© 1998 American Heart Association, Inc.
Original Contributions |
Angiotensin II Activates RhoA in Cardiac Myocytes
A Critical Role of RhoA in Angiotensin II–Induced Premyofibril Formation
From the Cardiovascular Research Center, Division of Cardiology, University of Michigan Medical Center, Ann Arbor.
Correspondence to Junichi Sadoshima, Cardiovascular and Pulmonary Research Institute, Allegheny University of the Health Sciences, 15th Floor, South Tower, 320 East North Ave, Pittsburgh, PA 15212-4772.
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Abstract |
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Abstract—The organization of actin into striated fibers (myofibrils) is one of the major features of cardiac hypertrophy. However, its signal transduction mechanism is not well understood. Although Rho-family small G proteins have been implicated in actin organization in many cell types, it is not fully elucidated whether Rho mediates the organization of actin fibers by hypertrophic stimuli in cardiac myocytes. Therefore, we examined (1) whether Rho is activated by the hypertrophic stimulus, angiotensin II (Ang II), and (2) whether Rho mediates the Ang II–induced organization of actin fibers in cultured neonatal rat cardiac myocytes. Treatment of myocytes with Ang II caused a rapid formation of both striated (mature myofibrils) and nonstriated (premyofibrils) actin fibers within 30 minutes, as determined by phalloidin stainings of the polymerized actin and troponin T stainings. Immunoblot analyses and immunostainings have indicated that cardiac myocytes express RhoA, but RhoB is undetectable. In the control state, RhoA was observed predominantly in the cytosolic fraction, but it was translocated in part to the particulate fraction in response to Ang II, consistent with activation of RhoA by Ang II. Incubation of myocytes with exoenzyme C3 for 48 hours completely ADP-ribosylated Rho in vivo. The C3 treatment abolished formation of premyofibrils induced by Ang II, suggesting that Ang II causes premyofibril formation via a Rho-dependent mechanism. The Ang II–induced mature myofibril formation was only partly abolished by C3. Expression of constitutively active RhoA (V14RhoA) caused the formation of premyofibrils but not mature myofibrils. The C3 treatment inhibited Ang II–induced atrial natriuretic factor induction, whereas it had no effect on c-fos induction. These results indicate that RhoA is activated by Ang II and mediates the Ang II–induced formation of premyofibrils and induction of a subset of genes. Distinct signaling mechanisms seem to be responsible for striated mature myofibril formation by Ang II.
Key Words: hypertrophy • small G protein • actin fiber • translocation • exoenzyme C3
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Small molecular weight GTP-binding proteins (small G proteins) are molecular switches that control various cell functions. More than 50 small G proteins have been found to date (see Reference 11 for review). The exchange of bound GDP for GTP converts these small G proteins to an active form, which returns to the GDP-bound inactive form by hydrolysis of GTP. GDP-GTP exchange reactions are positively regulated by guanine nucleotide exchange factors and negatively regulated by guanine nucleotide dissociation inhibitors, whereas hydrolysis of GTP is stimulated by GTPase-activating proteins (reviewed in Reference 11 ). Recent studies have shown that Rho-family small G proteins (Rho, Rac, and Cdc42) play a central role in the organization of the actin cytoskeleton (see Reference 22 for review). Each member of the Rho family controls the organization of different actin structures: Rho controls formation of stress fibers and focal adhesions; Rac and Cdc42 regulate formation of lamellipodia and filopodia, respectively.3 4 5 Recent studies have identified downstream targets of Rho, which physically associate with the GTP-bound form of Rho. These include serine/threonine kinases (Rho-kinase, Rho-associated coiled-coil–containing protein kinase, and protein kinase N), myosin phosphatase, and other novel proteins (rhophilin, rhotekin, and citron) (see Reference 22 for review). Recently, Rho-kinase has been shown to mediate stress fiber formation in Swiss 3T3 fibroblasts. Constitutively active forms of Rho-kinase cause stress fiber formation, whereas dominant-negative ones inhibit LPA-induced stress fiber formation.6
Organization of actin fibers into myofibrils is one of the major
characteristics of cardiac hypertrophy, a growth response
observed in terminally differentiated cardiac
myocytes.7 Cardiac hypertrophy is
also characterized by the induction of immediate-early genes (eg,
c-fos and c-jun), activation of the fetal gene
program (eg, ANF and skeletal 
-actin), and increases in protein
synthesis and cell size (reviewed in References 77 to 11). We and others
have previously shown that Ang II plays an essential role in mediating
load-induced cardiac hypertrophy12 13
and that Ang II is sufficient to cause many, if not all,
characteristics of the load-induced cardiac hypertrophy in
cultured neonatal rat and chick ventricular
myocytes.14 15
Ang II activates multiple second-messenger systems in cardiac myocytes,16 and each signaling molecule seems to mediate distinct hypertrophic responses. We have shown that protein kinase C mediates Ang II–induced c-fos immediate-early gene expression.16 The MEK/ERK pathway plays an essential role in Ang II–induced ANF expression,17 although the role of this pathway in ANF induction by other hypertrophic stimuli, such as phenylephrine, is controversial. We have also shown that a rapamycin-sensitive pathway, most probably involving 70-kD S6 kinase, is essential for Ang II–induced increases in protein synthesis.18 Although we have shown that Ang II causes organization of actin fibers into myofibrils in myocytes,18 the signal transduction pathway that controls this process is unknown.
During myofibrillogenesis, cardiac myocytes assemble nonstriated actin
fibers, which look similar in morphology to the prototypical stress
fiber in fibroblasts by phalloidin
stainings.19 20 21 These nonstriated actin fibers
in cardiac myocytes are termed premyofibrils, since they are composed
of not only nonmuscle myosin IIB but also muscle-specific (sarcomeric)
proteins, such as -actinin, tropomyosin, and
troponin20 22 and because they develop into
mature myofibrils.22 Since Rho controls stress
fiber formation in fibroblasts,3 we hypothesized
that Rho mediates the Ang II–induced organization of actin fibers in
cardiac myocytes.
Several growth factors have been suggested to activate Rho. These include agonists for receptor tyrosine kinases, such as hepatocyte growth factor23 and insulin,24 and agonists for GPCRs, including LPA, bombesin,3 norepinephrine,25 and endothelin.26 It is not known, however, whether Ang II activates Rho.
In the present study, we investigated whether Rho is activated by Ang II. We also examined the role of Rho in Ang II–induced actin organization in cardiac myocytes. Our results indicate that Ang II activates RhoA and that RhoA mediates the Ang II–induced formation of premyofibrils in cardiac myocytes. Interestingly, additional signaling mechanisms seem to be required for Ang II–induced mature striated myofibril formation in cardiac myocytes.
