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Myocardial expression and redistribution of GRKs in hypertensive hypertrophy and failure.

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THE ANATOMICAL RECORD PART A 282A:13–23 (2005)
Myocardial Expression and
Redistribution of GRKs in
Hypertensive Hypertrophy and
Failure
XIAN PING YI,1,2 JIBIN ZHOU,1 JUSTIN BAKER,1 XUEJUN WANG,1
A. MARTIN GERDES,1 AND FAQIAN LI1,3*
1
South Dakota Health Research Foundation-Cardiovascular Research Institute,
University of South Dakota School of Medicine, Sioux Falls, South Dakota
2
Department of Pathology, Zhongshan University the Fifth Affiliated Hospital,
Guangdong, China 3Department of Laboratory Medicine and Pathology, University
of South Dakota School of Medicine, Sioux Falls, South Dakota
ABSTRACT
G-protein-coupled receptor kinases (GRKs) are involved in cardiac hypertrophy and failure. But their temporal
expression and cellular localization during the development of hypertrophy and its transition to failure remains to be
investigated. In this study, we determined the expression and subcellular distribution of GRK2, GRK3, GRK5, and
GRK6 in cardiac myocytes of 2- to 24-month-old spontaneously hypertensive heart failure (SHHF) rats. GRK2 increased
in the intercalated disks in 6-, 12-, and 24-month-old SHHF rats, although total expression remained relatively
constant from 2 to 24 months in both SHHF and normotensive rats. GRK3 expression progressively increased in 6-, 12-,
and 24-month-old SHHF rats and was significantly higher than in age-matched controls. Immunolabeling of GRK3
showed a typical pattern of cross-striations that colocalized with ␣-actinin and G␣s at Z-lines in both SHHF and control
rats. GRK5 expression showed no change from 2 to 24 months in both SHHF and normotensive rats. Confocal analysis
revealed nuclear translocation of GRK5 in myocytes of SHHF rats. GRK6 had a striated pattern colocalized with
␣-actinin at Z-lines in the cytoplasm and was also present in the intercalated disks of cardiac myocytes from both SHHF
and control rats. GRK6 expression increased in 12- and 24-month-old SHHF rats and was significantly higher than in
age-matched controls. GRK6 labeling was reduced at the intercalated disks, but increased in the cytoplasm of cardiac
myocytes from SHHF rats compared to age-matched controls. The increased expression of GRK3 and GRK6 and
subcellular redistribution of GRK2, GRK5, and GRK6 in SHHF rats may be involved in abnormal remodeling of cardiac
myocytes in hypertensive hypertrophy and failure. © 2004 Wiley-Liss, Inc.
Key words: hypertrophy; hypertension; heart failure; G-proteins; kinase; cell signaling
Hypertension is often associated with the development
of cardiac hypertrophy, which frequently deteriorates into
congestive heart failure (CHF). Although mechanical load
appears to regulate directly cardiac mass and associated
phenotypic changes, the exact mechanisms that couple
load to the initiation of hypertrophic growth and its transition into CHF have yet to be delineated. Several signal
pathways including G-proteins (Akhter et al., 1998) and
G-protein-coupled receptor kinases (GRKs) (Anderson et
al., 1999; Yi et al., 2002), focal adhesion kinases (FAK)
(Laser et al., 2000; Yi et al., 2003), mitogen-activated
protein kinase family members (Aikawa et al., 2002), protein kinase C (Braz et al., 2002), and p70/85 S6 kinase
(Laser et al., 1998) have been implicated in the regulation
of cardiac hypertrophy and failure.
GRKs, a family of serine/threonine kinases, are comprised of seven family members, GRK1 to 7 (Penn et al.,
2000). They distribute widely in a variety of tissues and
©
2004 WILEY-LISS, INC.
are subdivided into three subgroups based on their sequence and functional homology: kinases subfamily for
Grant sponsor: the National Institutes of Health; Grant number: HL 62459; Grant sponsor: National Center for Research
Resources (NCRR), Grant number: P20 RR017662; Grant sponsor: the South Dakota Health Research Foundation (a partnership between the University of South Dakota School of Medicine
and Sioux Valley Hospital and Health Systems).
*Correspondence to: Faqian Li, South Dakota Health Research
Foundation-Cardiovascular Research Institute, 1100 East 21st
Street, 7th Floor, Sioux Falls, SD 57105. Fax: 605-328-1301.
E-mail: fli@usd.edu
Received 15 April 2004; Accepted 25 August 2004
DOI 10.1002/ar.a.20143
Published online 6 December 2004 in Wiley InterScience
(www.interscience.wiley.com).
