From the ¶ Department of Pharmacology and Graduate Program in
Biomedical Sciences and Department of Medicine and Center
for Molecular Genetics, University of California, San Diego,
La Jolla, California 92093
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ABSTRACT |
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The assembly of contractile proteins into
organized sarcomeric units is one of the most distinctive features of
cardiac myocyte hypertrophy. In a well characterized in
vitro model system using cultured neonatal rat ventricular
myocytes, a subset of G protein-coupled receptor agonists has been
shown to induce actin-myosin filament organization. Pretreatment of
myocytes with C3 exoenzyme ADP-ribosylated Rho and inhibited the
characteristic 1-adrenergic receptor agonist-induced myofibrillar organization, suggesting involvement of the Rho GTPase in
cardiac myofibrillogenesis. We used adenoviral mediated gene transfer
to examine the effects of activated Rho and inhibitory mutants of one
of its effectors, Rho kinase, in myocytes. Rho immunoreactivity was
increased in the particulate fraction of myocytes infected with a
recombinant adenovirus expressing constitutively activated Rho.
Rho-infected cells demonstrated a striking increase in the assembly and
organization of sarcomeric units and in the expression of the atrial
natriuretic factor protein. These Rho-induced responses were markedly
inhibited by co-infection with adenoviruses expressing putative
dominant negative forms of Rho kinase. A parallel pathway involving
Ras-induced myofibrillar organization and atrial natriuretic factor
expression was only minimally affected.
1-Adrenergic receptor agonist-induced myofibrillogenesis was inhibited by some but
not all of the Rho kinase mutants. Our data demonstrate that activated
Rho has profound effects on myofibrillar organization in cardiac
myocytes and suggest that Rho kinase mediates Rho-induced hypertrophic
responses.
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INTRODUCTION |
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Agonists that stimulate Gq-coupled receptors induce
hypertrophy in neonatal rat ventricular myocytes. Prominent features of the in vitro hypertrophic response include altered
expression of contractile protein genes such as that for myosin light
chain (MLC)1-2 and the
embryonic isoforms of myosin heavy chain and actin, as well as
increased assembly of contractile proteins into organized sarcomeric
units (myofibrillogenesis, myofibrillar organization) (1). These
responses have been demonstrated to be dependent on the function of the
subunit of the heterotrimeric Gq protein as well as the
low molecular weight GTPase Ras (2, 3). We recently showed that another
low molecular weight GTPase, Rho, was also a mediator of increased
MLC-2 and atrial natriuretic factor (ANF) gene expression induced
by the Gq-coupled
1-adrenergic receptor
(4).
There is accumulating evidence that Rho GTPases play crucial roles in cytoskeletal regulation, mediating cellular events such as changes in cell morphology, cell motility, and cytokinesis (reviewed in Refs. 5 and 6). Specifically, Rho is required for actin stress fiber and focal adhesion complex formation (5). Putative downstream effectors of Rho have been identified, including Rho kinase (7, 8), p160 ROCK (9), protein kinase N (10, 11), and phosphatidylinositol 4-phosphate 5-kinase (12). Recently, it was demonstrated that Rho-induced morphological responses in non-muscle cells were mediated through Rho kinase (13, 14). Rho and Rho kinase have also been shown to regulate the phosphorylation of MLC (15-18), thereby altering the sensitivity of smooth muscle myosin to calcium (17-19). Although myosin phosphorylation is not an obligatory or initiating step in activating the contractile response in cardiac muscle, it may modulate contractile function, as occurs in striated muscle (20, 21). Contractile function may in turn be important for cardiac myofibrillogenesis (22, 23). Thus, we reasoned that Rho and Rho kinase might play key roles in regulating the organization of actin-myosin myofibrils in cardiomyocytes.
In this study, we have tested the involvement of Rho and Rho kinase in
the regulation of myofibrillar organization induced by the
1-adrenergic receptor agonist phenylephrine (PE) and the ability of activated Rho to mimic this response. Our findings demonstrate that Rho- and Rho kinase-dependent signaling
pathways regulate myofibrillar organization and ANF expression in
myocardial cells.
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EXPERIMENTAL PROCEDURES |
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Cell Culture-- Neonatal rat ventricular myocytes were prepared from hearts of 2-3-day-old Sprague-Dawley rat pups as described previously (24, 25). Briefly, hearts were digested with collagenase and myocytes purified over a Percoll gradient. Myocytes were cultured overnight in 4:1 Dulbecco's modified Eagle's medium:medium 199 containing 10% horse serum, 5% fetal calf serum, and antibiotics (100 units/ml penicillin, 100 µg/ml streptomycin).
