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THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 3, Issue of January 25, pp. 2244-2249,1993 Printed in U.S.A.

HRas-dependent Pathways Can Activate Morphological and Genetic Markers of Cardiac Muscle Cell Hypertrophy* (Received for publication, March 31, 1992, and, in revised form, September 11, 1992)

Andrew Thorburn$Qll, Jackie ThorburnQII, Sei-Yu Chen**, Scott Powers**, Huda E. ShubeitaQII, James R. Feramisco$$$$,and Kenneth R. ChienQII$$ From the $Cancer Center, Departmentsof §Medicine and $$Pharmacology, IICenter for Molecular Genetics, and American Heart Association-Eugher Foundation Center for Molecular Biology, University of California at S a n Diego, La Jolla, California 92093 and the **Department of Biochemistry, Uniuersityof Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854

We have investigated the role of the proto-oncogene tractile protein gene expression is mediated byMyoD and HRas in cardiac cell growth and hypertrophy. By di- related helix-loop-helix factors (4, 5). The expression of Ras rect needle microinjectionof activated Ras protein into in skeletal muscle myoblasts leads to the rapid induction of primary neonatal rat ventricular cardiac myocytes, we cell proliferation and preventsdifferentiation into the mature find that, unlike many other cell types, Ras does not muscle phenotype (6-8). Accordingly, we have designed a induce DNA synthesis inthese cells. However, injection series of experiments to test the role of the HRas protein in of activated Ras does induce expression of both the c- the growth and hypertrophy of primary ventricular cardiac Fos and atrial natriuretic factor (ANF) genes. Expres- muscle cells. Wefind that active Ras protein does not induce sion of both these genes is associated with the hyper- proliferation in these cells, but rather activates several phetrophic response in ventricular myocytes suggesting that Ras is involved in the hypertrophic signalling notypic features of muscle cell hypertrophy. Furthermore, we pathway. Ras injection also causes morphological demonstrate that a dominantly actingnegative HRas mutant changes in the cells so that they increase in profile and significantly inhibits the hypertrophic effect of the a-adreshow changes in the organization of the contractile nergic agonist phenylephrine, suggesting that a Ras-dependapparatus. Further support for a role for Ras in the ent pathway, at least in part, mediates hypertrophy following hypertrophic response was obtained from studies stimulation of a classical G protein-coupled receptor. showing that activated Ras stimulates ANF promoter EXPERIMENTALPROCEDURES activity in transient transfection assays. We also show that a dominant interfering Ras mutant inhibits the Cell Culture and Microinjection-Primary neonatal rat ventricular hypertrophic stimulation of the ANFpromoterby myocytes were isolated from tissue by collagenasedigest.ion and plated phenylephrine, indicating a role for Ras in the hyper- in serum-containing media onto laminin-coated chamber slides. Introphic effect of an a-adrenergic agonist. jections were carried out by standard methods using modifications

In response to a wide variety of mechanical, hormonal, and pathological stimuli, the myocardium is able toadapt to increased workloads through the hypertrophy of individual muscle cells. The hypertrophic response is characterized by a number of phenotypic changes, which include an increase in cell size and contractile protein content without cellular proliferation, activation of an embryonic gene program, and induction of a panel of immediate early genes (for review, see Ref. 1). Although the precise signalling mechanisms which activate cardiac hypertrophy remain unclear, a cultured myocardial cell model in which several features of hypertrophy are activated following stimulation with either a-adrenergic agonists or endothelin has served as a valuable model system to study this complex adaptive response (2,3). During skeletal muscle myogenesis, the acquisition of the differentiated muscle cell phenotype and theconcomitant up-regulation of con* This work was supported by grants from the National Institutes of Health and theAmerican Heart Association (to K. R. C.) and from the National Cancer Institute (to J. R. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact. 7 Supported by a long term fellowship from the Human Frontiers Science Program. Present address: Dept. of Medicine, University of Utah, Salt Lake City, UT 84132. $$ To whom reprint requests should be addressed 0613-C, UCSD, Dept. of Medicine, La Jolla, CA 92093.