14
YI ET AL.
the photoreceptor rhodopsin, including GRK1 and GRK7;
␤-adrenergic receptor (AR) kinases (␤-ARKs) subfamily,
consisting of GRK2 (␤-ARK1) and GRK3 (␤-ARK2); and
GRK4 subfamily, containing GRK4, GRK5, and GRK6.
The major GRKs expressed in the heart are GRK2, GRK3,
GRK5, and GRK6 (Penn et al., 2000; Ferguson, 2001).
GRKs are able to phosphorylate and desensitize agonistoccupied cell surface G-protein-coupled receptors (GPCRs)
at both serine and threonine residues leading to functional
uncoupling and altered downstream signal transduction
(Penn et al., 2000; Brady and Limbird, 2002). GRK-mediated ␣- and ␤-AR desensitization has been shown to occur
in cardiac hypertrophy and CHF (Eckhart et al., 2000;
Rapacciuolo et al., 2001) and is considered to be an important alteration leading to cardiac contractile dysfunction
(Pitcher et al., 1999; Ferguson, 2001). The temporal
change and cellular localization of individual GRK during
the development of hypertrophy and its transition to failure, however, have not been systematically investigated.
The spontaneously hypertensive heart failure (SHHF)
rat is a genetic rat model developing hypertension at an
early age (Gerdes et al., 1996), cardiac hypertrophy by 4
months of age, and eventually CHF (Tamura et al., 1998).
SHHF rats exhibit numerous pathologic, morphologic, and
biochemical changes that parallel documented changes in
patients with hypertension. The evaluation of SHHF rats
at different pathological stages should facilitate the elucidation of mechanisms involved in the progression from
cardiac hypertrophy to CHF. Our previous studies have
shown that significant subcellular redistribution of GRK2
and GRK5 occurred in cardiac myocytes of 6-month-old
SHHF rats without obvious changes in their expression
levels. The accumulation of GRK2 in the intercalated
disks and the nuclear translocation of GRK5 in SHHF rat
myocytes suggest different roles of GRK2 and GRK5 in
G-protein signaling, RNA biogenesis, and hypertrophic
gene transcription during cardiac hypertrophy resulting
from chronic hypertension (Yi et al., 2002). In the present
study, we further determined the expression and subcellular distribution of GRK2, GRK3, GRK5, and GRK6 in
cardiac myocytes of SHHF rats during the progression
from hypertrophy to failure using Western blots, immunolabeling, and confocal microscopy.
Fluor 488- or 568-labeled goat antirabbit IgG or goat antimouse IgG antibodies were purchased from Molecular
Probes (Eugene, OR). Horseradish peroxidase-linked donkey antirabbit IgG antibody was obtained from Amersham
Pharmacia Biotech (Piscataway, NJ).
Myocyte Isolation, Immunofluorescent
Labeling, and Confocal Microscopy
Cardiac myocytes were enzymatically isolated using a
standard procedure as described previously (Li et al.,
1996). The isolated cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min. After
quenching paraformaldehyde with 0.1 mol/L glycine in
PBS for 30 min, myocytes were suspended in PBS for
immunofluorescent labeling. Cardiac myocyte suspensions were aliquoted onto positively charged slides, permeated with 0.5% Triton X-100 for 30 min, and washed
with PBS. After blocking with 1% bovine serum albumin,
the attached myocytes were incubated with a primary
antibody at 4°C overnight and washed with PBS. Then
myocytes were incubated with fluorescence-conjugated
secondary antibody for 1 hr at room temperature and
washed with PBS. The same procedure was repeated with
a second set of antibodies for double labeling. The nucleus
was counterstained with propidium iodide. The slides
were mounted in 60% glycerol in PBS and sealed with nail
polish for observation using an Olympus Fluoview Confocal Laser Scanning Microscope System (Olympus America, Melville, NY). Negative controls were incubated with
the same primary antibody solution after neutralization
with specific blocking peptides or with the omission or
substitution of primary antibodies with rabbit serum under the same conditions. Cardiac myocytes labeled without primary antibody or with substitution of primary antibodies with rabbit serum only showed weak diffuse
background fluorescence. The striation and specific labeling of GRK2, GRK3, GRK5, and GRK6 in the intercalated
disk and nucleus were not present when primary antibodies of GRK2, GRK3, GRK5, and GRK6 were neutralized
with specific blocking peptides.
Tissue Lysate Preparation
MATERIALS AND METHODS
Animals
Two-, 6-, 12-, and 24-month-old lean female SHHF rats
were acquired from Charles River (Wilmington, MA) and
Genetic Models (Indianapolis, IN). Age-matched female
Wistar-Kyoto (WKY) rats from Harlan (Indianapolis, IN)
were used as normotensive controls. All procedures were
performed in accordance with the Guide for the Care and
Use of Laboratory Animals published by the U.S. Department of Health, Education, and Welfare, Department of
Health and Human Services, and the protocols were approved by the University of South Dakota Animal Care
and Use Committee.