Preparation of Purified Recombinant C3
Exoenzyme--
Clostridium botulinum C3 exoenzyme cDNA
in the PGEX vector expression system was a gift from Dr. Judy Meinkoth
(University of Pennsylvania). The recombinant protein was purified from
Escherichia coli as described previously (26). Briefly,
expression of a glutathione S-transferase-C3 fusion protein
was induced with isopropyl -D-thiogalactopyranoside, and
the fusion protein purified by binding to glutathione-agarose beads.
Thrombin was used to cleave the C3 protein from the beads.
Generation of Recombinant Adenoviruses-- Expression plasmids encoding dominant negative mutants of Rho kinase (RB, Rho-binding domain; PH, pleckstrin homology domain; CAT-KD, kinase deficient catalytic domain) were from Dr. Kozo Kaibuchi (Nara Institute of Science and Technology, Japan) and have been described previously (14). Generation of recombinant adenoviruses expressing hemagglutinin (HA)-tagged activated Rho (L63Rho), activated H-Ras (V12Ras) (27), or Myc-tagged Rho kinase mutants driven by the cytomegalovirus promoter was carried out through homologous recombination between co-transfected pJM17 and the shuttle plasmids in 293 cells as described previously (28). Integration of the transgene into the adenoviral genome was determined by a polymerase chain reaction and restriction analysis. High titer adenovirus was prepared as described previously (28).
Adenoviral Infection--
Following overnight incubation in
serum-containing medium, myocytes cells were washed and infected at a
titer of 50-100 viral particles/cell. 24 h later, cells were
washed and serum-free medium was replaced. Cells were further incubated
at 37 °C for 36 h prior to stimulation with 40 µM
PE supplemented with 0.8 µM propranolol (to block
-adrenergic receptors). For co-infection experiments, myocytes were
infected with adenoviruses expressing the Rho kinase mutants 9 h
before infection with adenovirus expressing L63Rho or V12Ras.
Actin and ANF Staining-- Myocytes were fixed in 3% paraformaldehyde, permeabilized in 0.3% Triton X-100, and blocked in 10% serum in phosphate-buffered saline. Cells were then incubated with polyclonal rabbit anti-ANF antibody (Peninsula Labs) for 1 h at 37 °C, washed, and subsequently incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit antibody and rhodamine-conjugated phalloidin (Molecular Probes) for 1 h at 37 °C.
Cell Fractionation-- Myocytes were lysed in a hypotonic lysis buffer (10 mM Hepes, pH 8.0, 2 mM EDTA, 1 mM MgCl2, 10 mM Na4P2O7, 10 mM NaF, 500 µM Na3VO4, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) by shearing 15 times through a 27-gauge needle. Nuclei and unbroken cells were pelleted by low speed (500 × g) centrifugation. The supernatant was then subjected to a 10-min high speed spin (37,000 × g). The pellet obtained from this centrifugation was designated as the particulate fraction, and was resuspended in the hypotonic lysis buffer. This fraction contains membrane as well as cytoskeleton. The supernatant was further clarified by a 30-min high speed (37,000 × g) spin, and the resultant supernatant was designated as the soluble or cytosolic fraction. Protein content in particulate and cytosolic fractions was determined by Bradford analysis.
Western Blotting-- 10-50 µg of protein samples were boiled in Laemmli buffer and separated by 15% SDS-polyacrylamide gel electrophoresis. Proteins were then electrotransfered onto Immobilon-P membranes (Millipore). Membranes were blocked in phosphate-buffered saline, 0.1% Tween 20 containing 3% bovine serum albumin, and incubated for 1 h at room temperature with monoclonal mouse anti-RhoA antibody (Santa Cruz Biotechnologies, Inc.) or with monoclonal mouse anti-HA antibody (Boehringer Mannheim) in blocking buffer. After several washes with phosphate-buffered saline, 0.1% Tween 20, membranes were incubated with horseradish peroxidase-conjugated anti-mouse antibody (Sigma) for 1 h at room temperature. Enhanced chemiluminescence was then performed using the supersignal chemiluminescent detection system (Pierce).
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RESULTS |
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C3 Exoenzyme Inhibits 1-Adrenergic Receptor
Agonist-induced Myofibrillar Organization--
C3 exoenzyme has been
previously demonstrated to specifically ADP-ribosylate and inactivate
Rho (29). A 24-h pretreatment of myocytes with 40 µg/ml C3
ribosylated Rho, as indicated by a ~80% decrease in the amount of
Rho available for the back-ribosylation reaction (data not shown). The
organization of contractile proteins into sarcomeric units is a
characteristic feature of the hypertrophic phenotype. Phenylephrine
treatment resulted in an increase in myocyte cell size and induced a
marked increase in the organization of actin myofibrils, as assessed by
phalloidin staining (Fig. 1c).