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Materials and Methods |
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Primary Culture of Neonatal Rat Ventricular Myocytes
Primary cultures of cardiac ventricular myocytes from 1-day-old Wistar rats were prepared as described previously.27 Briefly, ventricular myocytes were enzymatically dissociated and preplated for 1 hour to enrich for myocytes. Cells were plated onto either gelatin-coated 60-mm culture dishes or coverslips and cultured in cardiac myocyte culture media that contained DMEM/F-12 supplemented with 5% horse serum, 4 µg/mL transferrin, 0.7 ng/mL sodium selenite (GIBCO BRL), 2 g/L bovine serum albumin (fraction V), 3 mmol/L pyruvic acid, 15 mmol/L HEPES (pH 7.6), 100 µmol/L ascorbic acid, 100 µg/mL ampicillin, 5 µg/mL linoleic acid, and 100 µmol/L 5-bromo-2'-deoxyuridine (Sigma Chemical Co). Culture media were changed to serum-free media after 24 hours. Myocytes were further cultured under serum-free conditions for 48 hours before the experiments.
Immunoprecipitation
For immunoprecipitation of RhoA and RhoB, myocytes were lysed
with 1 mL lysis buffer containing 1% NP-40, 150 mmol/L NaCl,
50 mmol/L Tris (pH 8.0), 1 mmol/L AEBSF, 20 µg/mL
aprotinin, and 100 µmol/L leupeptin. Cell debris was pelleted by
centrifugation (14 000 rpm at 4°C for 20 minutes).
Immunoprecipitation was carried out by adding 1 µg/mL of anti-RhoA or
anti-RhoB antibody (Santa Cruz Biotechnology) for 3 hours, followed by
the addition of 40 µL of protein A–Sepharose (50% slurry)
(Pharmacia Biotech) for 1 hour at 4°C. Immunoprecipitates were washed
three times with the lysis buffer and subjected to further
analyses.
Subcellular Fractionation
Cell-free lysates were prepared by adding 100 µL hypotonic
lysis buffer (per 60-mm dish) containing 20 mmol/L Tris (pH 8.0),
3 mmol/L MgCl2, 0.4 mmol/L AEBSF, 5
µg/mL aprotinin, 2 µg/mL trypsin inhibitor, and 20
µmol/L leupeptin. After three cycles of freeze and thaw, samples were
centrifuged at 100 000g at 4°C for 60 minutes.
The supernatant was saved as a "soluble" fraction. Pellets were
washed twice by the same lysis buffer and resuspended in 100 µL of
the lysis buffer supplemented with 1% Triton X-100 and 0.1% SDS. Cell
debris was separated by centrifugation (14 000 rpm at
4°C, 20 minutes), and supernatant was saved as a "particulate"
fraction. The protein content of each fraction was determined by the
Lowry method.
Immunoblot Analysis of Rho Proteins
Either cell lysates containing equal amounts of protein or
immunoprecipitates were subjected to SDS-PAGE on 15% gels. Proteins
were transferred onto polyvinylidene difluoride membranes
(Immobilon-P, Millipore) and probed with 1 µg/mL of either anti-RhoA
or anti-RhoB antibody (Santa Cruz Biotechnology), followed by
horseradish peroxidase–conjugated protein A (Zymed) at 1:10 000
dilution. The protein probed was then visualized by the enhanced
chemiluminescence system (ECL, Amersham) in the linear range of x-ray
films.
ADP-Ribosylation Assay of Rho by Exoenzyme C3
Cell lysates containing equal amounts of protein were
incubated with 4 µg/mL exoenzyme C3 from Clostridium
botulinum (List Biological Laboratories) or recombinant C3 (see
below) and 1 µCi [32P]NAD (Amersham) in
20 µL of buffer A containing 10 µmol/L NAD, 50 mmol/L
triethanolamine hydrochloride (pH 7.5), 2 mmol/L
MgCl2, 1 mmol/L EDTA, 1 mmol/L
dithiothreitol, 0.2 mmol/L AEBSF, 10 mmol/L thymidine, and
100 µmol/L GTP at 37°C for 60 minutes. Samples were resolved
by SDS-PAGE on 15% gels, and the ADP-ribosylated Rho was visualized by
autoradiography in the linear range of x-ray films.
Overexpression of Constitutively Active RhoA (V14RhoA)
Myocytes grown on coverslips were transiently transfected with a
plasmid encoding Myc-tagged V14RhoA (pEXV/V14RhoA, 2 µg/coverslip;
courtesy of Dr Alan Hall, London, England) using lipofectamine (GIBCO
BRL). Forty eight hours after transfection, cells were fixed in 3.7%
formaldehyde for 10 minutes and subjected to
immunostainings.
ADP-Ribosylation of Rho in Intact Cardiac Myocytes
Recombinant C3 was expressed in Escherichia coli
using a plasmid encoding C botulinum exoenzyme C3 (pET3a/C3;
courtesy of Dr Shu Narumiya, Kyoto, Japan) and purified as
described.28 To ADP-ribosylate
endogenous Rho, cardiac myocytes grown on 60-mm dishes were
incubated with various concentrations of C3 for 48 hours. We used
48-hour treatment since it has been reported that a 24- to 48-hour
incubation time is required for complete ADP-ribosylation of C3 in
various cell types28 and because we observed
complete ADP-ribosylation of Rho at 48 hours but not at 24 hours in
cardiac myocytes (Fig 5
and data not shown). The effectiveness of in
vivo ADP-ribosylation of Rho by the C3 treatment was confirmed as
follows: After the C3 treatment, cells were extensively washed with PBS
to remove bound C3 on the cell surface. Cells were lysed by three
cycles of freeze and thaw in 100 µL of the hypotonic lysis buffer
(see above) per 60-mm dish. Cell debris was separated by
centrifugation (14 000 rpm at 4°C for 20 minutes),
and the supernatant was subjected to the ADP-ribosylation assay as
described above. After 48 hours of treatment with the highest
concentration of C3 (200 µg/mL), IGF-1–induced c-fos
expression, which is known to be mediated through Rho-independent
signaling mechanisms,29 30 was preserved (see Fig 9). This suggests that the myocytes remained viable and that the
response to growth factor stimulation was preserved after C3
treatment.
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Immunofluorescent Staining
Cells were fixed in 3.7% formaldehyde for 10 minutes.