Antibodies
Anti-GRK2, -GRK3, -GRK5, -GRK6, and G␣s rabbit
polyclonal antibodies and their blocking peptides were
obtained from Santa Cruz Biotechnology (Santa Cruz,
CA). Anti-␣-actinin and anti-N-cadherin monoclonal antibodies were acquired from Sigma (St. Louis, MO). Alexa
Whole tissue lysates from left ventricles (LVs) were
prepared and separated into Triton X-100-soluble fractions (TSFs) and -insoluble fractions (TIFs) as described
(Laser et al., 2000). LV free wall tissues were homogenized
using a PT1200C Polytron in ice-cold Tris/Triton extraction buffer (10 mmol/L Tris-HCl, 50 mmol/L Na2Cl, 5
mmol/L EGTA, 1% Triton X-100, pH 7.4) containing phosphatase inhibitors (0.1 mmol/L Na3VO4, 30 mmol/L
Na4P2O7, 50 mmol/L NaF) and proteinase inhibitors (10
mg/ml phenylmethylsulfonyl fluoride, 1 ␮g/ml aprotinin).
The homogenates were centrifuged at 15,000 g for 15 min
at 4°C. The supernatants were saved as TSF for further
analysis. The pellet was suspended, vortexed, then boiled
in 2 ⫻ stock Laemmli sodium dodecyl sulfate (SDS) sample buffer (0.125 mmol/L Tris-HCl, pH 6.8, 10% SDS, 20%
sucrose) for 5 min. After centrifuging for 5 min at 15,000
g at 4°C, the supernatants were used as the TIF for Western blots. The TSF contains both cytosolic and membraneassociated proteins while the TIF only cytoskeletal proteins.
GRK IN HYPERTENSIVE HYPERTROPHY AND FAILURE
15
Protein Separation, Electrophoresis, and
Western Blots
One-dimensional SDS-polyacrylamide gel electrophoresis of Laemmli was used to separate proteins on the basis
of their molecular weight. Protein content was determined
for each sample using the method of Bradford. Fifty micrograms of protein from each LV sample were mixed with
Laemmli SDS sample buffer (2 ⫻ stock Laemmli SDS
sample buffer, 5% 2-mercaptoethenol, 0.005% bromophenol blue), then the samples were boiled and electrophoresed on 4 –20% linear gradient Ready Gels (Bio-Rad, Hercules, CA). The proteins were then transferred onto
nitrocellulose membranes. The membranes were blocked
with 3% BSA and 5% dry milk in PBS for 1 hr and probed
with primary antibodies to GRK2, GRK3, GRK5, and
GRK6. After thorough washing, the bound antibodies
were visualized with horseradish peroxidase-conjugated
antirabbit IgG antibody and the enhanced chemiluminescence technique (Amersham life Science). To confirm the
specificity of migrating band, specific GRK2, GRK3,
GRK5, and GRK6 blocking peptides (Santa Cruz Biotechnology) were used in a neutralization assay. A 10-fold
excess of peptide to antibody (wt/wt) was incubated with
the corresponding diluted antibody for 2 hr and the neutralized antibody was used as primary antibody. Densities
of protein bands with specific antibody were scanned and
quantified with NIH image software. The mean density of
each band was regarded as a density unit (du) for relative
GRK protein content.
RNA Isolation and RT-PCR
Total RNA of the LV from 6- and 12-month-old SHHF
and WKY rats was isolated with the RNeasy Midi Kit from
Qiagen (Valencia, CA) according to the manufacturer’s
protocol. RT-PCR was carried out with primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), GRK2,
and GRK3 as published (Ungerer et al., 1996; Xiao et al.,
1998). Quantification of band density was performed as
described for Western blot.
Statistics
Data are expressed as mean ⫾ SEM. Student’s t-test
was performed to examine significant differences between
age-matched SHHF and WKY rats. Differences between
different age groups were compared with one-way analysis of variance (ANOVA). Bonferroni t-test was performed
to examine significant differences observed with ANOVA.
Probability less than 5% was regarded as significant.
RESULTS
GRK2 and GRK3 Expression in LV of SHHF
Rats
Our previous study showed no significant change of
GRK2 expression in the LV between 6-month-old SHHF
and WKY rats (Yi et al., 2002). Anderson et al. (1999)
found that GRK2/3 expression progressively increased
during progression to failure in SHHF rats using an antibody against both GRK2 and GRK3. To characterize
expression pattern of GRKs in the ␤-ARK subfamily during the transition from hypertrophy to failure, we used
antibodies specific to GRK2 or GRK3 to perform Western
blots in the LV of 2-, 6-, 12-, and 24-month-old SHHF and
age-matched WKY rats. The results demonstrated that
Fig. 1. GRK2 and GRK3 expression in the LV of SHHF and WKY rats.
