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-arrestin isoforms in congestive heart failure in
rats
1 Institute for Surgical Research, 2 MerckSharp & Dohme Cardiovascular Research Center, and 3 Department of Pharmacology, Rikshospitalet University Hospital, University of Oslo, N-0027 Oslo, Norway
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Myocardial G
protein-coupled receptor kinase 2 (GRK2) has been shown to be involved
in the pathophysiology of congestive heart failure (CHF). However, the
cellular distribution of this isoform, as well as the other isoforms of
the GRK-arrestin system, has not been studied in myocardial tissue.
Thus myocardial expression and cellular distribution of the different
GRK and arrestin isoforms were investigated in a rat model of CHF. Rats
subjected to ligation of the left coronary artery or sham operation
were euthanized 2, 7, or 42 days after the surgical procedure.
Myocardial GRK2, GRK5, 
-arrestin-1, and -arrestin-2 mRNA levels,
but not that of GRK3, were induced in the failing hearts. Consistently,
Western blot analysis of tissue extracts from the nonischemic
region of the left ventricle revealed 3.0-, 2.6-, and 1.5-fold
elevations of GRK2, GRK5, and -arrestin-1, respectively, 7 days
after induction of myocardial infarction compared with the
sham-operated rats (P < 0.05). Immunohistochemical
analysis of myocardial tissue sections and Western blot analysis of
isolated cells revealed localization of GRK2 and -arrestin-1
predominantly in endothelial cells. Conversely, GRK3 was confined to
cardiac myocytes. GRK5 immunostaining appeared to be homogeneously
distributed in the cellular elements of the myocardium. In conclusion,
myocardial mRNA and protein levels of GRK2, GRK5, and -arrestin-1
are induced in postinfarction failure in rats. The immunohistochemical
analysis suggests that GRK2 and -arrestin-1 may act as primary
regulators of endothelial function. Conversely, the cellular
distribution of GRK3 and GRK5 implicates these isoforms as putative
regulators of cardiac myocyte function.
gene expression; immunohistochemistry; G protein-coupled receptor kinase
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THE G PROTEIN-COUPLED
RECEPTOR KINASE (GRK)-arrestin system is considered to play a
pivotal role in desensitization and downregulation of G protein-coupled
receptors (GPCRs). Desensitization of -adrenergic receptors is an
important contributor to the reduced myocardial contractility
associated with congestive heart failure (CHF). Furthermore,
substantial data now implicate GRK2 in the pathophysiological mechanisms of CHF. First, myocardial GRK2 is upregulated in several experimental models of CHF as well as in patients with severe CHF
(12, 19, 22, 23). Second, transgenic animal models with
cardiac myocyte-specific overexpression of GRK2 or GRK2-CT, a
competitive inhibitor of GRK2, demonstrate decreased or increased isoproterenol-stimulated myocardial contractility, respectively (11). Recent evidence also indicates that inhibition of
GRK2 activity may impair adverse cardiac remodeling in dilated
cardiomyopathy and CHF (19). However, the
pathophysiologically relevant substrate of GRK2 in CHF still remains
unresolved because the substrate specificity of GRK2 for GPCRs in the
cardiovascular system is only partially known. Myocardial levels of
GRK5 mRNA and protein content have also been reported to be upregulated
in some models of experimental CHF (3, 17). However,
several aspects concerning the time course and the site of regulation
of the different GRK isoforms after induction of myocardial infarction
(MI) remain to be investigated. Furthermore, the cellular distribution
of the different GRK isoforms in myocardial tissue is not known. The
latter issue may provide new insights into the functional roles of the
different GRK isoforms in myocardial tissue. Even less is known about
myocardial regulation and distribution of the nonvisual arrestin
isoforms, -arrestin-1 and -arrestin-2, in CHF. Indeed, studies on
Drosophila mutants defective in retinal arrestin function
indicate that arrestin receptor binding may be the rate-limiting step
in receptor desensitization and inactivation (18). Thus,
hypothetically, regulation of myocardial arrestin may be a nodal point
in the receptor desensitization and uncoupling associated with CHF.