Pretreatment of myocytes with 40 µg/ml purified recombinant C. botulinum C3 exoenzyme disrupted the ability of PE to induce this
response (Fig. 1e). Consistent with our previous data
suggesting that Rho regulates expression of the cardiac-specific
embryonic gene ANF, PE-induced ANF protein expression was also
significantly reduced in C3-pretreated myocytes (Fig. 1, d
and f).
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Infection of Myocytes with Activated Rho-expressing Adenovirus Increases Rho Immunoreactivity in the Particulate Fraction-- We recently demonstrated that adenoviral infection results in a high efficiency of gene delivery into neonatal rat ventricular myocytes (30). A recombinant adenovirus expressing HA-tagged activated Rho (L63Rho) was generated to determine whether expression of constitutively activated Rho would activate hypertrophic responses in cardiomyocytes. The level of expression of Rho protein was examined by immunoblotting lysates from uninfected myocytes and myocytes infected with control (LacZ) or L63Rho adenovirus with an anti-RhoA antibody. Myocytes infected with LacZ/Adv did not have altered Rho content relative to uninfected cells (data not shown), but infection with L63Rho/Adv resulted in a significant increase in Rho protein (Fig. 2a). Two immunoreactive bands were detected in L63Rho-infected myocytes. The upper band was absent in LacZ-infected cells, and is presumed to be a result of differential processing of Rho upon overexpression of the protein. These data demonstrate that infection of myocytes with a control virus does not change the endogenous Rho content and that infection with L63Rho virus markedly increases Rho immunoreactivity.
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Activated Rho Induces Myofibrillar Organization and ANF Expression in Myocytes-- Since inhibition of Rho function prevented PE-induced myofibrillar organization in cardiomyocytes, we hypothesized that expression of a constitutively activated mutant of Rho would lead to the organization of myofibrils into sarcomeric units. Indeed, as shown in Fig. 3c, infection of myocytes with L63Rho/Adv induced a marked increase in the assembly and organization of sarcomeric units as compared with LacZ/Adv-infected control cells (Fig. 3a). Vinculin, a protein involved in the myofibril-sarcolemma attachment, was also found by immunostaining to be diffusely localized in control-infected cells, but localized to the Z-band of L63Rho/Adv-induced sarcomeres (data not shown). A slight but consistent increase in myocyte cell size was observed in the L63Rho/Adv-infected cells. In addition, L63Rho/Adv-infected cells also showed increased ANF immunoreactivity (Fig. 3d), consistent with our previous observations of transcriptional activation of the ANF reporter gene (4). The remarkable ability of activated Rho to induce sarcomere formation, together with the inhibition of myofibrillar organization in C3-treated cells, implicates Rho as a regulator of myofibrillogenesis in cardiac myocytes.
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Dominant Negative Mutants of Rho Kinase Inhibit Rho-induced Myofibrillar Organization and ANF Expression-- Among the various Rho targets identified thus far, Rho kinase is of particular interest because it has been suggested to be involved in both Rho-dependent stress fiber formation and gene expression (13, 14, 37). To further dissect the downstream pathway for Rho-dependent hypertrophic responses, three types of dominant negative mutants of Rho kinase were cloned into an adenovirus expression system. The RB/Adv expresses the Rho binding domain for Rho kinase; PH/Adv expresses the pleckstrin homology domain of Rho kinase; CAT-KD/Adv expresses a catalytically inactive form of Rho kinase. These mutant Rho kinase proteins have been shown to suppress Rho-induced actin stress fiber and focal adhesion complex formation in Swiss 3T3 and Madin-Darby canine kidney cells (14). Adenoviral expression of the RB mutant completely abolished L63Rho/Adv-induced myofibrillar organization and ANF expression (Fig. 4 c and d). The PH or CAT-KD mutants, while less efficacious than RB, also markedly inhibited myofibrillar organization (Fig. 4, e and g) and ANF expression (Fig. 4, f and h) in L63 Rho/Adv-infected myocytes.