A: Representative Western blots (n ⫽ 6; top) show similar levels of GRK2
in the TSF of the LV from 2- (2M), 6- (6M), 12- (12M), and 24- (24M)
month-old SHHF and WKY rats. Densitometry data (bottom) also demonstrate that there are no significant differences in GRK2 content between SHHF and WKY rats. B: Representative Western blots (n ⫽ 6; top)
show that GRK3 expression remains at stable low levels in 2-, 6-, 12-,
and 24-month-old WKY rats, but progressively increases with age in 2-,
6-, 12-, and 24-month-old SHHF rats. Densitometry data (bottom) reveal
that there are no significant differences in GRK3 content between SHHF
and WKY rats at 2 months, but GRK3 content in 6-, 12-, and 24-monthold SHHF rats increases with age and is significantly greater than that in
age-matched WKY rats (asterisk, P ⬍ 0.05 between age-matched SHHF
and WKY rats; dagger, P ⬍ 0.05 compared with 2-month-old SHHF rats;
double dagger, P ⬍ 0.05 compared with 6-month-old SHHF rats).
both GRK2 and GRK3 were expressed mainly in the TSF
of the LV without detectable levels in the TIF. There were
no obvious changes of GRK2 expression among 2-, 6-, 12-,
and 24-month-old SHHF and WKY rats (74 ⫾ 8 vs. 79 ⫾ 7,
77 ⫾ 6 vs. 75 ⫾ 7, 78 ⫾ 7 vs. 74 ⫾ 7, and 77 ⫾ 8 vs. 73 ⫾
7 du; n ⫽ 6; P ⬎ 0.05; Fig. 1A). GRK3 expression did not
change with age in WKY rats (10 ⫾ 2, 10 ⫾ 2, 12 ⫾ 2, and
11 ⫾ 2 du; n ⫽ 6; P ⬎ 0.05; Fig. 1B), but increased
16
YI ET AL.
progressively with age in SHHF rats (13 ⫾ 2, 34 ⫾ 1, 54 ⫾
5, and 57 ⫾ 5 du; Fig. 1B) and was significantly greater
than that in age-matched control rats at 6, 12, and 24
months (34 ⫾ 1 vs. 10 ⫾ 2, 54 ⫾ 5 vs. 12 ⫾ 2, and 57 ⫾ 5
vs. 11 ⫾ 2 du; n ⫽ 6; P ⬍ 0.05; Fig. 1B).
The expressional change of GRK2 and GRK3 was confirmed by RT-PCR on RNA isolated from 6- and 12-monthold SHHF and WKY rats. There is no change of GRK2
expression among 6- and 12-month-old SHHF and WKY
rats (4.08 ⫾ 0.59 vs. 4.55 ⫾ 0.79 and 4.35 ⫾ 0.42 vs. 4.40 ⫾
0.46; n ⫽ 4; P ⬎ 0.05; Fig. 2A). On the other hand, GRK3
increased significantly in 6- and 12-month-old SHHF rats
compared to age-matched WKY rats (6.57 ⫾ 0.98 vs.
4.46 ⫾ 0.63 and 7.31 ⫾ 1.08 vs. 4.36 ⫾ 0.58; n ⫽ 4; P ⬍
0.05; Fig. 2B). The further increase of GRK3 in 12-monthold SHHF rats is not statistically significant compared
with 6-month-old SHHF rats (P ⬎ 0.05).
Subcellular Distribution of GRK2 and GRK3 in
Cardiac Myocytes of SHHF Rats
Consistent with our previous results (Yi et al., 2002),
GRK2 showed a typical striated pattern in the cytoplasm
of 2-, 6-, 12-, and 24-month-old SHHF and WKY rats.
GRK2 fluorescence was minimal in the intercalated disks
of myocytes from 2-, 6-, 12-, and 24-month-old WKY and
2-month-old SHHF rats, but remarkably increased in the
intercalated disks of myocytes from 6-, 12-, and 24-monthold SHHF rats, suggesting that GRK2 redistributed to the
intercalated disks in these age groups of SHHF rats (Fig.
3). The labeling intensity of GRK2 in the intercalated
disks was similar among 6-, 12-, and 24-month-old SHHF
rats.