Therefore, the aims of the present study were to investigate the
myocardial mRNA levels and protein contents of GRK and arrestin
isoforms after induction of postinfarction failure in rats.
Furthermore, to elucidate the preferential sites of action of the
various GRK and -arrestin isoforms, the cellular distribution of
these regulators was investigated by immunohistochemical analysis. The
latter issue has not yet been investigated and is of crucial importance
in providing insights into the functional roles of the different GRK
and arrestin isoforms in myocardial tissue.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animal preparation and study protocol.
Rats (Wistar, males 
250 g body wt) were subjected to
ligation of the left coronary artery as previously described (14, 16). The procedure generally resulted in transmural infarction of the left ventricular (LV) free wall comprising 40-50% of the ventricular circumference (as assessed by perimetry of LV tissue sections). Except for ligation of the coronary artery, sham-operated rats underwent a similar procedure. Assessment of hemodynamic function
and tissue sampling procedures were performed as previously described
(13, 14). The animal experiments and housing were in
accordance with institutional guidelines and national legislation conforming to the European Convention for The Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes of 18 March 1986.
Isolation of cardiac myocytes and fibroblasts and maintenance of cell cultures. Cardiac myocytes were isolated from the hearts of adult Wistar rats by Ca2+-free retrograde perfusion and enzymatic digestion using trypsin (60 U/ml) and collagenase (90 U/ml) as described previously (6, 24). Ca2+ was reintroduced in successive steps to 0.5 mmol/l. The cardiac myocytes were then purified by repeated centrifugation (45 g) and subsequently sedimented by gravity through a solution containing 1% BSA. This cell population contained >95% elongated cardiac myocytes.
Rat cardiac fibroblasts were isolated from the supernatants removed during purification of the cardiac myocytes. After additional low-speed centrifugation to remove cardiac myocytes contaminating the supernatant, this nonmyocyte fraction was plated onto noncoated cell culture dishes (100 mm). The cells were maintained and propagated in Dulbecco's modified Eagle's medium (Life Technologies; Gaithersburg, MD) supplemented with 10% fetal calf serum (Bio Whitaker) and 40 µg/ml garamycin in a humidified atmosphere at 37°C. The cells were split at confluence and used after three passages. The homogeneity of this primary culture was assessed by immunocytochemistry using a monoclonal anti-mouse vimentin antibody (Zymed Laboratories) as a fibroblast marker and a anti-rat von Willebrand factor antibody as an endothelial cell marker. At the third passage, >99% of the cells stained positive for vimentin and <1% displayed immunoreactivity against von Willebrand factor. Rat microvascular endothelial cells (American Type Culture Collection No. CRL-2222) were maintained in minimal essential medium (Life Technologies) supplemented with 5% fetal calf serum, 30 µg/ml heparin, and 40 µg/ml garamycin in a humidified atmosphere at 37°C.Isolation of total RNA, Northern blot analysis, and RPA. Total RNA from rat myocardial tissues was prepared by acid-phenol-chloroform extraction in the presence of chaotropic salts and subsequently precipitated with ethanol as previously described (14). Northern blot analysis was performed as previously described (13) using 30 µg of total RNA per sample and a rat atrial natriuretic peptide (ANP) cDNA probe.
The RPA was performed as previously described (13, 14). The cDNA sequenses used as templates for synthesis of the riboprobes are provided in Table 1. Best fit analysis of the nucleotide sequences of the GRK2 riboprobe and the corresponding GRK3 cDNA revealed 47.6% identity. Similarly, best fit analysis of the nucleotide sequences of the GRK3 riboprobe and the corresponding GRK2 cDNA revealed 42.2% identity. The profound divergence of the nucleotide sequences of GRK2 and GRK3 in the region employed for riboprobe synthesis does not allow for cross-reactivity in the RPA.
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Western blot analysis.