Immunofluorescent stainings were performed as described
previously.27 Endogenous RhoA was
detected by anti-RhoA antibody (1:40 dilution) followed by
FITC-conjugated donkey anti-rabbit IgG antibody (1:50 dilution). Cells
transfected with Myc-tagged V14RhoA were stained with either mouse
monoclonal antibody (9E10, Santa Cruz Biotechnology) (1:50 dilution) or
rabbit polyclonal anti–Myc-tag antibody (Pan Vera) (1:100 dilution)
followed by Texas Red–conjugated donkey anti-mouse IgG (1:50 dilution)
or rhodamine-conjugated donkey anti-rabbit IgG antibody (1:100
dilution), respectively. Troponin T was detected by mouse monoclonal
anti–troponin T antibody (JLT-12, Sigma) (1:100 dilution) followed by
either FITC-conjugated or Texas Red–conjugated donkey anti-mouse IgG
antibody (1:50 dilution). Actin fibers were stained with 40 µg/mL of
FITC-conjugated phalloidin (Sigma) as described
previously.18 In some experiments, cells were
triple-stained with anti–Myc-tag rabbit polyclonal antibody,
anti–troponin T mouse monoclonal antibody, and FITC-conjugated
phalloidin. Texas Red–conjugated donkey anti-mouse IgG and
7-amino-4-methylcoumarin-3-acetic acid–conjugated
donkey anti-rabbit IgG antibodies were used as secondary antibodies for
troponin T and Myc-tag staining, respectively. Cells were observed
either by an epifluorescence microscope (Microphoto SA, Nikon)
or a laser scanning confocal microscope (MRC600, Bio-Rad
Laboratories).
Analysis of mRNA
Total RNA was extracted from myocytes using Trizol (1 mL/60-mm
dish, GIBCO BRL). The RNA was resolved by electrophoresis on 1%
agarose gels and transferred onto nitrocellulose membranes (Schleicher
& Schuell). The probes for c-fos and ANF were used as
described previously.15
Statistics
Data are given as mean±SE. Statistical analyses were
performed using ANOVA. A post hoc test was performed by the method of
Bonferroni. Significance was accepted at the P<.05
level.
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Results |
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Ang II Causes a Very Rapid Formation of Nonstriated and Striated Actin Fibers in Cardiac Myocytes
We have previously shown that Ang II causes sarcomeric actin organization in cardiac myocytes.18 We first examined the time course of Ang II–induced sarcomeric actin organization in cultured cardiac myocytes. Myocytes were stimulated with 100 nmol/L Ang II, fixed at the indicated times, and stained with FITC-conjugated phalloidin. Before stimulation with Ang II, the phalloidin staining showed a punctated pattern (Fig 1A
), indicating that the sarcomere was
poorly organized. Only 5 minutes after stimulation, cells already
started to form thin striated actin fibers (Fig 1C, arrows). At 30
minutes, striated actin fibers became more prominent and thicker (Fig 1E, arrows). Besides striated actin fibers, Ang II also caused
formation of nonstriated actin fibers at the periphery of myocytes
(indicated by arrowheads in Fig 1C and 1E).
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Double staining with phalloidin and anti–troponin T (a striated
muscle–specific contractile regulatory protein) antibody indicated
that both striated (Fig 2, arrows) and
nonstriated (Fig 2, large arrowheads) actin fibers in cardiac myocytes
are positive for troponin T. Thus, nonstriated actin fibers are
probably premature forms of myofibrils (premyofibrils), as proposed
previously,20 22 but not authentic stress fibers
observed in nonmyocytes, which are negative for troponin T (Fig 2, small arrowheads). These results indicate that Ang II induces a
rapid organization of both premyofibrils and striated myofibrils in
cardiac myocytes. Ang II–induced sarcomeric actin organization was
inhibited by an AT1 receptor
antagonist, losartan, indicating that this response
is mediated by the AT1 receptor (data not
shown).
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RhoA but Not RhoB Is Expressed in Cardiac Myocytes
Since Rho-family small GTP-binding proteins play an important role
in actin organization in other cell types (see Reference 22 for review),
we investigated whether Rho is involved in Ang II–induced actin
organization in cardiac myocytes. We examined the expression of Rho
proteins in cultured cardiac myocytes. Immunoprecipitations using
anti-RhoA antibody followed by immunoblottings with the
same antibody showed a 21-kD band (Fig 3A, lane 1). This band was not observed
when the immunoprecipitation was performed either without the primary
antibody (Fig 3A, lane 2) or with nonimmune serum (not shown).
Immunoprecipitations using anti-RhoB antibody followed by
immunoblottings with the same antibody showed no
specific band (Fig 3B), indicating that RhoB was undetectable in
cardiac myocytes. We also examined the subcellular localization of RhoA
in cardiac myocytes using indirect immunofluorescent stainings.
Stainings with anti-RhoA antibody showed a diffuse cytoplasmic pattern
(Fig 3C, a). The staining was not observed when the antibody was
preabsorbed with 10-fold excess of RhoA antigen peptide (Fig 3C, c),
indicating that the staining was RhoA specific. These results indicate
that cardiac myocytes express RhoA but not RhoB and that RhoA is
located predominantly in the cytoplasm.
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Ang II Induces Partial Translocation of RhoA From the Soluble to
the Particulate Fraction in Cardiac Myocytes
Rho and Rac have been shown to partially translocate from the
soluble to the particulate fraction on their
activation.31 To obtain evidence that RhoA is
activated by Ang II in cardiac myocytes, the subcellular
localization of RhoA before and after Ang II stimulation was examined.
Cardiac myocytes were stimulated with Ang II for the indicated times,
and subcellular fractionations were performed. Samples were subjected
to SDS-PAGE on 15% gels and analyzed by
immunoblotting. Before stimulation, the majority of
RhoA was detected in the soluble fraction (Fig 4A), consistent with the result
of the immunostaining (Fig 3C
) and a previous report
involving fibroblasts.32 Although a weak signal
of RhoA was observed in the particulate fraction before Ang II
stimulation, treatment with Ang II caused a rapid and statistically
significant increase in the RhoA content of the particulate fraction
(Fig 4A and 4B) (1.94±0.33-fold increase at 15 minutes versus control,
P<.05 by ANOVA). A significant decrease in RhoA content in
the soluble fraction was not detected in our assay conditions, most
likely because of the relative abundance of RhoA in the soluble
fraction. Parallel immunoblot analyses indicated
that the amount of RhoA detectable in total cell lysate did not
significantly change before and after Ang II stimulation. These results
indicate that a part of RhoA translocates from the soluble to the
particulate fraction in response to Ang II. Exact cellular localization
of RhoA in the particulate fraction remains to be elucidated.
