WJ. 2010. Level of
Gprotein-coupled receptor kinase-2 determines myocardial ischemia/reperfusion
injury via pro- and anti-apoptotic mechanisms. Circulation research. 107:1140-9
2
Online Supplemental Figure Legends
Online Fig. I Representative Echocardiogram tracings from WT and GRK5gKO mice 12
weeks following sham or TAC operations
Online Fig. II GRK5Flox/αMHC-Cre+ mice have a greater than 50% knockdown of
GRK5. (A) To determine GRK5 expression in our αMHC-Cre system, hearts from
GRK5Flox/αMHC-Cre- (WT) and GRK5Flox/αMHC-Cre+ (cKO) were homogenized and
immunoblotted for GRK5 and GAPDH. (B) Quantitative analysis of immunoblots for
GRK5 expression in the hearts of (A). n=4, *p<0.01. (C) Semi-quantitative PCR was
used to assess GRK5 mRNA levels in the hearts of WT or cKO mice. n=6, *p<0.05
Online Fig. III Representative Echocardiogram tracings from WT and GRK5cKO mice
12 weeks following sham or TAC operations.
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Online Supplemental Figure I
4
Online Supplemental Figure II
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Online Supplemental Figure III
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