developed previously for primary cardiac myocytes (9). Immediately prior to injection, the cells were treated with 20 WM 2,3-butanedione monoxime (Sigma) which prevents hypercontraction after the influx of calcium caused by puncture of the cell membrane, and cells were injected for 10 min using a semiautomated microinjection apparatus (Eppendorf 5170). In 10 min we can inject about 80 to 100 cardiac cells of which we routinely find 30 to 60% survive to be analyzed later. The variation in survival is due to the variability of different preparations of primary cells and is also affected by the particular experiment, for example, cellsmaintained in serum-containingmedia survive better thancells maintained andinjected in serum-freemedia. After injection, the 2,3-butanedionemonoxime was removed by washing with serum-free media, and the cells were incubated for various times in either serum-free media or (for DNA synthesis analysis) serum-containing media. Activated Ras protein was expressed and purified fromEscherichia colias described previously(IO)and injected at a concentration of 1 mg/ml in an injection buffer consisting of 40 mM Hepes, pH 7.4, 50 mM NaCI. In all the experiments, a rabbit, mouse, or sheep antibody (Sigma) at a concentration of 3-4 mg/ml wasco-injected along with the experimental material so that the injected cells could be unequivocally identified. Expression plasmids were injected directly into the nucleus a t concentrations of 0.2 mg/ ml each. For these experiments, the cantrol for successful injection was to inject a constitutively active reporter plasmid (RSV-luciferase)’ along with the plasmid being tested. After staining for luciferase, the cells which expressed this plasmid were scored for ANF expression. D N A Synthesis Assay-After injection, the thymidine analog BrdU (Amersham) was added to themedia at a lOOOX dilution of the stock The abbreviations usedare:RSV,Rous sarcoma virus;ANF, atrial natriuretic factor; MLC-2, myosinlight chain-2; BrdU, bromodeoxyuridine;CMV,cytomegalovirus; PBS,phosphate-buffered saline.

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HRas-induced Markers of Cardiac Cell Hypertrophy as recommended by the manufacturer. For immunofluorescence analysis, the cells were fixed in 95% ethanol, 5% acetic acid, blocked with 10% goat serum in PBS, 0.1% Tween 20, and stained. BrdU was detectedusing amonoclonal antibody from Amersham which contains DNase (the DNase partially digests the chromatin inthe cell allowing the antibody to gain access to theBrdU). After the primary antibody incubation, the cells were washed thoroughly with PBS, 0.1% Tween 20, and then incubated with a cascade blue anti-mouse (Molecular Probes) to identify the incorporated BrdU and Texas Red-conjugated anti-rabbit antibody to identify the co-injected rabbit IgG. After staining, the pattern of immunofluorescence was analyzed using a Zeiss fluorescent microscope. Photographs were taken on TMax 400 film (Kodak) pushed to 800 ASA. Immunofluorescence Analysis-Cells were starved for 18-24 h in serum-free medium, prior to injection as before. All the injections were carried out into cells in nonconfluent areas, which were either not touching or were in small groups of no more than three or four cells. This is particularly important in the assay of ANF expression, since larger groups of cells often express the gene in the absence of any other stimuli. Since c- Fos expression was detected using a rabbit anti-Fos antibody (Oncogene Science), a marker mouse antibody was used to identify injected cells. ANF was detected with a mouse antibody (generouslyprovided by Dr. C. Glembotski, San Diego State University), while MLC-2 was detected with a rabbit antibody. For analysis of ANF expression alone, a marker rabbit antibody was injected, while for analysis of both ANF and MLC-2, a sheepantibody was included to identify the injected cells. Analysis of ANF expression after injection of expression plasmids was carried out by staining for both luciferase and ANF using a rabbit antibody against luciferase (5’,3’) and the mouse antibody against ANF. After incubation for 1 h (for c-Fos analysis) or 30 h (for ANF and MLC-2 analysis), cells were fixed in 3.7% formaldehyde in PBS, lysed in 0.3%Triton X-100 in PBS, blocked in 10% goat serum, PBS, 0.1% Tween 20, and incubated with the primary antibodies. After washing, the primary antibodies were detected with fluorochrome-conjugated second antibodies as required and analyzed by fluorescence microscopy as before. Transfection Analysis-Transfections were carried out in 10-cm dishes as previously described (11) using the calcium phosphate method. 4 pgof a CMV-0-galactosidase plasmid was used in all transfections, along with 15 pg of the ANF or AP-1 luciferase plasmid and 5 pg of the required Ras expression plasmid. Inert DNA wasused t o make up the total DNA for the samples where Ras was not expressed. After incubation for 48 h in serum-free or phenylephrinecontaining medium as required, cells were harvested, and 0-galactosidase and luciferase activities were measured using standard enzymatic assays. Luciferase activities were normalized to their corresponding @-galactosidaseactivities to correct for variation in transfection efficiency. Plasmids-Plasmids for the transfection experiments were as follows. The P-galactosidase control plasmid used the CMV promoter for expression, while the Val-12 Ras expression plasmid (provided by Dr. A. Schonthal) used the native Ras promoter. The Ala-15 mutant was expressed from the SV40 promoter in control experiments and a parental SV40 promoter plasmid (pSVL, Pharmacia LKB Biotechnology Inc.) was used. The ANF-luciferase plasmid contains a 3.3kilobase upstream fragment fused to the luciferase gene (ll), while the AP-1 luciferase plasmid contained a synthetic AP-1 site and a basal tk promoter fused tothe luciferase gene. Inthe injection experiments, the same Ras expression plasmids or the pSVL control plasmid wereused along with, in all the injection experiments, a constitutively active RSV-luciferase plasmid which uses the RSV long term repeat promoter. RESULTSANDDISCUSSION