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To further confirm the translocation of RhoA, we used another specific
and sensitive detection method of Rho, the ADP-ribosylation of Rho with
C botulinum exoenzyme C3.33 As shown
in Fig 4C, a single band was detected at 21 kD by the ADP-ribosylation
assay in a C3-dependent fashion. In control myocytes, only faint
signals were observed in the particulate fractions (lanes 1, 4, and 7),
whereas a much stronger signal was observed in the soluble fraction
(data not shown). Fifteen minutes of treatment with Ang II caused a
2.6-fold increase in the Rho content of the particulate fraction (lane
3), consistent with the result of
immunoblotting. In addition, we compared the potency of
Ang II to cause Rho translocation with that of other agonists that have
been reported to activate Rho in other cell types, namely,
bombesin and LPA.3 The potency of 100 nmol/L Ang
II for inducing translocation of Rho was comparable to that of 100
nmol/L bombesin (2.9-fold) or 100 ng/mL LPA (2.6-fold).
Treatment of Myocytes with C3 ADP-Ribosylates Rho In Vivo
Exoenzyme C3 transfers the ADP-ribose from NAD to Rho at Asn41,
which is located in the putative effector domain of Ras-related
GTP-binding proteins.34 Because this reaction is
highly specific to Rho and inhibits the function of
Rho,35 C3 has been a useful and well-established
tool in studying the function of Rho.3 36 We
incubated cardiac myocytes with various concentrations (0 to 200
µg/mL) of C3 for 48 hours.37 Cell lysates were
prepared and subjected to in vitro ADP-ribosylation using
32P-labeled NAD. If Rho had already been
ADP-ribosylated in vivo, no further ADP-ribosylation would occur in
vitro; thus, Rho would not be labeled with
32P.28 As shown in Fig 5, incubation of myocytes with C3 in vivo
inhibited subsequent ADP-ribosylation of Rho in vitro. The effect of C3
was concentration dependent; treatment of myocytes with 200 µg/mL of
C3 for 48 hours completely abolished the following ADP-ribosylation of
Rho in vitro. These results indicate that incubation of myocytes with
C3 causes the ADP-ribosylation of Rho in vivo.
C3 Inhibits Formation of Premyofibrils but Does Not Inhibit
Formation of Striated Actin Fibers in Cardiac Myocytes
We next examined the effect of C3 treatment on Ang II–induced
actin organization in cardiac myocytes. Myocytes were treated with 200
µg/mL of C3 for 48 hours, a treatment that completely ADP-ribosylates
Rho in vivo (see above). Cells were then stimulated with 100 nmol/L Ang
II for 30 minutes and stained with FITC-labeled phalloidin. In the
control state, punctated actin staining (Fig 6A, small arrows) was observed in cardiac
myocytes. After a 30-minute stimulation with Ang II, a well-developed
sarcomeric pattern (Fig 6B, large arrows) and premyofibril formation
were observed (Fig 6B, large arrowheads). In myocytes treated with C3
for 48 hours, punctated actin staining was still observed (Fig 6D, small arrows). A 30-minute stimulation of C3-treated myocytes with Ang
II still caused prominent striated sarcomeric actin fiber formation
(Fig 6E, large arrows), although these actin fibers were less dense
compared with those in C3-untreated myocytes. Interestingly, the C3
pretreatment almost completely prevented Ang II–induced increases in
premyofibril formation in cardiac myocytes (Fig 6E). The same
concentration of C3 abolished stress fibers in nonmyocytes
(presumably fibroblasts) (compare Figs 6C [without C3] and F [with
C3]), indicating that the recombinant C3 used was functionally active.
C3-treated myocytes were viable because they assembled myofibrils and
revealed an increase in the rate of protein synthesis over 24 hours in
response to Ang II (data not shown).
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Constitutively Active RhoA Induces Premyofibril Formation but Not
Striated Sarcomeric Actin Organization in Cardiac Myocytes
We next examined whether activation of RhoA is sufficient to
induce sarcomeric actin organization in cardiac myocytes. Cardiac
myocytes were transiently transfected with a plasmid encoding
constitutively active RhoA (V14RhoA) with Myc-tag. Cells expressing
V14RhoA were identified by immunostaining with
anti–Myc-tag antibody. Myocytes were triple-stained with phalloidin,
anti–troponin T, and anti–Myc-tag antibodies. Cardiac myocytes
expressing V14RhoA (Fig 7A, large arrow)
revealed increased formation of fine actin fibers across the cytoplasm
(Fig 7B, large arrow) compared with untransfected myocytes (Fig 7A and 7B, large arrowheads). These actin fibers did not show the sarcomeric
pattern (Fig 7B, large arrow) but were rather similar to stress fibers
in nonmyocytes (Fig 7F). The same cells were found to have
increased formation of troponin T–positive fibers, which also lacked
the sarcomeric pattern (Fig 7C, large arrow). These phalloidin-positive
fibers, indicated by green in Fig 7D, were positive for troponin T
(red), as shown in the merged image (yellow) of phalloidin and troponin
T stainings (Fig 7D). This indicates that these fibers are not
authentic stress fibers but probably premyofibrils. Nonmyocytes
(presumably fibroblasts) expressing V14RhoA (Fig 7E, small arrow)
showed very dense actin stress fibers (Fig 7F) as
reported,3 indicating that the V14RhoA construct
we used was functionally active.
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The structure of fibers stained by either troponin T or phalloidin was
further characterized using confocal microscopy. Cardiac myocytes
expressing V14RhoA (Fig 8A, arrow) showed
an increase in formation of actin fibers (Fig 8B, arrow) compared with
untransfected cells (Fig 8B, arrowheads). Myocytes expressing V14RhoA
(Fig 8C, arrow) also had increased formation of troponin T–positive
fibers (Fig 8D, arrow). Both phalloidin-positive and troponin
T–positive fibers showed a very similar staining pattern, which lacked
significant sarcomeric (striated) patterns. These results suggest that
V14RhoA induces formation of nonstriated actin fibers that are probably
premyofibrils, but it is not sufficient to cause mature striated
sarcomere formation in cardiac myocytes.