The tissue or cell samples were homogenized, denatured, and separated
by 10% SDS-PAGE. The gels were subsequently electroblotted onto
nitrocellulose filter membranes. For immunoblot analysis of GRK2 and
-arrestin-1, the membranes were blocked with 2.5% nonfat dry milk
(Carnation; Nestle, CA) and 2.5% BSA in Tris-buffered saline-Tween 20 [50 mmol/l Tris · HCl (pH 8.0), 100 mmol/l NaCl, and 0.1%
Tween 20] for 1 h at room temperature (RT). The GRK2 blots were
probed with a polyclonal anti-GRK2 antibody (Santa Cruz Biotechnology)
in blocking solution at a concentration of 0.67 µg/ml (4°C
overnight). The anti--arrestin-1 immunserum was prepared by
immunization against a GST--arrestin-1 fusion protein in rabbits
(4) and used at a dilution of 1:1,500 in blocking buffer
(4°C overnight). For immunoblot analysis of GRK5, the filters were
initially blocked in PBS with 5% nonfat dry milk (4°C overnight) and
further incubated with a polyclonal anti-GRK5 antibody (Santa Cruz
Biotechnology) using 0.1 µg/ml in blocking solution containing 0.05%
Tween 20 (1 h at RT). The blots were subsequently incubated with a horseradish peroxidase-conjugated anti-rabbit IgG (dilution of
1:3,000) for 20 min at RT and visualized using the enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech, Buckinghamshire, UK).
Immunohistochemistry.
Immunohistochemical analysis was performed using 7-µm myocardial
sections as previously described (14). The antibodies used were either purified rabbit polyclonal anti-GRK2, anti-GRK3, or anti-GRK5 IgG (2 µg/ml; Santa Cruz Biotechnology) or rabbit
polyclonal anti--arrestin-1 antiserum (dilution of 1:1,000)
(4). A commercially available avidin-biotin-peroxidase
system (Vectastain Elite kit, Vector Laboratories; Burlingame, CA) was
used according to the manufacturer's instructions to amplify the
signals. To test the specificity of the immunostaining and to provide
negative controls, the different anti-GRK antibodies were preincubated
with their respective antigenic peptides to neutralize the antibodies
before immunostaining. Nonimmune normal rabbit serum was used as a
negative control for the anti--arrestin-1 antiserum.
Immunoprecipitation. Cell samples were homogenized in homogenization buffer [25 mmol/l Tris · HCl (pH 7.5), 5 mmol/l EDTA, 5 mmol/l EGTA, 20 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.2 mmol/l phenylmethylsulfonyl fluoride (PMSF)] and centrifuged at 800 g for 5 min. The supernatant was solubilized by adding an equal volume of 2× RIPA buffer [50 mmol/l Tris · HCl (pH 7.5), 5 mmol/l EDTA, 5 mmol/l EGTA, 300 mmol/l NaCl, 4% Triton X-100, 2% Nonidet P-40, 0.2% SDS, 20 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.1 mmol/l PMSF] with subsequent incubation at 4°C for 30 min and cleared by centrifugation (20,000 g for 10 min). GRK2 and GRK3 were immunoprecipitated from the extracts using anti-GRK2 (0.5 µg/ml) or anti-GRK3 (0.5 µg/ml) antibodies (Santa Cruz Biotechnology) and protein A agarose. The immunoprecipitated proteins were separated by 10% SDS-PAGE and subjected to immunoblot analysis using a monoclonal anti-GRK2/GRK3 antibody (Upstate Biotech) according to the manufacturer's instructions.
Statistical analysis.
All data are presented as means ± SE. Between-treatment group
variations were assessed by a two-tailed unpaired t-test.
P values 
0.05 were considered to be statistically significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Hemodynamic measurements.
Table 2 shows LVEDP and LV systolic
pressure (LVSP) of sham-operated and CHF rats at various time points
after the surgical procedures. The hemodynamic measurements demonstrate
LV dysfunction in the CHF groups with LVSP significantly decreased and
LVEDP significantly increased compared with sham-operated animals
(P < 0.05).
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Myocardial expression of ANP mRNA in CHF. Northern blot analysis of failing nonischemic LV tissue displayed 4.9-, 11.1-, and 12.6-fold elevations of ANP mRNA levels at 2, 7, and 42 days after MI (P < 0.05; data not shown), respectively, compared with sham-operated rats, consistent with a heart failure phenotype at all time points studied.