Ras Does Not Activate Cardiac Muscle Cell ProliferationSince Ras is known to cause proliferation in many cell types (6, 7, 12, 13) and previous studies have established that the expression of another oncogene protein (SV40 large T antigen) can exert a mitogenic effect on cardiac myocytes (14, 15), we initially investigated whether cardiac muscle cells would undergo a proliferative response following the microinjection of purified, recombinant oncogenic Ras protein. Primary cultures of neonatal rat ventricular myocardial cells were prepared and the cells were injected with an oncogenic mutant Ras protein (Val-12), along with an inert marker

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rabbit antibody which allows us to unambiguously identify the injected cells. In order to determine if any DNA synthesis had been induced by the injected Ras protein, we included the thymidine analog BrdU in the culture media so that any proliferating cells would incorporate the analog which is detected by indirect immunofluorescence using a mouse monoclonal antibody against BrdU. As a positive control to ensure that the Ras protein was active, quiescent fibroblasts, which are known to undergo DNA synthesis in response to Ras injection (12, 13), were injected with the same preparation. Fig. 1 shows the result of this experiment. Cells were fixed24 hafter injection, and the injected cells were detected by staining with a Texas Red-conjugated anti-rabbit antibody which detects the co-injected marker protein, while those cells which incorporated BrdU during the incubation were identified by staining with the anti-BrdU followed by a cascade blue anti-mouse antibody. Panel A shows a field of injected fibroblasts stained for the marker IgG. Panel B is the same field stained for BrdU, indicating that theinjected cells have undergone DNA synthesis and showing that the Ras protein is active. Panels C and D show the Texas Red and cascade blue fluorescence of a field of injected cardiac cells, indicating that these cells have not undergone DNA synthesis, and, therefore, that the active Ras protein does not induce proliferation in ventricular myocytes. In a set of separate experiments, we were unable to detect DNA synthesis in Rasinjected cardiac cells, even after long incubations of up to 72 h. In these longer incubations, we did detect some speckled BrdU fluorescence, which may have been indicative of repair synthesis in both injected and uninjected cells. This further suggested that the lack of labeling with BrdU is not due simply to aninability of these cells to incorporate this analog into DNA. Ras Activates Morphological and Genetic Markersof Hypertrophy-Since the Ras oncogene protein did not induce proliferation in ventricular myocytes, we decided to investigate whether it was stimulating the hypertrophic response in these cells. It is known from previous work that a number of cell types including neuronal (16, 17), endocrine (18), andlymphoid (19) cells are differentiated inresponse to Ras expression or injection, suggesting the possibility of an analogous role for Ras in cardiac cells. In the cultured neonatal ventricular cell system which was used, it has been shown that the hypertrophic response involves activation of a number of immediate early genes including the proto-oncogene c-fos (20) as well as activation of the ANF gene. The ANF gene is expressed in both the atrium and theventricle of the embryonic heart, but is down-regulated in neonatal and adult ventricular cells. Reactivation of ventricular ANF expression is dependent on hypertrophic stimuli and hasbecome a hallmark of the onset of an embryonic gene program in hypertrophied ventricular myocardium (21). A series of separate injection experiments was performed to test whether the Val-12 Ras protein was capable of activating expression of these genes. Cultured ventricular myocytes were starved for 24 h in serumfree media andthen injected with either the Val-12 Ras protein plus an inert marker antibody or with the marker antibody alone as a control. After injection, the cells were maintained in serum-free media to prevent any serum-induced hypertrophy and fixed 1h (for analysis of c-Fos expression) or 30 h (for analysis of ANF expression) after injection. Injected cells were identified by staining with a fluorescein isothiocyanate-conjugated second antibody against the coinjected marker, while c-Fos was detected with a rabbit polyclonal anti-Fos antibody and ANF expression was assayed with a mouse monoclonal anti-ANF antibody. The expressed