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RhoA Mediates Ang II–Induced ANF Expression
It has been shown that RhoA mediates
phenylephrine-induced ANF
expression,38 one of the important
characteristics of cardiac hypertrophy, in neonatal rat
cardiac myocytes. Thus, we examined the effect of C3 treatment on Ang
II–induced gene expression, namely, c-fos and ANF. A
30-minute treatment of cardiac myocytes with Ang II caused
c-fos expression, and this was not affected when myocytes
were pretreated with C3 (200 µg/mL) (Fig 9A, lanes 2 and 5). As an additional
control, when a separate agonist, IGF-1, was used, c-fos
induction was not affected by C3 treatment (Fig 9A, lanes 3 and 6). The
same C3 treatment, however, abolished Ang II–induced ANF expression
(Fig 9B), suggesting that Rho is involved in Ang II–induced ANF
expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results suggest that Ang II activates RhoA in cardiac myocytes. RhoA mediates Ang II–induced formation of nonstriated sarcomeric actin fibers (premyofibrils), which are distinct from stress fibers, in cardiac myocytes. Considering the existence of multiple downstream target molecules and the pleiotropic functions of Rho as reported in other cell types (References 30, 39, and 4030 39 40 ; see Reference 22 for review), RhoA may play an important role in remodeling processes of the heart caused by Ang II.
Since Rho is a member of the small GTP-binding proteins, activation of Rho could be shown by its increased binding of GTP. However, the guanine nucleotide binding assay or determination of the guanine nucleotide exchange activity for Rho has been technically difficult because the available antibody is not suitable for immunoprecipitating the native form of Rho41 in the presence of Mg2+ (authors' unpublished data, 1997). Since Mg2+ is essential for preserving the guanine nucleotide binding of Rho,36 it is difficult to immunoprecipitate Rho while preserving its guanine nucleotide binding. However, the following results strongly suggest that Ang II activates RhoA in cardiac myocytes. First, Ang II causes translocation of a fraction of Rho from the cytosolic to the particulate fraction as determined by both immunoprecipitation/immunoblotting and the C3/ADP-ribosylation assay. It was recently shown that several growth factors, including norepinephrine,42 LPA, endothelin,43 hepatocyte growth factor,44 and insulin,24 cause translocation of a fraction of Rho from the cytosolic to the particulate fractions in various cell types, leading to a 1.5- to 2-fold increase in the RhoA content in the particulate fraction. The level of Ang II–induced RhoA translocation was comparable to that in those known stimulators of RhoA. Second, Ang II–induced premyofibril formation was inhibited by the C3 treatment, which was confirmed to ADP-ribosylate most of Rho in situ. Third, expression of constitutively active RhoA mimicked Ang II–induced premyofibril formation in cardiac myocytes. Taken together, these results are consistent with RhoA activation by Ang II in cardiac myocytes.
Ang II–induced RhoA activation seems to be mediated by an AT1 receptor–dependent mechanism, since Ang II–induced actin organization was inhibited in the presence of the AT1 receptor antagonist losartan. When the C3-inhibitable cellular responses and membrane translocation were used as markers for Rho activation, agonists for other GPCRs, including LPA, bombesin,3 endothelin,26 43 and norepinephrine,25 were shown to activate Rho. However, the mechanism of Rho activation by these GPCR agonists is not well understood. It has been shown that tyrphostin-sensitive (unidentified) tyrosine kinases mediate the activation of Rho by LPA in fibroblasts.45 Since Ang II activates tyrosine kinases, including the Src family,46 47 48 49 it is possible that these tyrosine kinases may mediate Ang II–induced RhoA activation in cardiac myocytes. Recent evidence suggests that Dbl-related proteins, such as Lbc,50 Lfc, and Lsc,51 have Rho-specific guanine nucleotide exchange activities. However, it is unknown how these guanine nucleotide exchange factors for Rho are regulated by growth factor stimulation.
We found that Ang II causes sarcomeric actin organization quite
rapidly. Formation of both premyofibrils and mature myofibrils is
observed within 5 minutes and is completed within 30 minutes of Ang II
stimulation. Quantification of the total actin content by
immunoblot analyses indicated that the amount of
total (both unpolymerized and polymerized) actin did not change
significantly before and after stimulation with Ang II for 30 minutes
(data not shown). This suggests that posttranslational mechanisms play
an important role in Ang II–induced actin organization. The formation
of premyofibrils by Ang II is likely to be mediated by Rho. First, the
time course of Ang II–induced actin organization (shown in Fig 1) is
very similar to that of the Ang II–induced translocation of RhoA into
the particulate fraction (which presumably reflects activation of RhoA)
(shown in Fig 4). Second, Ang II–induced premyofibril formation was
inhibited by pretreatment with C3 and was mimicked by expression of
constitutively active RhoA in cardiac myocytes. It should be noted that
premyofibrils formed after Ang II stimulation and expression of
constitutively active RhoA in cardiac myocytes are not authentic stress
fibers, because they colocalize with troponin T. Nonstriated actin
fibers formed at the periphery of myocytes have been shown to develop
into mature myofibrils and are thus termed
premyofibrils.19 20 22 These premyofibrils are
composed of nonmuscle myosin IIB as well as striated muscle–specific
isoforms of cytoskeletal proteins, and this property make them distinct
from authentic stress fibers seen in other cell
types.20 22 Recently, Wang et
al52 also reported that C3 treatment abolished
ß-actin–positive nonstriated fibrils in unstimulated chicken
embryonic cardiac myocytes. Taken together, these results indicate that
RhoA stimulates organization of not only stress fibers in nonmuscle
cells but also premyofibrils in cardiac myocytes.
Another interesting observation concerning the mechanism of sarcomeric organization in cardiac myocytes is that formation of the mature myofibrils by Ang II was only partially affected in the presence of C3, although the C3 treatment effectively abolished Ang II–induced premyofibril formation. Constitutively active RhoA failed to induce the mature striated sarcomeric actin. Recently, Thorburn et al53 have also reported that C3 treatment does not affect phenylephrine-induced actin reorganization in cardiac myocytes, although they did not describe the effect of C3 on nonstriated actin fiber formation. These results suggest that additional signaling mechanisms are required for mature sarcomere formation by Ang II or phenylephrine stimulation in cardiac myocytes. Recent evidence suggests that the Z-band region of titin, a 3 000 000-D protein, plays an essential role in organizing and maintaining the structure of the myofibril.54 Thus, it will be interesting to examine how this protein is modulated by Ang II.
Dabiri et al22 have reported that premyofibrils
labeled with green fluorescent protein–linked sarcomeric
-actinin become mature myofibrils in the same living cardiac
myocytes, indicating that the formation of premyofibrils and the
formation of mature myofibrils are sequential events.
Consistent with this observation, the total amount of striated
mature actin fibers formed was partially reduced when the formation of
premyofibrils was blocked by C3 in Ang II–stimulated myocytes (Fig 6).