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Dominant Negative Mutants of Rho Kinase Display Variable Effects on
PE-induced Myofibrillar Organization--
To evaluate the role of Rho
kinase in agonist-induced myofibrillar organization, myocytes were
infected with the various dominant negative Rho kinase mutants and then
stimulated with PE. While the RB Rho kinase mutant inhibited PE-induced
myofibrillogenesis (Fig. 6b),
the PH and CAT-KD mutants displayed little inhibitory effect (Fig. 6,
c and d). The RB mutant should interfere with Rho
function by binding Rho and preventing its interaction with its
downstream effectors. Inhibition by the RB mutant is thus consistent
with our results demonstrating inhibition by C3-mediated inactivation
of Rho (Fig. 1) and further supports a role for Rho in
1-adrenergic receptor-induced cardiac
myofibrillogenesis. The inability of the other Rho kinase mutants to
block suggests that additional downstream effectors are required for
the response to PE (Fig. 7).
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DISCUSSION |
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Little is known regarding the involvement of Rho in myofibrillar organization in cardiac myocytes. Studies examining the effects of microinjection of C3 transferase protein into neonatal rat ventricular myocytes suggested that Rho function was not required for regulation of actin myofibrillar organization (38). On the other hand, we observed that treatment of rat cardiomyocytes with the Clostridium limosum C3-like exoenzyme blocks agonist-stimulated myofibrillar organization (39). This finding is consistent with a more recent report demonstrating that introduction of C3 transferase into chicken embryonic cardiomyocytes by electroporation leads to the disruption of developing myofibrils (40). Data presented here extend these findings, demonstrating that C3 transferase inhibits myofibrillogenesis induced by agonist in mammalian cardiomyocytes. Furthermore, the Rho-binding domain of Rho kinase (RB), which behaves as a putative competitive inhibitor of Rho, also inhibits PE-induced myofibrillar organization. These findings support a role for Rho in the regulation of cardiac myofibrillogenesis induced by agonist treatment.
While a requirement for Rho in cardiac myofibrillogenesis may be deduced from studies using C3 exoenzyme to inactivate Rho (38-40), no published studies have determined whether Rho activation is a sufficient signal to effect myofibrillar organization. By taking advantage of adenoviral expression to achieve complete infection efficiency of cardiomyocytes, we demonstrate that expression of a constitutively activated mutant of Rho results in dramatic increases in the organization of myofibrils into sarcomeric units. In addition, a significant increase in cellular Rho content is observed, and the heterologously expressed Rho is preferentially localized in the particulate (membrane/cytoskeleton) fraction. This latter finding is not unexpected, as increases in membrane-localized Rho have been associated with its activation (31-36). However this, to our knowledge, is the first direct demonstration that an activated form of Rho does in fact preferentially localize to the particulate fraction. These experiments therefore establish that activated Rho associates with the membrane and/or cytoskeleton and serves as a stimulus for myofibrillogenesis in cardiac muscle cells.
Rho has been shown to regulate transcriptional activation of the c-fos SRE as well as participate in actin-based cytoskeletal organization in fibroblasts (37, 41, 42). The relationship between these genetic and morphological effects of Rho has yet to be elucidated. Hypertrophy of cardiac myocytes is characterized by transcriptional activation of immediate early, embryonic, and contractile protein genes and by myofibrillar organization. Using C3 transferase and a dominant negative mutant of Rho, we previously showed that Rho function was required for PE-induced transcriptional activation of ANF and MLC-2 reporter genes (4). We demonstrate here that inactivation of Rho by C3 pretreatment, or sequestration of Rho by the RB Rho kinase mutant, also inhibits PE-induced ANF protein expression, and that activated Rho stimulates ANF protein expression. Activated Rho was recently shown to stimulate c-fos gene expression via SRE and AP-1 binding sites in myocardial cells (43). The ANF promoter contains several canonical SREs, and preliminary experiments suggest that these are activated in a Rho-dependent manner.2
It is not known how the observed changes in cardiac gene expression relate to the accompanying changes in myofibrillar organization seen in hypertrophy. Changes in cardiac gene expression as well as myofibrillar assembly have been suggested by some investigators to occur secondary to changes in cardiomyocyte contractile function. Specifically, ventricular myosin heavy chain gene expression, and the synthesis and organization of actin into striated myofibrils are prevented when contractile activity is arrested with a calcium channel blocker (44-46). In addition, we have observed that cytochalasin D, which inhibits actin polymerization, prevents ANF and MLC-2 gene expression and myofibrillar organization induced by PE stimulation of cardiac myocytes.3 Since PE-induced gene expression (4, 38) and myofibrillar organization are also both inhibited by blockade of Rho function, Rho-mediated effects on the actin cytoskeleton may coordinate these responses in cardiomyocytes.