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HRas-induced Markers of Cardiac Cell Hypertrophy

to Ras. Panels E, F, G,and H show the resultsobtained when ANF expression is monitored. Once again, while the control injections (panel E ) have not induced expression (panel F ) , injection of Rasprotein (panel G) has resultedin ANF expression, which is detected as perinuclear staining in the injected cells (panel H).We also noted that Ras injection may activate other phenotypic changes associated with hypertrophy. By comparing Fig. 2, E and G, it appears that the Ras-injected cells display a largercell profile and that thecoinjected antibody which associates with dense protein in the cytoplasm reveals more highly organized myofibrils compared with the controlinjections. This is similar toprevious studies examining the effect of bona fide hypertrophic stimuli on myocyte size and assembly of contractile proteins into organized units (3, 20). To extend these observations, injection experiments were carriedoutto analyze bothANF expression and MLC-2 organization in the same cells. Cells were injected as before, incubatedinserum-free mediafor 30 h, andstained for injected sheep antibody, as well as ANF and MLC-2. Fig. 3 shows a typical example. Panels A, B, and C show cells from a field injected with Ras; panels D,E, and F display control injected cells. Panels G and H show uninjected cells which were treated with the bona fide hypertrophic agent phenylephrine, while panels I and J show uninjected, untreated cells. FIG. 1. Val-12 Has induces DNA synthesis in fibroblasts but By comparing the pattern of ANF expression and MLC-2 notincardiacmyocytes. Quiescent C3H 10T1/2 fibroblasts arrangement, as well as the size of the cells, it is clear that (panels A and H ) or cardiac myocytes (panels C and D )were injected the Ras-injected cell has acquired morphological and genetic with Val-12 Ras plus a rabbit IgG and incubated in media containing markersconsistent with thephenylephrine-treated cells RrdU. Twenty-fourh later, thecells were fixed and analyzed by which have entered the hypertrophicpathway. It is also clear indirect immunofluorescence to detect the injected cells ( A and C) and incorporated BrdU ( B and D),indicating that the Ras protein that the controlinjected cells maintain the phenotypeof the induces DNAsynthesis in the fibroblasts but not in the cardiac uninjected, untreated cells, indicating thatinjection alonehas myocytes. The magnification is 400X, the bar represents 25 pm. not induced these phenotypic changes. In order to quantitatively characterizethese changes,injected cells from four separate experimentswere analyzed and scored for both ANF expression and cell area. The data from these experiments are summarized in Table 11, which indicates that injection of activated Ras protein causes both activation of ANF gene expression and an increase in the apparent area of the cells, in a manner similar to the a-adrenergicagonist, phenylephrine. The increase in area of about 50% which we observe afterRas injection compares favorably with otheragents which inducehypertrophy in these cells, for example, agrowth factor secreted by nonmyocytes increases the area by about 30% (22). We also assayed the effect of microinjecting Ras expressionplasmids onANF expression. Since efficient expression of injectedplasmidsrequiresnuclearinjection which is less efficientthan cytoplasmic injection,we therefore used as a control a co-injected constitutively active reporter plasmid (RSV-luciferase) which was used to score for successful nuclear injection. After staining for both luciferase and ANF, the effect of injection of either wild type Ras or Val-12 Ras expression plasmidswas measured. Table I1 shows that while the wild type expression plasmid had little effect on ANF expression, the Val-12 mutant induced ANF expression similarly to thatwhich we observed with the protein. In order to extend theseobservations with an independent FIG. 2. Val-12 Has induces c-Fos and ANF expression in cardiac myocytes.Cardiac cells were starved for 24 h in serum-free experimental approach, we carried out a series of transient media before injection with either a marker antibody alone (panels transfection experiments to test whether an ANF-luciferase A, R, E, and F ) or with Val-12 Ras plus the marker antibody (panels fusion gene, which is activated by hypertrophic agonists (3, C, D,G, and H).Slides were fixed after 1 h (A, B, C, and D )or 30 h ll),would be trans-activated by expression of Val-12 Ras. ( E , F, G, and H )and stained with an antibody against Fos or ANF. The ANF-luciferase reporter plasmid was co-transfected with Each pair of images shows the same field stained for injected cells aplasmid which expresses the Val-12 Rasmutant. As a ( A , C, E, and G) or expressed fos ( B and D ) or ANF ( F and H ) demonstrating that the Ras- injected cells have expressed both these control for transfection efficiency, a CMV-p-galactosidase proteins. Magnifications are200X (panels A through D ) or 400X plasmid was included, and the luciferase levels were normal(panels E through H),the bars represent 25 gm. ized to the p-galactosidase activity within each dish of cells.