Our present hypothesis regarding the mechanism of Ang II–induced
sarcomeric organization is depicted in Fig 10. Since actin is a major component of
thin filaments in the sarcomere, it is possible that the reorganization
of actin fibers could alter the contractile function of cardiac
myocytes. It should be noted, however, that cytoskeletal reorganization
by hypertrophic agonists has been observed predominantly in in vitro
models of cardiac hypertrophy and that its role in
hypertrophy in vivo remains to be elucidated.
|
At present, we do not know how actin is modulated when premyofibrils are formed or when they later become striated actin fibers. We also do not know which signaling molecules cause formation of mature striated actin fibers in response to Ang II. We have shown that a rapamycin-sensitive mechanism (presumably p70 S6 kinase) is unlikely to be involved in the formation of striated actin fibers.18 Our preliminary results showed that inhibition of ERK1/2 by a specific MEK inhibitor, PD98059, failed to inhibit Ang II–induced striated actin fiber formation,17 suggesting that the MEK/ERK pathway is not involved in this process. Since Ras,4 other members of the Rho family, namely, Rac and Cdc42,4 5 and other kinases, such as protein kinase C,55 PI3-kinase,56 and p38RK,57 are known to cause actin organization in other cell types, it will be intriguing to see whether or not these molecules can cause mature striated actin fiber formation in cardiac myocytes. Very recently, p38RK has been reported to cause myofibril formation when an activated form of p38RK kinase, MKK6, is overexpressed in cardiac myocytes.58 The relationship between p38RK and Rho and whether Ang II activates p38RK remain to be elucidated.
Recent evidence suggests that the activated form of RhoA directly associates with various downstream signaling molecules and regulates their activity. However, the functional role of these molecules remains unclear, with the exception of Rho-kinase, which plays an important role in agonist-induced stress fiber formation in fibroblasts.6 Rho is also known to induce integrin-dependent tyrosine phosphorylation of actin-associated proteins, namely, FAK and paxillin, via a genistein-sensitive mechanism.59 FAK and paxillin further interact with various signaling molecules, such as Src, Grb2, PI3-kinase, p130Cas, and Crk.60 Since Ang II activates FAK, Src, and PI3-kinase and tyrosine-phosphorylates paxillin in cardiac myocytes46 49 61 (authors' unpublished data, 1997), it is possible that Rho may mediate Ang II–induced activation of these signaling events. Furthermore, Rho mediates c-fos expression by agonists for GPCRs via the serum response element,30 and constitutively active RhoA induces c-fos expression in cardiac myocytes.62 Our result suggests, however, that an additional mechanism seems to exist for Ang II–induced c-fos expression. Recent evidence suggests that Rho plays an important role in phenylephrine-induced ANF expression in cardiac myocytes.38 53 We have shown evidence that RhoA mediates Ang II–induced ANF expression. Further studies are required to elucidate the signaling mechanism involving RhoA in Ang II–induced cardiac hypertrophy.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
|---|
This study was supported in part by a Grant-in-Aid (Dr Sadoshima) and a Postdoctoral Fellowship (Dr Aoki) from the American Heart Association, Michigan Affiliate, Inc, and by a grant from the National Institutes of Health (Dr Izumo). We thank Drs A. Hall and S. Narumiya for plasmids and Dr J.M. Metzger for helpful suggestions.
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Footnotes |
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This manuscript was sent to Leslie A. Leinwand, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Received November 25, 1997; accepted January 28, 1998.
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References |
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K. Kuwahara, T. Barrientos, G. C. T. Pipes, S. Li, and E. N. Olson Muscle-Specific Signaling Mechanism That Links Actin Dynamics to Serum Response Factor Mol. Cell. Biol., April 15, 2005; 25(8): 3173 - 3181. [Abstract] [Full Text] [PDF]
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S. Vahebi, T. Kobayashi, C. M. Warren, P. P. de Tombe, and R. J. Solaro Functional Effects of Rho-Kinase-Dependent Phosphorylation of Specific Sites on Cardiac Troponin Circ. Res., April 15, 2005; 96(7): 740 - 747. [Abstract] [Full Text] [PDF]
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W. G. Barnes, E. Reiter, J. D. Violin, X.-R. Ren, G. Milligan, and R. J. Lefkowitz {beta}-Arrestin 1 and G{alpha}q/11 Coordinately Activate RhoA and Stress Fiber Formation following Receptor Stimulation J. Biol. Chem., March 4, 2005; 280(9): 8041 - 8050. [Abstract] [Full Text] [PDF]
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A. Yatani, K. Irie, T. Otani, M. Abdellatif, and L. Wei RhoA GTPase regulates L-type Ca2+ currents in cardiac myocytes Am J Physiol Heart Circ Physiol, February 1, 2005; 288(2): H650 - H659. [Abstract] [Full Text] [PDF]
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C. M. Filipeanu, F. Zhou, W. C. Claycomb, and G. Wu Regulation of the Cell Surface Expression and Function of Angiotensin II Type 1 Receptor by Rab1-mediated Endoplasmic Reticulum-to-Golgi Transport in Cardiac Myocytes J. Biol. Chem., September 24, 2004; 279(39): 41077 - 41084. [Abstract] [Full Text] [PDF]
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 D. G. Romero, M. Plonczynski, G. R. Vergara, E. P. Gomez-Sanchez, and C. E. Gomez-Sanchez Angiotensin II early regulated genes in H295R human adrenocortical cells Physiol Genomics, September 16, 2004; 19(1): 106 - 116. [Abstract] [Full Text] [PDF]
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M. J. Ryan, S. P. Didion, S. Mathur, F. M. Faraci, and C. D. Sigmund Angiotensin II-Induced Vascular Dysfunction Is Mediated by the AT1A Receptor in Mice Hypertension, May 1, 2004; 43(5): 1074 - 1079. [Abstract] [Full Text] [PDF]