GRK3 also had a similar pattern of cross-striations in
the cytoplasm in 2-, 6-, 12-, and 24-month-old SHHF and
WKY rats with no significant difference in distribution
among different groups (Fig. 4). The cross-striations of
GRK3 colocalized with sarcomeric ␣-actinin as demonstrated in Figure 4A–F. Many G-proteins such as G␣s have
also been shown in transverse tubules at Z-lines
(Laflamme and Becker, 1999). Previously we demonstrated that G␣s had a typical striation pattern colocalizing with GRK2 (Yi et al., 2002). The double labeling of
GRK3 and G␣s verified their colocalization at Z-lines (Fig.
4G–I). GRK3 appeared to be much more abundant within
the transverse tubular membrane than in the peripheral
sarcolemma (Fig. 4G) when comparing with G␣s distribution (Fig. 4H).
GRK5 and GRK6 Expression in LV of SHHF
Rats
GRK5 and GRK6 form another subfamily of the GRKs
family that is present in cardiac tissues. The expression of
GRK5 was identified in both TSF and TIF of the LV with
no difference at 6 months between SHHF and WKY rats in
our previous study (Yi et al., 2002). In this study, we
further confirmed that the expression levels of GRK5 did
not change with age in both TSF (52 ⫾ 5, 50 ⫾ 3, 52 ⫾ 7,
and 50 ⫾ 3 du; Fig. 5A) and TIF (46 ⫾ 2, 47 ⫾ 5, 47 ⫾ 5,
and 48 ⫾ 4 du; Fig. 5B) in 2-, 6-, 12-, and 24-month-old
SHHF rats. Similar levels of GRK5 were present in 2-, 6-,
12-, and 24-month-old SHHF and WKY rats in TSF (52 ⫾
5 vs. 50 ⫾ 5, 50 ⫾ 3 vs. 51 ⫾ 6, 52 ⫾ 7 vs. 51 ⫾ 5, and 50 ⫾
3 vs. 51 ⫾ 7 du; n ⫽ 6; P ⬎ 0.05; Fig. 5A) and in TIF (46 ⫾
2 vs. 46 ⫾ 3, 47 ⫾ 5 vs. 47 ⫾ 5, 47 ⫾ 5 vs. 48 ⫾ 4, and 48 ⫾
4 vs. 48 ⫾ 3 du; P ⬎ 0.05; n ⫽ 6; Fig. 5B).
GRK6 is expressed throughout the body. Its role in
hypertensive cardiac hypertrophy and failure remains unclear. Similar to GRK5, GRK6 was present in both TSF
and TIF of the LV in SHHF and WKY rats. In TSF, there
were no significant changes of GRK6 expression among 2-,
6-, 12-, and 24-month-old SHHF and WKY rats (13 ⫾ 4 vs.
12 ⫾ 5, 14 ⫾ 4 vs. 13 ⫾ 2, 14 ⫾ 3 vs. 15 ⫾ 2, and 14 ⫾ 2
vs. 15 ⫾ 2 du; n ⫽ 6; P ⬎ 0.05; Fig. 5C). GRK6 expression
remained relatively stable in TIF with age in WKY rats.
There were also no significant difference in TIF between 2and 6-month-old SHHF and WKY rats (12 ⫾ 4 vs. 13 ⫾ 4
and 12 ⫾ 5 vs. 12 ⫾ 4 du; n ⫽ 6; P ⬎ 0.05; Fig. 5D). In
contrast, GRK6 expression was significantly increased in
TIF in 12- and 24-month-old SHHF rats (Fig. 5D) and was
significantly higher than age-matched controls (55 ⫾ 6 vs.
17 ⫾ 7 and 60 ⫾ 6 vs. 12 ⫾ 5 du; n ⫽ 6; P ⬍ 0.05; Fig. 5D).
Subcellular Distribution of GRK5 and GRK6 in
Myocytes of SHHF Rats
Confocal microscopy demonstrated that GRK5 had a
weak and diffuse fluorescence in the cytoplasm with similar staining intensity among SHHF and WKY rats. In
contrast to the weak nuclear fluorescence in 2-, 6-, 12-, and
24-month-old WKY rats (Fig. 6A, C, E, and G), a remarkably bright fluorescence was observed in the nucleus of 2-,
6-, 12-, and 24-month-old SHHF rats (Fig. 6B, D, F, and
H), suggesting nuclear accumulation of GRK5 in SHHF
rats.
GRK6 was present in the intercalated disks and cytoplasm in 2-, 6-, 12-, and 24-month-old WKY rats (Fig. 7A,
C, E, and G). Fluorescent intensity in the intercalated
disks and cytoplasm had no significant changes with age
in WKY rats. In 2-month-old SHHF rats (Fig. 7B), GRK6
fluorescence in the intercalated disks had similar intensity to age-matched WKY rats. The staining of GRK6 in
the intercalated disks was obviously weaker in 6-, 12-, and
24-month-old SHHF rats (Fig. 7D, F, and H) when compared to age-matched WKY rats. In contrast, the cytoplasmic staining of GRK6 increased in 12- and 24-month-old
SHHF rats (Fig. 7F and H) and showed a typical crossstriation pattern. These results suggest that GRK6 relocates away from the intercalated disks to the cytoplasm in
cardiac myocytes of SHHF rats.