proteins were detected by staining with biotin-labeled antirabbit or anti-mousefollowed by Texas Red streptavidin. Fig. 2 shows a representative example of a typical experiment. Panel A shows cells injected with the control antibody,while panel B reveals Fos staining for this field of cells, indicating that injection alone does not induce c-Fos expression in these cells. Panels C and D show a similar field injected with Val12 Ras, which has induced c-Fos expression. Table I shows the quantitation of two separate experiments indicating that there is a significant increase in c-Fos expression in response

HRas-induced Markers

of Cardiac Cell Hypertrophy

To confirm the validity of this approach in these cells, w e also performed experiments with an AP-1-dependent luciferase construct which, as expected, was transactivated by the mutant Ras. Fig. 4 shows the result of three separate transfection experiments, each performed in triplicate. From this result, it is clear that co-transfection with the oncogenic Ras expression vector results in activation of transcription from the ANF promoter by at least 4-fold, providing independent evidence that theactivated Ras induces this response. HRas Is Required forMaximal Activation of A N F Expression during cu-Adrenergic Receptor-mediated Hypertrophy-While these results demonstrate that an active Ras molecule can stimulate transcription from the ANF promoter, it does not necessarily follow from this that Ras is involved in the normal

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TABLE I1 Ras induces ANF expression and an increase in myocyte area while dominant inhibitory Ras prevents phenylephrine-induced ANFexpression Cells were injectedwith either Val-12 Ras protein plusmarker antibody or with the marker antibody alone. As further controls, uninjected cellswere either maintainedin serum-free media or treated with 100 pM phenylephrine (PE). After incubation for 30 h, the cells were fixed andstained forinjection andANF expression.After photographing the cells, the area of the cells was measured. Nuclear injection of the Ala-I5 Ras expression plasmid plus RSV-luciferase or, as a control, the parental SV40 expression plasmid plus RSVluciferase was followed by P E treatment for 30 h and stained for luciferase and ANF expression. Data from three or four separate exDeriments are included for each samDle. ANF

Injection expressing Cells

TABLE I

%

Ras induces fos expression in cardiac myocytes Starved cells were injected with either Val-12 Ras protein plus the marker antibody or with the marker alone. As a further control, uninjected cells were maintainedintheserum-free media.After incubation for 1 h, the cells were fixed and stained for both the injected antibody and Fos. exmessing Cells Iniection

Fos %

Uninjected Val-I2 protein Control urotein " p < 0.01 by Student's t test.

Mean area (arbitrary units)

32 f 3 ( n = 95)" 30 f 3 ( n = 42)" 38 ? 4 ( n = 35)"

14 ? 7 ( n= 423)' Uninjected 85 f 2 ( n= 110)" Val-12 Ras protein 21 ? 9 ( n = 75)" Control protein 84 f 5 ( n = 572)" Uninjected PE Wild type Ras plasmid 26 +. 11 ( n = 38)" 62 f 8 ( n= 84)" Val-12 Ras plasmid Ala-15 Ras plasmid P E 18 +. 2 ( n = 92)" 77 f 7 ( n = 48)'' Control Dlasmid PE " p < 0.01 by Student's t test. ND, not determined.