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S. E. Hardt, H. Tomita, H. A. Katus, and J. Sadoshima Phosphorylation of Eukaryotic Translation Initiation Factor 2B{epsilon} by Glycogen Synthase Kinase-3{beta} Regulates {beta}-Adrenergic Cardiac Myocyte Hypertrophy Circ. Res., April 16, 2004; 94(7): 926 - 935. [Abstract] [Full Text] [PDF]
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K. Ito, Y. Hirooka, T. Kishi, Y. Kimura, K. Kaibuchi, H. Shimokawa, and A. Takeshita Rho/Rho-Kinase Pathway in the Brainstem Contributes to Hypertension Caused by Chronic Nitric Oxide Synthase Inhibition Hypertension, February 1, 2004; 43(2): 156 - 162. [Abstract] [Full Text] [PDF]
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M. C. Heidkamp, A. L. Bayer, B. T. Scully, D. M. Eble, and A. M. Samarel Activation of focal adhesion kinase by protein kinase C{epsilon} in neonatal rat ventricular myocytes Am J Physiol Heart Circ Physiol, October 1, 2003; 285(4): H1684 - H1696. [Abstract] [Full Text] [PDF]
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 J. Galle, A. Mameghani, S.-S. Bolz, S. Gambaryan, M. Gorg, T. Quaschning, U. Raff, H. Barth, S. Seibold, C. Wanner, et al. Oxidized LDL and its Compound Lysophosphatidylcholine Potentiate AngII-Induced Vasoconstriction by Stimulation of RhoA J. Am. Soc. Nephrol., June 1, 2003; 14(6): 1471 - 1479. [Abstract] [Full Text] [PDF]
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H. Akazawa and I. Komuro Roles of Cardiac Transcription Factors in Cardiac Hypertrophy Circ. Res., May 30, 2003; 92(10): 1079 - 1088. [Abstract] [Full Text] [PDF]
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A. J. Putnam, J. J. Cunningham, B. B. L. Pillemer, and D. J. Mooney External mechanical strain regulates membrane targeting of Rho GTPases by controlling microtubule assembly Am J Physiol Cell Physiol, March 1, 2003; 284(3): C627 - C639. [Abstract] [Full Text] [PDF]
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 J. Duan, H.-Y. Zhang, S. D. Adkins, B. H. Ren, F. L. Norby, X. Zhang, J. N. Benoit, P. N. Epstein, and J. Ren Impaired cardiac function and IGF-I response in myocytes from calmodulin-diabetic mice: role of Akt and RhoA Am J Physiol Endocrinol Metab, February 1, 2003; 284(2): E366 - E376. [Abstract] [Full Text] [PDF]
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C. Depre, M. Hase, V. Gaussin, A. Zajac, L. Wang, L. Hittinger, B. Ghaleh, X. Yu, R. K. Kudej, T. Wagner, et al. H11 Kinase Is a Novel Mediator of Myocardial Hypertrophy In Vivo Circ. Res., November 29, 2002; 91(11): 1007 - 1014. [Abstract] [Full Text] [PDF]
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A. Arai, J. A. Spencer, and E. N. Olson STARS, a Striated Muscle Activator of Rho Signaling and Serum Response Factor-dependent Transcription J. Biol. Chem., June 28, 2002; 277(27): 24453 - 24459. [Abstract] [Full Text] [PDF]
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T. Yanazume, K. Hasegawa, H. Wada, T. Morimoto, M. Abe, T. Kawamura, and S. Sasayama Rho/ROCK Pathway Contributes to the Activation of Extracellular Signal-regulated Kinase/GATA-4 during Myocardial Cell Hypertrophy J. Biol. Chem., March 1, 2002; 277(10): 8618 - 8625. [Abstract] [Full Text] [PDF]
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U. Laufs, H. Kilter, C. Konkol, S. Wassmann, M. Bohm, and G. Nickenig Impact of HMG CoA reductase inhibition on small GTPases in the heart Cardiovasc Res, March 1, 2002; 53(4): 911 - 920. [Abstract] [Full Text] [PDF]
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C. Kataoka, K. Egashira, S. Inoue, M. Takemoto, W. Ni, M. Koyanagi, S. Kitamoto, M. Usui, K. Kaibuchi, H. Shimokawa, et al. Important Role of Rho-kinase in the Pathogenesis of Cardiovascular Inflammation and Remodeling Induced by Long-Term Blockade of Nitric Oxide Synthesis in Rats Hypertension, February 1, 2002; 39(2): 245 - 250. [Abstract] [Full Text] [PDF]
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 M. Souchet, E. Portales-Casamar, D. Mazurais, S. Schmidt, I. Leger, J.-L. Javre, P. Robert, I. Berrebi-Bertrand, A. Bril, B. Gout, et al. Human p63RhoGEF, a novel RhoA-specific guanine nucleotide exchange factor, is localized in cardiac sarcomere J. Cell Sci., January 2, 2002; 115(3): 629 - 640. [Abstract] [Full Text] [PDF]
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 F. Charron, G. Tsimiklis, M. Arcand, L. Robitaille, Q. Liang, J. D. Molkentin, S. Meloche, and M. Nemer Tissue-specific GATA factors are transcriptional effectors of the small GTPase RhoA Genes & Dev., October 15, 2001; 15(20): 2702 - 2719. [Abstract] [Full Text] [PDF]
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N. Suematsu, S. Satoh, S. Kinugawa, H. Tsutsui, S. Hayashidani, R. Nakamura, K. Egashira, N. Makino, and A. Takeshita {alpha}1-Adrenoceptor-Gq-RhoA signaling is upregulated to increase myofibrillar Ca2+ sensitivity in failing hearts Am J Physiol Heart Circ Physiol, August 1, 2001; 281(2): H637 - H646. [Abstract] [Full Text] [PDF]
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 B. Kovacic-Milivojevic, F. Roediger, E. A.C. Almeida, C. H. Damsky, D. G. Gardner, and D. Ilic Focal Adhesion Kinase and p130Cas Mediate Both Sarcomeric Organization and Activation of Genes Associated with Cardiac Myocyte Hypertrophy Mol. Biol. Cell, August 1, 2001; 12(8): 2290 - 2307. [Abstract] [Full Text] [PDF]
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Y. Funakoshi, T. Ichiki, H. Shimokawa, K. Egashira, K. Takeda, K. Kaibuchi, M. Takeya, T. Yoshimura, and A. Takeshita Rho-Kinase Mediates Angiotensin II-Induced Monocyte Chemoattractant Protein-1 Expression in Rat Vascular Smooth Muscle Cells Hypertension, July 1, 2001; 38(1): 100 - 104. [Abstract] [Full Text] [PDF]
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H. Shimokawa, K. Morishige, K. Miyata, T. Kandabashi, Y. Eto, I. Ikegaki, T. Asano, K. Kaibuchi, and A. Takeshita Long-term inhibition of Rho-kinase induces a regression of arteriosclerotic coronary lesions in a porcine model in vivo Cardiovasc Res, July 1, 2001; 51(1): 169 - 177. [Abstract] [Full Text] [PDF]