Regulation of myocardial GRK2, GRK3,
and GRK5 mRNA levels in
CHF.
As shown in Fig. 1B,
myocardial GRK2 mRNA levels in the LV of CHF rats were elevated
compared with sham-operated rats throughout the study period (1.3- and
1.2-fold elevations at 2 and 7 days after MI, respectively,
P < 0.05). Although the GRK2 mRNA levels declined from
day 2 to day 42 after the surgical procedure in both CHF rats as well as sham-operated rats, the levels remained higher
in the CHF rats compared with the sham-operated rats. Analysis of RNA
from right ventricular (RV) tissue samples demonstrated 1.7-, 1.7-, and
1.6-fold elevations of GRK2 mRNA levels at 2, 7, and 42 days after MI,
respectively, compared with sham-operated rats (P < 0.05; data not shown). Transient elevations of GRK2 mRNA expression
could also be seen in the ischemic region, with peak values of
expression 7 days after induction of MI (data not shown). Myocardial
GRK3 mRNA (Fig. 1C), on the other hand, did not display
evidence of regulation in the nonischemic region of the LV
during the timespan of the study. Myocardial GRK5 mRNA displayed a
distinctive pattern of regulation after induction of ischemic
heart failure different from the other GRK isoforms. Myocardial GRK5
mRNA levels in the nonischemic region of the LV (Fig.
1D) were slightly elevated at days 2 and
7 and continued to increase at day 42 after
induction of MI. Forty-two days after induction of MI, the GRK5 mRNA
levels in the nonischemic region of the LV were 1.9-fold above
the levels in sham-operated rats (P < 0.05). Similar
results were obtained from RV tissue samples (data not shown)
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Myocardial expression of -arrestin-1 and -arrestin-2
mRNA in CHF.
Myocardial -arrestin-1 mRNA levels in the nonischemic region
of the LV displayed a time course similar to that of GRK2 mRNA after
induction of MI (Fig. 2B).
Although -arrestin-1 mRNA levels declined from day 2 to
day 42 in both the CHF rats as well as in the sham-operated
rats, the levels in the CHF rats were elevated compared with the
sham-operated rats (1.5- and 1.6-fold above the sham-operated rats at
days 2 and 42, respectively, P < 0.05). Similar elevations of -arrestin-1 mRNA levels were observed
in the RV (data not shown). Myocardial -arrestin-2 mRNA levels were increased in both the LV (Fig. 2C) and RV (data not shown)
2 days after MI (1.9-fold, P < 0.05 in the LV) and
declined toward the levels in sham-operated rats at days 7 and 42.
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Western blot analysis of myocardial GRK2,
GRK5, and -arrestin-1 levels in CHF.
Figure 3, A, C, and
E, shows photographs of Western blot analysis of myocardial
GRK2, GRK5, and -arrestin-1, respectively. The results of subsequent
densitometric analysis of the immunoreactive (IR) bands are shown in
Fig. 3, B, D, and F, demonstrating
3.0-, 2.6-, and 1.6-fold upregulation of myocardial GRK2, GRK5, and -arrestin-1 levels, respectively, in the nonischemic LV 7 days after MI compared with the levels observed in the corresponding sham-operated groups (P < 0.05). GRK2-IR and GRK5-IR
migrated similar to the immunoreactive bands in extracts from Sf9 cells infected with recombinant baculovirus encoding GRK2 or GRK5,
respectively. -Arrestin-1-IR migrated similar to the immunoreactive
band in extracts from COS-7 cells transfected with a recombinant
plasmid encoding -arrestin-1. We were not able to detect
-arrestin-2-IR in myocardial tissue extracts, although
-arrestin-2-IR could readily be detected in transfected COS-7 cells.
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Cellular distribution of GRK isoforms and
-arrestin-1 in myocardial tissue.
Immunohistochemical analysis of myocardial tissue sections of
sham-operated rats revealed the presence of immunoreactivities toward
GRK2, -arrestin-1, GRK5, and GRK3 (Figs.
4, B, F, and J, and 5B,
respectively). The distribution of the cellular immunostaining of the
different GRK isoforms and -arrestin-1 revealed different patterns.