,

100 +. 10 ( n= 50)" 167 f 13 ( n = 40)"

111 +. 6 ( n = 25)" 155 f 12 ( n = 75)" NDb ND ND ND

8 luciferaseactivity

I

ANF-luc exot 1

alone

T

Val12 Ras

FIG.4. Val-12 Ras transactivates the ANF promoter. Transient transfectionswere carried out inprimary cardiac myocytes with either anANF-luciferase or an AP-1-luciferase plasmid alone or after co-tranfection with a Val-12 Ras expression plasmid. All the luciferase activities shown are normalized relative to a co-transfected CMV@-galactosidase plasmid.Each bar represents the mean plusor minus the standard deviation from three plates of cells, while experiments 1, 2, and 3 represent three experiments performed a t different times using different preparations of cells and plasmids. From these experiments, it is clear the Val-12 Ras expression stimulates transcription from the ANF promoter.

FIG. 3. Val-12 Ras induces both ANF expression and morphological changes in cardiac myocytes in a manner similar to phenylephrine. Cardiac cells were starved and injected as before with either Val-12 Ras plus a marker sheep IgG (panels A , E , and C) or the sheep IgG alone (panels D,E, and F) or uninjected cells were treated with 100 PM phenylephrine (panels G and H ) or keptin serum-free media as a control (panek; I and J).After fixation, slides were stained for the injected antibody ( A and D),MLC-2 ( E , E, G, and I ) and ANF (C, F, H , and J) by simultaneously detecting the various antibodies with fluorescein isothiocyanate anti-sheep, Texas Red anti-rabbit, and cascade blue anti-mouse. From this experiment, it is clear that the Ras-injected cells appear like the phenylephrinetreated cells both in terms of morphology, MLC-2 arrangement, and ANF gene expression. Magnificationsare 400X, the bar represents 25

m.

.u

ij/ 20

n

SVL

SVL PE Aim15 Rss PE

FIG.5. Ala-15 Ras inhibits phenylephrine-induced activation of the ANF promoter. Transient transfections were carried out in primary cardiac myocytes with the ANF-luciferase reporter plasmid plus the pSVL control plasmid or the SV40 Ala-15 expression plasmid and a CMV-@-galactosidase plasmid. After treatment with 100 W M phenylephrine ( P E )as required, cells were harvested and the luciferase activity was normalized to @-galactosidaselevels. Each bar represents the mean plus or minus the standard deviation from five plates of cells. The results indicate that the Ala-15 mutant inhibits phenylephrine-induced ANF promoter activity.

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HRas-induced Markers of Cardiac Cell Hypertrophy Ras antibody(24) or by overexpression of another dominantly interfering Ras mutant (25). However, an important implication of the current study is that a-adrenergic receptors, which are normally coupled to G,-dependent signalling pathways, can utilize a Ras-dependent pathway to activate the transcription of a muscle-specific gene in fully differentiated cardiac cells. The intriguing question can be raised as to the mechanisms by which a classic G-protein-coupled receptor andRasinteractduringtheactivation of thisimportant adaptive response. SUMMARY

FIG. 6. Ala- 15 Kas inhibits phenylephrine-induced activation of endogenous ANF expression. Suclear microinjection of IiSV-luciferase plus the parental pSVL plasmid ( p a n e k A and R ) or IZSV-luciferase plus the Ala-15 expression plasmid (panels C and D ) was followed by incubation, in the presence of 100 p~ phenylephrine, for 30 h. After fixation, the cells were stained for both luciferase (panekA and C) and ANF (panels R and D).From these experiments, we conclude that the Ala-15 Ras, but not thecontrol plasmid, inhibits endogenous ANF expression. Arrows indicate luciferase expressing (i.e. successfully injected with the expression plasmids) cells.