Colocalization of GRK6 With N-Cadherin and
Sarcomeric ␣-Actinin
N-cadherin is a marker for the intercalated disks (Wang
and Gerdes, 1999). The double labeling revealed that the
staining of GRK6 in the intercalated disks colocalized
with N-cadherin (Fig. 8A–F). The abundance of N-cadherin was similar among SHHF and WKY rats, so it can
be used as a reference standard for analyzing other intercalated disk-associated proteins. Because of the remarkable accumulation of GRK6 in the intercalated disks of
WKY rats, a bright yellow color was observed in the intercalated disks of WKY rats (Fig. 8C) when the bright
green fluorescence of GRK6 was overlaid with the red
fluorescence of N-cadherin. In contrast, only a red or orange color was seen in the intercalated disks of SHHF rats
when the weak green fluorescence of GRK6 was overlaid
with the red fluorescence of N-cadherin (Fig. 8D–F), reflecting the reduction of GRK6 fluorescence in the intercalated disks in 6-, 12-, and 24-month-old SHHF rats. In
the cytoplasm, GRK6 demonstrated a typical cross-stria-
Fig. 2. RT-PCR of GRK2 (A) and GRK3 (B) in the LV of SHHF and
WKY rats. A: Similar levels of GRK2 (expected size, 334 bp) are present
among 6- and 12-month-old SHHF and WKY rats. B: GRK3 (expected
size, 617 bp) increases significantly in 6- and 12-month-old SHHF rats
compared to WKY controls. There is a further increase of GKR3 in
12-month-old SHHF rats, but the difference is not statistically significant
among 6- and 12-month-old SHHF rats. GPDH (expected size, 617 bp)
remains constant among SHHF and WKY rats. bp, base pair; CTL,
control without reverse transcription; M, DNA molecular weight marker.
Asterisk, P ⬍ 0.05 between age-matched SHHF and WKY rats.
18
YI ET AL.
Fig. 3. Representative confocal images of GRK2 in cardiac myocytes. GRK2 has a striated pattern in the cytoplasm of 2-, 6-, 12-, and
24-month-old SHHF and WKY rats. GRK2 fluorescence is weak in the
intercalated disks in 2-, 6-, 12-, and 24-month-old WKY (A, C, E, and G,
respectively), and 2-month SHHF rats (B), but remarkably increased in
the intercalated disks in 6-, 12-, and 24-month-old SHHF rats (D, F, and
H, respectively). Scale bar ⫽ 50 ␮m.
Fig. 4. Double labeling of GRK3 (green; A, D, and G) with ␣-actinin (red; B and E) or G-protein ␣s (G␣s;
red; H) in cardiac myocytes from 6-month-old WKY (A–C) and SHHF rats (D–I). The cross-striations of GRK3
colocalize with that of ␣-actinin (B and E) or G␣s (H). Right column, overlays (C and F, GRK3 ⫹ ␣-actinin; I,
GRK3 ⫹ G␣s). Scale bar ⫽ 50 ␮m.
GRK IN HYPERTENSIVE HYPERTROPHY AND FAILURE
19
Fig. 5. GRK5 and GRK6 expression in the LV of 2- (2M), 6- (6M), 12(12M), and 24- (24M) month-old SHHF and WKY rats. A: Representative
Western blots (n ⫽ 6; top) show similar levels of GRK5 in the TSF of the
LV from 2-, 6-, 12-, and 24-month-old SHHF and WKY rats. Densitometry data (bottom) also demonstrate that there are no significant differences in GRK5 content between SHHF and WKY rats. B: Representative
Western blots (n ⫽ 6; top) show similar expression of GRK5 in the TIF of
the LV from 2-, 6-, 12-, and 24-month-old SHHF and WKY rats. Densitometry data (bottom) reveal that there are no significant differences in
GRK5 content between SHHF and WKY rats. C: Representative Western
blots (n ⫽ 6; top) show similar expression of GRK6 in the TSF of the LV
from 2-, 6-, 12-, and 24-month-old SHHF and WKY rats. Densitometry
data (bottom) demonstrate that there are no significant differences in
GRK6 content between SHHF and WKY rats. D: Representative Western
blots (n ⫽ 6; top) show that similar low levels of GRK6 are present in the
TIF of the LV from 2-, 6-, 12-, and 24-month-old WKY and 2- and
6-month-old SHHF rats, but its levels significantly increased in 12- and
24-month-old SHHF rats. Densitometry data (bottom) demonstrate that
there are no significant differences in GRK6 content in the TIF between
2- and 6-month-old SHHF and WKY rats, but GRK6 content in 12- and
24-month-old SHHF rats is significantly greater than that in agematched WKY rats. (asterisk, P ⬍ 0.05 between age-matched SHHF and
WKY rats; dagger, P ⬍ 0.05 compared with 2- or 6-month-old SHHF
rats).