The Rho effector, Rho kinase is a serine-threonine kinase that binds
and is regulated by GTP-bound Rho (7). We used adenoviral expression of
three previously characterized mutants of Rho kinase to examine the
involvement of Rho kinase in cardiac myofibrillogenesis. The RB mutant
is predicted to act as a dominant interfering protein by titrating out
upstream activators of Rho kinase including Rho; the PH mutant may
inhibit proper localization of Rho kinase; the CAT-KD mutant consists
of a kinase-deficient catalytic domain that may inhibit the interaction
of Rho kinase with its substrates (14). We demonstrate that Rho-induced
myofibrillar organization and ANF protein expression are completely
extinguished by expression of the RB mutant, and markedly attenuated by
coexpression of the PH or CAT-KD mutants. In contrast, these mutants
displayed little inhibitory effect on the Ras-induced responses,
consistent with our previous data suggesting that Ras and Rho define
separate and complementary pathways in cardiomyocyte signaling to
hypertrophic gene expression (4). Myofibrillar organization induced by
1-adrenergic receptor stimulation with PE was also
attenuated by expression of the RB mutant. However, the PH and CAT-KD
mutants displayed little inhibitory effect. Together, these data
indicate that while Rho kinase is a mediator of the Rho-generated
signals leading to myofibrillar organization and ANF expression, it is
not required for Ras-induced hypertrophic responses, and may not be the
only Rho effector utilized in
1-adrenergic
receptor-mediated activation of myofibrillar organization (Fig. 7).
Kinase cascades involving other putative Rho targets, including protein
kinase N or protein kinase C-related PRK2, may therefore exist
downstream of, and mediate the effects of Rho.
Further studies are needed to determine how Rho kinase modulates Rho-induced cardiac myofibrillogenesis. Known substrates for Rho kinase are myosin light chain and the myosin binding subunit of MLC phosphatase (15, 16). Evidence for the presence of the myosin-binding subunit of myosin phosphatase in bovine heart myofibrils has recently been presented (47). In smooth muscle and non-muscle cells, activated Rho kinase increases the level of MLC phosphorylation by direct phosphorylation of MLC and by inactivation of the MLC phosphatase. Rho kinase can thereby induce calcium-independent smooth muscle contraction (17).
The role of MLC phosphorylation in cardiac muscle contraction is less clear. However, cardiac contractile function in diabetic rats is depressed in association with decreased MLC content and MLC phosphorylation (48). Missense mutations in MLC have also been associated with a rare variant of cardiac hypertrophy in humans (49). Finally, mouse embryos which harbor a selective ablation of the ventricular myosin light chain (MLC-2v) gene display embryonic heart failure despite adequate levels of atrial MLC-2, implying that there may be an essential role for MLC-2v in the maintenance of ventricular muscle function (50). We are currently examining the importance of MLC-2v phosphorylation using mice harboring a genetically modified MLC-2v gene which lacks the serine phosphorylation site (by conversion to an alanine residue). This should establish whether there is a specific role for MLC-2v phosphorylation in myofibrillar organization and cardiac contractile function. The effects of Rho and Rho kinase on cardiac muscle MLC phosphorylation and contraction are also currently being examined.
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ACKNOWLEDGEMENTS |
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We thank Drs. Kozo Kaibuchi, Judy Meinkoth, Gary Bokoch, and Klaus Aktories for reagents, Dr. David Becker for helpful discussions, and David Goldstein, Mahmoud Itani, and Anh Le for technical assistance.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants HL28143 (to J. H. B.) and HL46345 (to J. H. B. and K. R. C.).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.
§ These authors contributed equally to this manuscript.
Supported by an American Heart Association-California
Affiliate predoctoral fellowship. Work was in partial fulfillment of the Ph.D. degree in the Biomedical Sciences Graduate Program.
** To whom correspondence should be addressed: Dept. of Pharmacology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0636. Tel.: 619-534-2595; Fax: 619-822-0041; E-mail: jhbrown{at}ucsd.edu.
1 The abbreviations used are: MLC, myosin light chain; MLC-2v; ventricular MLC-2; ANF, atrial natriuretic factor; PE, phenylephrine; SRE, serum response element; HA, hemagglutinin; RB, Rho-binding domain; PH, pleckstrin homology domain; CAT-KD, kinase deficient catalytic domain.
2 M. R. Morissette, V. P. Sah, C. C. Glembotski and J. H. Brown, unpublished data.
3 V. P. Sah and J. H. Brown, unpublished data.
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REFERENCES |
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