GRK2-IR and -arrestin-1-IR were predominantly seen in vascular
endothelial cells, and only very weak GRK2-IR could be observed in the
cardiac myocytes. On the other hand, myocardial GRK3-IR could only be
observed in the cardiac myocytes, whereas fairly strong GRK5-IR could
be seen in several cellular elements, including cardiac myocytes and
vascular endothelial cells. In CHF rats 7 days after MI, strong
anti-GRK2 and anti--arrestin-1 immunostaining were observed in the
ischemic region (Fig. 4, D and H,
respectively), predominantly confined to vascular endothelial cells.
Although GRK5-IR could readily be detected in several cellular elements, immunostaining of the cardiac myocytes was very distinct. Interestingly, anti-GRK5-IR of the cardiac myocytes contiguous to the
granulation tissue appeared substantially increased compared with
distally located cardiac myocytes (Fig. 4L). Tissue sections of hearts incubated with peptide-neutralized antibodies or nonimmune rabbit serum (-arrestin-1) did not demonstrate immunostaining of any
of the cellular elements of the myocardial tissue, demonstrating specificity of the antibodies (Figs. 4, A, E, and
I, and 5A for GRK2, -arrestin-1, GRK5, and
GRK3, respectively). We also performed immunohistochemical analysis of
GRK2-IR in sections from human and porcine myocardial tissue. Indeed, a
similar distribution was found in these species (data not shown).
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Contents of GRK isoforms and -arrestin-1 in
isolated cells and cell lines.
To strengthen the immunohistochemical observation of the distinct
cellular distribution of GRK2 and -arrestin-1, we performed immunoprecipitation and Western blot analysis of extracts from isolated
rat cardiac myocytes, rat microvascular endothelial cells, and primary
cultures of rat cardiac fibroblasts. Transfected COS-7 cells expressing
high levels of GRK2 or GRK3 were subjected to similar analysis to
assess the specificity of the polyclonal anti-GRK2 and anti-GRK3
antibodies. As shown in Fig. 6,
A and B, the anti-GRK2 and anti-GRK3 antibodies
displayed a fairly high degree of specificity. Furthermore, as shown in
Fig. 6C, immunoprecipitation and Western blot analysis of
extracts from rat cardiac myocytes, rat microvascular endothelial
cells, and rat cardiac fibroblasts demonstrated substantially higher
anti-GRK2-IR and anti--arrestin-1-IR in endothelial cells compared
with cardiac myocytes. However, both GRK2 and -arrestin-1 could be
detected in cardiac myocytes, albeit at a much lower level.
Anti-GRK2-IR and anti--arrestin-1-IR could also be detected in
cardiac fibroblasts. We have previously performed experiments assessing the levels of GRK2, GRK3, GRK5, and -arrestin-1 in extracts from human aortic endothelial cells (HAEC) compared with extracts from rat cardiac myocytes. In these experiments, we found prevailing expression of GRK2 and -arrestin-1 in endothelial cells
(data not shown). In addition, we found that GRK3 was present in
cardiac myocytes. However, no detectable anti-GRK3-IR could be
discerned in HAEC. GRK5, on the other hand, could readily be detected
in both cardiac myocytes and HAEC (data not shown).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Although CHF is associated with elevated levels of myocardial GRK2 (1, 19, 22), lack of information on the cellular distribution of GRK2, as well as of the other members of this receptor kinase family, has largely precluded investigators from drawing conclusions regarding their sites of action in myocardial tissue and therefore the role of these kinases in cardiac physiology and pathophysiology. The present report provides novel information on the cellular distribution and sites of action of the different GRK isoforms in myocardial tissue. Furthermore, the temporal profile of GRK regulation during the time span of infarct healing and development of myocardial dysfunction was investigated. As shown in the present study, induction of myocardial GRK2 and GRK5 mRNA after MI displayed different temporal patterns. Myocardial GRK2 mRNA levels increased early after induction of MI coincidentally with the development of cardiac dysfunction and myocardial hypertrophy. Consistent with this finding, a report (2) on regulation of myocardial GRK2 levels in spontaneously hypertensive rats demonstrated that elevation of GRK2 preceded the development of heart failure, suggesting that GRK2 may be a precipitating factor in the transition from hypertension-induced cardiac hypertrophy to heart failure. On the other hand, as shown in the present study, myocardial GRK5 mRNA levels are induced at a later stage subsequent to development of LV dysfunction and dilatation of the LV cavity. Thus myocardial GRK2 and GRK5 may be involved at different steps of the pathophysiological mechanisms of CHF and myocardial remodeling. Although induction of GRK2 expression appears to be a consistent finding in different experimental models of CHF, species-related differences in regulation of myocardial GRK5 expression in CHF appear to exist (17). Such species-related differences may limit our conclusions as to the involvement of GRK5 in the pathophysiology of CHF.