tion pattern colocalizing with ␣-actinin (Fig. 8G–I) as revealed by the double labeling of GRK6 with sarcomeric
␣-actinin.
are coupled to the modulation of intracellular signals
that are responsible for changes in myocyte size and
shape. In an effort to understand the roles of GRK
signaling events in cardiac hypertrophy and failure resulting from genetic hypertension, we have investigated
the expression and subcellular distribution of four
members of the GRK family, GRK2, GRK3, GRK5, and
GRK6, that are present in the heart during the transition from concentric to eccentric hypertrophy and to
CHF in 2-, 6-, 12-, and 24-month-old SHHF rats.
GRK activity can be regulated by either changes in
expression levels or subcellular localization in addition
DISCUSSION
Previous studies have shown that remodeling of cytoskeleton and intercalated disks is the cellular basis
for myocyte lengthening, ventricular dilatation, and
contractile dysfunction during pressure-overloaded cardiac hypertrophy (Tagawa et al., 1996; Wang and Gerdes, 1999; Wang et al., 1999). A key question that has
not yet been answered is how changes in cardiac load
20
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Fig. 6. Immunofluorescent labeling of GRK5 in LV myocytes from 2-,
6-, 12-, and 24-month-old WKY and SHHF rats. Very weak green fluorescence distributes diffusely in the cytoplasm of 2-, 6-, 12-, and 24month-old WKY (A, C, E, and G, respectively) and SHHF rats (B, D, F,
and H, respectively). Remarkably bright green fluorescence is present in
nuclei of 2-, 6-, 12-, and 24-month-old SHHF rats (B, D, F, and H,
respectively), in contrast with very weak green fluorescence in nuclei of
control WKY rats (A, C, E, and G). Scale bar ⫽ 50 ␮m.
to the modulation in intrinsic kinase activity (Penn et
al., 2000). GRK2 and GRK3 mRNA expression level and
activity increase during CHF in human hearts (Ungerer
et al., 1994). GRK2, however, shows no change in a
porcine model of heart failure (Ping et al., 1997). In
SHHF rats, GRK2 and GRK3 expression progressively
increases with age and is significantly greater than that
in control rats at both 14 and 20 months (Anderson et
al., 1999) when an anti-GRK2/3 (C5/1) monoclonal antibody, which can react with both GRK2 and GRK3
(Oppermann et al., 1996), was used to examine GRK2/3
protein levels. We have shown here that GRK2 protein
level remains relatively unchanged with age in SHHF
rats consistent with our previous report (Yi et al., 2002).
GRK2, however, redistributes into the intercalated
disks in SHHF rats at 6 months of age when cardiac
myocytes begin to lengthen. On the other hand, GRK3
expression is increased with age especially after 12
months of age in SHHF, but not WKY rats. Therefore,
increased levels of GRK2/3 in 14- and 20-month-old
SHHF rats reported in an earlier study (Anderson et al.,
1999) are most likely due to higher levels of GRK3
rather than GRK2. The distributional pattern of GRK3,
however, is not altered in either animal. GRK2 and
GRK3 have been considered isozymes because they
share an overall 85% homology in amino acid with approximate 95% homology in the catalytic domain. Recent studies, however, have shown that GRK2 and
GRK3 may play distinct roles in the regulation of myocardial function in vivo (Iaccarino et al., 1998; Eckhart
et al., 2000). Both GRK2 and GRK3 colocalize with
␣-actinin and G␣s at Z-lines. The approximation of
GRKs, ␣-actinin, and G␣s in cardiac myocytes may regulate GRK activity and G-protein signaling since ␣-actinin inhibits GRK activity (Freeman et al., 2000). Interestingly, GRK2, but not GRK3, associates with the
intercalated disks, which also contain abundant G␣s
(Laflamme and Becker, 1999). The accumulation of
GRK2 in the intercalated disks becomes apparent in
6-month-old SHHF rats during myocyte lengthening,
which also starts at about 6 months of age. The role of
GRK2 redistribution to the intercalated disks during
myocyte lengthening is presently unknown. Other cell
signaling molecules, such as FAK and p130Cas, have
been shown to accumulate at intercalated disks during
cardiac hypertrophy both in vitro and in vivo (KovacicMilivojevic et al., 2001; Yi et al., 2003). The accumulation of signaling molecules in the intercalated disks
might promote preferential addition of sarcomeres in
series rather than parallel.