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K. Takeda, T. Ichiki, T. Tokunou, N. Iino, S. Fujii, A. Kitabatake, H. Shimokawa, and A. Takeshita Critical Role of Rho-Kinase and MEK/ERK Pathways for Angiotensin II-Induced Plasminogen Activator Inhibitor Type-1 Gene Expression Arterioscler Thromb Vasc Biol, May 1, 2001; 21(5): 868 - 873. [Abstract] [Full Text] [PDF]
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K. Morishige, H. Shimokawa, Y. Eto, T. Kandabashi, K. Miyata, Y. Matsumoto, M. Hoshijima, K. Kaibuchi, and A. Takeshita Adenovirus-Mediated Transfer of Dominant-Negative Rho-Kinase Induces a Regression of Coronary Arteriosclerosis in Pigs In Vivo Arterioscler Thromb Vasc Biol, April 1, 2001; 21(4): 548 - 554. [Abstract] [Full Text] [PDF]
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 L. WEI, L. WANG, J. A. CARSON, J. E. AGAN, K. IMANAKA-YOSHIDA, and R. J. SCHWARTZ {beta}1 integrin and organized actin filaments facilitate cardiomyocyte-specific RhoA-dependent activation of the skeletal {alpha}-actin promoter FASEB J, March 1, 2001; 15(3): 785 - 796. [Abstract] [Full Text] [PDF]
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G. P. van Nieuw Amerongen and V. W.M. van Hinsbergh Cytoskeletal Effects of Rho-Like Small Guanine Nucleotide-Binding Proteins in the Vascular System Arterioscler Thromb Vasc Biol, March 1, 2001; 21(3): 300 - 311. [Abstract] [Full Text] [PDF]
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P. M. Kang, A. Haunstetter, H. Aoki, A. Usheva, and S. Izumo Morphological and Molecular Characterization of Adult Cardiomyocyte Apoptosis During Hypoxia and Reoxygenation Circ. Res., July 21, 2000; 87(2): 118 - 125. [Abstract] [Full Text] [PDF]
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D. E. Vatner and D. L. Kunze Prologue: low-molecular-weight GTPases in the heart and circulation Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H1733 - H1735. [Full Text] [PDF]
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M. R. Morissette, V. P. Sah, C. C. Glembotski, and J. H. Brown The Rho effector, PKN, regulates ANF gene transcription in cardiomyocytes through a serum response element Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H1769 - H1774. [Abstract] [Full Text] [PDF]
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A. Clerk and P. H. Sugden Small Guanine Nucleotide-Binding Proteins and Myocardial Hypertrophy Circ. Res., May 26, 2000; 86(10): 1019 - 1023. [Abstract] [Full Text] [PDF]
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D. M. Eble, J. B. Strait, G. Govindarajan, J. Lou, K. L. Byron, and A. M. Samarel Endothelin-induced cardiac myocyte hypertrophy: role for focal adhesion kinase Am J Physiol Heart Circ Physiol, May 1, 2000; 278(5): H1695 - H1707. [Abstract] [Full Text] [PDF]
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T. K Borg, E. C Goldsmith, R. Price, W. Carver, L. Terracio, and A. M Samarel Specialization at the Z line of cardiac myocytes Cardiovasc Res, May 1, 2000; 46(2): 277 - 285. [Full Text] [PDF]
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 S. Kim and H. Iwao Molecular and Cellular Mechanisms of Angiotensin II-Mediated Cardiovascular and Renal Diseases Pharmacol. Rev., March 1, 2000; 52(1): 11 - 34. [Abstract] [Full Text] [PDF]
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T. Yamakawa, S.-i. Tanaka, K. Numaguchi, Y. Yamakawa, E. D. Motley, S. Ichihara, and T. Inagami Involvement of Rho-Kinase in Angiotensin II-Induced Hypertrophy of Rat Vascular Smooth Muscle Cells Hypertension, January 1, 2000; 35(1): 313 - 318. [Abstract] [Full Text] [PDF]
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R. R. Baliga, D. R. Pimental, Y.-Y. Zhao, W. W. Simmons, M. A. Marchionni, D. B. Sawyer, and R. A. Kelly NRG-1-induced cardiomyocyte hypertrophy. Role of PI-3-kinase, p70S6K, and MEK-MAPK-RSK Am J Physiol Heart Circ Physiol, November 1, 1999; 277(5): H2026 - H2037. [Abstract] [Full Text] [PDF]
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K. C Wollert and H. Drexler The renin-angiotensin system and experimental heart failure Cardiovasc Res, September 1, 1999; 43(4): 838 - 849. [Abstract] [Full Text] [PDF]
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K. Numaguchi, S. Eguchi, T. Yamakawa, E. D. Motley, and T. Inagami Mechanotransduction of Rat Aortic Vascular Smooth Muscle Cells Requires RhoA and Intact Actin Filaments Circ. Res., July 9, 1999; 85(1): 5 - 11. [Abstract] [Full Text] [PDF]
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T. M. Seasholtz, M. Majumdar, and J. H. Brown MINIREVIEW: Rho as a Mediator of G Protein-Coupled Receptor Signaling Mol. Pharmacol., June 1, 1999; 55(6): 949 - 956. [Full Text]
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T. M. Seasholtz, M. Majumdar, D. D. Kaplan, and J. H. Brown Rho and Rho Kinase Mediate Thrombin-Stimulated Vascular Smooth Muscle Cell DNA Synthesis and Migration Circ. Res., May 28, 1999; 84(10): 1186 - 1193. [Abstract] [Full Text] [PDF]
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P. H. Sugden Signaling in Myocardial Hypertrophy : Life After Calcineurin? Circ. Res., April 2, 1999; 84(6): 633 - 646. [Full Text] [PDF]
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J. Sadoshima Versatility of the Angiotensin II Type 1 Receptor Circ. Res., June 29, 1998; 82(12): 1352 - 1355. [Full Text] [PDF]
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W. Ni, K. Egashira, C. Kataoka, S. Kitamoto, M. Koyanagi, S. Inoue, and A. Takeshita Antiinflammatory and Antiarteriosclerotic Actions of HMG-CoA Reductase Inhibitors in a Rat Model of Chronic Inhibition of Nitric Oxide Synthesis Circ. Res., August 31, 2001; 89(5): 415 - 421. [Abstract] [Full Text] [PDF]
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T. M. Seasholtz, T. Zhang, M. R. Morissette, A. L. Howes, A. H. Yang, and J. H. Brown Increased Expression and Activity of RhoA Are Associated With Increased DNA Synthesis and Reduced p27Kip1 Expression in the Vasculature of Hypertensive Rats Circ. Res., September 14, 2001; 89(6): 488 - 495. [Abstract] [Full Text] [PDF]
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