The specificities of the GRK isoforms towards different GPCRs are still
not well characterized. The most extensively studied isoform is GRK2,
or -adrenergic receptor kinase 1 (ARK1). Although this kinase was
originally termed according to its first known substrate, the
2-adrenergic receptor, it was later shown to catalyze phosphorylation of several GPCRs in vitro (7, 8, 15). Even
though the specific cellular localization of the different GRK isoforms
in myocardial tissue strongly indicates that the various isoforms
participate in regulation of different receptor systems, such confined
cellular distribution per se does not provide cues as to which receptor
systems are subject to regulation. However, specificity in cellular
distribution suggests that the different isoforms participate in
regulation of different cellular functions. The predominant
localization of GRK2 in rat endothelial cells indicates that this
isoform participates in regulation of vascular endothelial function.
This conclusion is corroborated by several additional findings in this
study. First, similar cellular distribution with predominant anti-GRK2
immunostaining of endothelial cells was found in sections of human and
porcine myocardial tissue samples. Second, Western blot analysis of
extracts from the isolated myocardial cells and cell lines demonstrated
highest expression levels of GRK2 in the endothelial cells. Third,
transient induction of GRK2 mRNA in the ischemic area with peak
levels of expression coinciding with neovascularization in the
granulation tissue was found. However, we also demonstrated
anti-GRK2-IR in extracts from isolated rat cardiac myocytes, albeit at
a much lower expression level. Therefore, our data do not contradict
the findings by other authors but rather indicate a hitherto unknown
role for the GRK2 isoform in regulation of endothelial function. On the
other hand, GRK3 was predominantly expressed in cardiac myocytes,
indicating that this isoform may be a putative regulator of cardiac
myocyte function. However, the GRK3 content in myocardial tissue
appeared to be minute, because only a weak band representing
anti-GRK3-IR could be detected in cardiac myocytes after
immunoprecipitation and immunoblot analysis. It should be emphasized
that GRK3-IR could not be detected in HAEC, an observation strongly
supported by a recent report (21) in which expression of
the GRK3 isoform could not be detected by immunoblotting or by RT-PCR
in several different endothelial cell lines. On the other hand, as
shown in the present study, GRK5 was fairly abundant in cardiac
myocytes, implicating GRK5 as another putative regulator of cardiac
myocyte function. However, GRK5-IR was more homogeneously distributed
in myocardial tissue, suggesting this isoform to be involved in
regulation of diverse cell types in myocardial tissue. In this respect,
GRK5-IR could also be demonstrated in smooth muscle cells. This finding
is also consistent with previous studies demonstrating that GRK5
participates in the desensitization of angiotensin II type 1 receptors
(10, 15) on smooth muscle cells. Data from
cardiac-specific overexpression of GRK2 and GRK5 using the 
-myosin
heavy chain promoter indicate that both receptor kinases may
participate in the regulation of myocardial contractility in vivo
(11, 20). Myocardial overexpression of GRK3, on the other
hand, did not alter signaling through -adrenergic receptors, nor did
it affect hemodynamic function in response to -adrenergic agonists
(9). As shown in the present study, induction of
myocardial GRK5 in CHF indicates that this isoform may mediate the
attenuation of myocardial contractility in response to -adrenergic
receptor agonists generally observed in CHF. Indeed, GRK5 has been
shown to be a potent regulator of myocardial -adrenergic receptor
signaling (20). Previous reports of cardiac
myocyte-specific overexpression of ARK1-CT, i.e., the COOH-terminal
region of GRK2 conferring competitive inhibition of GRK2, have
indicated that endogenous GRK2 activities in cardiac myocytes may be
involved in the diminished sensitivity to -adrenergic agonists
associated with CHF (19). However, ARK1-CT may also
interfere with signaling through GPCR by tying up G
subunits in
the cardiac myocytes. Thus the beneficial effects of cardiac
myocyte-specific overexpression of ARK1-CT in experimental models of
CHF, attenuating the progression of dilated cardiomyopathy, may also be
due to altered signaling through other GPCR systems not yet defined.