GRK IN HYPERTENSIVE HYPERTROPHY AND FAILURE
21
Fig. 7. Immunofluorescent labeling of GRK6 in LV myocytes from 2-,
6-, 12-, and 24-month-old WKY and SHHF rats. GRK6 localizes in the
intercalated disks and cytoplasm with the typical cross-striation in 2-, 6-,
12-, and 24-month-old WKY (A, C, E, and G, respectively) and SHHF rats
(B, D, F, and H). The fluorescent intensity is similar in different age
groups of WKY rats. However, GRK6 fluorescence at the intercalated
disks obviously weakens in 6-, 12-, and 24-month-old SHHF rats (D, F,
and H, respectively) when comparing with age-matched WKY rats (C, E,
and G) except GRK6 fluorescence at the intercalated disks in 2-monthold SHHF rats (B) has similar intensity to age-matched WKY rats (A). In
addition, the fluorescent intensity of GRK6 progressively increased in
cytoplasm with aging in SHHF rats (B, D, F, and H). Scale bar ⫽ 50 ␮m.
GRK5 expression shows no change with age in both
SHHF and WKY rats. Immunolabeling demonstrates
nuclear redistribution of GRK5 in cardiac myocytes of
SHHF rats. Interestingly, the nuclear redistribution of
GRK5 is detected as early as 2 months of age before
myocyte lengthening begins, indicating it might not be
directly involved in myocyte elongation. Nuclear GRK5
colocalizes with Cajal bodies (Yi et al., 2002), which are
involved in transcription and processing of nuclear RNA
(Gall, 2000). Thus, GRK5 may be a potential candidate
for hypertrophic gene transcription and RNA processing
in the nucleus during cardiac hypertrophy in chronic
hypertension. The early nuclear translocation of GRK5
before myocyte lengthening actually correlates with the
early increase of hypertrophic genes such as atrial natriuretic peptide in SHHF rats during the development
of concentric hypertrophy (Carraway et al., 1999).
Both GRK5 and GRK6 belong to the GRK4 subgroup
of the GRK family. GRK6 has been shown to be expressed in the heart. Its role in cardiac hypertrophy and
failure remains unclear. In this study, we have shown
that GRK6 is present in both the TSF and TIF fractions.
In the TSF, GRK6 shows no changes with age in either
SHHF or WKY rats. In contrast, GRK6 increases with
age in the TIF in SHHF, but not WKY rats. The increase
of GRK6 becomes significantly greater in 12- and 24month-old SHHF rats than in age-matched control rats.
The TIF represents the cytoskeletal fraction that contains not only many sarcomeric and cytoskeletal proteins, but also a variety of cell signaling molecules, such
as c-Src, FAK, and ␤-integrin (Laser et al., 2000). GRKs
are also able to phosphorylate cytoskeletal proteins
(Brady and Limbird, 2002). The increase of GRK6 in the
TIF suggests that GRK6 might be involved in cytoskeletal remodeling and myocyte shape regulation during
cardiac hypertrophy and failure. Confocal analysis has
revealed that GRK6 is located in the cytoplasm and the
intercalated disks. GRK6 fluorescence shows a typical
cross-striation pattern in the cytoplasm colocalizing
with ␣-actinin at Z-lines. In SHHF rats, GRK6 is reduced in the intercalated disks, but progressively increases in the cytoplasm of SHHF rats when compared
to age-matched WKY rats. Similar redistribution of
other intercalated disk molecules has been reported
22
YI ET AL.
Fig. 8. Double labeling of GRK6 with N-cadherin or ␣-actinin in the
myocytes of left ventricle of 6-month-old WKY and SHHF rats. The green
fluorescence of GRK6 at the intercalated disks colocalizes with red
fluorescence N-cadherin (A–F). Because of remarkable accumulation of
GRK6 in intercalated disks in WKY rats (A), when overlaid with the red
fluorescence of N-cadherin (B), bright yellow color has been seen in the
intercalated disks in the merged images (C). In contrast, because the
GRK6 fluorescence at the intercalated disks is obviously weaker in
SHHF rats (D), when overlaid bright red fluorescence of N-cadherin (E),
only bright red fluorescence of N-cadherin or orange color fluorescence
is shown at the intercalated disks in the merged images (F). In the
cytoplasm, the cross-striations of GRK6 (green; G) colocalize with that of
␣-actinin (red; H) in 6-month-old SHHF (I; overlay). Scale bar ⫽ 50 ␮m.
during pressure overload-induced hypertrophy and failure. The role of this GRK6 translocation in myocyte
elongation remains to be elucidated.
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