Little is known about the regulation of myocardial arrestin isoforms in
CHF. However, GRK and arrestin are considered to be coactors in a
system aimed at modifying signaling through GPCRs. Thus concerted
regulation of both GRK and arrestin isoforms would conceivably enhance
the effect on the targeted receptor. In the present study, we provide
novel data demonstrating that myocardial -arrestin-1 is also
substantially induced both at the mRNA and protein levels after
induction of CHF. A recent report (5) of gene targeting of
-arrestin-1 in mice provided evidence of enhanced sensitivity to
-adrenergic agonist-stimulated increases in ejection fraction,
indicating that -arrestin-1 participates in the regulation of
-adrenergic receptor-mediated cardiac responses. The present study
demonstrates that -arrestin-1 is predominantly localized to vascular
endothelial cells. However, -arrestin-1-IR could also be detected in
other cellular constituents of myocardial tissue, including cardiac
myocytes. The increased sensitivity to -adrenergic
agonist-stimulated enhancements in the LV ejection fraction observed in
the -arrestin-1 knockout mice could conceivably be due to impaired
desensitization of -adrenergic receptors in either the vasculature
or in cardiac myocytes.
The concerted induction of myocardial GRK and arrestin isoforms during
CHF indicates that these receptor activity-modifying systems operate in
the pathophysiology of CHF. However, the precise role of -arrestin
in the pathophysiology of CHF is not known because the consequences of
transgenic modulation of myocardial -arrestin levels in CHF have not
been investigated. On the other hand, evidence from visual signaling
experiments in Drosophila indicates that arrestin may be a
rate-limiting factor in adaptation or desensitization of rhodopsin
signaling (18). Thus modulation of nonvisual arrestin
isoforms may be a crucial point of regulation of several GPCRs.
In conclusion, the present study demonstrates that the different GRK
and -arrestin isoforms expressed in myocardial tissue display
different cellular localization. Whereas GRK2 was predominantly confined to vascular endothelial cells, GRK3 was primarily localized to
cardiac myocytes. Fairly strong GRK5 immunostaining could be identified
both in cardiac myocytes and in endothelial cells. Myocardial GRK2,
GRK5, and -arrestin-1 mRNA levels, as well as protein levels, were
induced in heart failure in rats. The concerted induction of both GRK
and -arrestin-1 in the nonischemic LV indicates that this
receptor activity-modifying system is activated and may be involved in
the pathophysiology of CHF. Furthermore, the distinct distribution of
the different GRK isoforms in myocardial tissue calls for diverse roles
of the GRK isoforms in cardiac cellular biology and pathophysiology.
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ACKNOWLEDGEMENTS |
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We thank Dr. Dag Sørensen, Chief Veterinarian, Rikshospitalet University Hospital, for expert assistance with the animal experiments.
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FOOTNOTES |
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This study was supported by grants from the National Research Council, the Norwegian Council on Cardiovascular Diseases, the Medinnova Research Fund, and the Dr. Alexander Malthes Foundation.
Address for reprint requests and other correspondence: H. Attramadal, Institute for Surgical Research, Rm. A3.1013, Rikshospitalet, N-0027 Oslo, Norway (E-mail: havard.attramadal{at}klinmed.uio.no).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11 August 2000; accepted in final form 22 August 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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) and 6 CHF rats (
). Data are
representative of 3 independent assays. *P < 0.05, 
P < 0.01.
P < 0.001 vs. sham.