In conclusion, we have shown that active Ras protein is able to activate several features of the hypertrophic phenotype in primary neonatal ventricular myocytes and that the aadrenergic receptor-dependent signallingpathways require the wild type Ras protein to maximally activate theexpression of a geneticmarker of the hypertrophic response. Recent work from another group has shown that adifferent embryonic gene (skeletal a-actin) is also stimulated by Ras in thesecells, thussupporting our conclusions." Inatrial cells, theRas protein is involved in the regulation of potassium channel signaling pathways during cu-adrenergic-mediated hypertro- interaction with muscarinic receptors (26), and itis therefore phy of cardiac muscle cells. In order to test this point more possible that we have detected different functions for the Ras directly, additional transfection experimentswere performed protein in atrial and ventricularcells. It is interesting to note in which the effect of a different Ras mutant (Ala-15), which that theresponse to active Ras proteinin cardiac muscle cells acts as a negative interfering mutant (23) and inhibits the is quite different from that in skeletal muscle, in which Ras growth-stimulatory activity of the wild type molecule in fibro- stimulates proliferation and inhibits the differentiated pheblasts was analyzed.2 Fig. 5 shows the results obtained. As notype (6, 7). It is not clear whether this difference reflects expected ( l l ) , treatment of the cells with 100 PM phenyleph- inherent differences between the two muscle types orwhether rine caused a large transactivation of the ANF promoter.Co- it reflects different responsesto Rasin fully differentiated (as transfection with the Ala-15 mutant significantly inhibits this the cardiac cells are) versus determined but not completely effect. The inhibition which is observed is unlikely to be due differentiated muscle cells (as the skeletalmyoblasts are). t o nonspecific promoter competition since the luciferase ac- Nevertheless,our results emphasize that the response to tivity was again normalized to P-galactosidase activity from activation of a signalling pathway, in this case through Ras, the CMV-P-galactosidase. Specific promoter competition be- is dependent upon the cellular context. In the future, it will tween the ANF promoter and the SV40 promoter is also be of interest to investigate the molecular mechanisms which excluded since the parental SV40 plasmid (SVL) was used in mediate thedifferent responses of skeletal and cardiac muscle the control transfections. These data therefore suggest that cells to Ras. In addition, the cultured myocardial cell model the transcriptional activation of the ANF gene by a-adrener- providesa novel system todetermine how Ras signalling gic agonists is mediated, at least in part, through the endog- pathways interactwith those which are activated by classical enous wild type Ras protein. These results also show that the G protein-coupled adrenergic receptors. inhibition is not complete, which may indicate that phenylephrine can activate ANF expression through different sigAcknowledgments-We are grateful t o our colleagues in both the nalling pathways, not all of which involve Ras. To further Chien and Feramisco laboratories for providing plasmids and for extend these observations to the endogenous ANF gene, mi- discussion during the course of this work. Inparticular,we are grateful croinjection experiments were performedwith the Ala-15 to Art Alberts for help with the statistical analysis. expression plasmid. As before, the plasmid was co-injected REFERENCES with an RSV-luciferase plasmid which was used to identify K: R., Knowlton, K. U., Zhu, H., and Chien, S. (1991) FASEB J. 5 , 1. Chien, cells which had been successfully injected in the nucleus. Fig. 3037-3046 6 shows the result of this analysis indicating that cells which 2.Lee,H. R., Henderson, S., Reynolds,R.,Dunnmon,R.,Yuan,D.,and Chien, K. R. (1988) J. Biol. Chem. 263,7352-7358 had been injected with the RSV-luciferase plus the Ala-15 3. Shuheita, H. E., McDonough, P. M., Harris, A., Knowlton, K. U., Glembotski, C., Brown, J. H., and Chien, K.R. (1990) J. Elof. Chem. 2 6 5 , expression plasmiddid not show phenylephrine-induced ANF 20555-20562 expression (panels C and D).Conversely, the control injec4. Olson, E. N. (1990) Genes & Deu. 4 , 1454-1461 5. Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M., Benezra, tions with RSV-luciferase plus the parental SV40 expression R.. Blackwell. T. K.. Turner. D.. RUDD.R., Hollenberg, S., Zhuana, Y., plasmid (panels A and B ) did not show any inhibition of and Lassar, A. (1991) Science 251,7'6'1-766 phenylephrine-inducedexpression of the endogenous ANF 6. 0I:y:E. N.. Spizz, G., and Tainsky, M.A. (1987) Mol. Cell. Biol. 7,2104Zlll gene. As expected, uninjected cells in both fields expressed 7. Payne, P. A., Olson, E. N., Hsiau, P., Roberts, R., Perryman, M. B., and Schneider, M. D. (1987) Proc. Natl. Acod. Sci. U.S. A. 84,8956-8960 ANF as a result of the phenylephrine treatment. The data 8. Lassar, A. B., Thayer, M. J., Overell, R. W . , and Weintraub, H. (1989)Cell from these experiments arealso summarized in Table 11. 58.659-667 - - , - - - - .. 9. Shuheita, H. E., Thorhurn, J., andChien, K. R. (1992) Circulation 8 5 , The response of cardiac muscle cells to oncogenic Ras is 2236-2246 reminiscent of the situation found in the pheochromocytoma 10. Gross , M., Sweet, R., Sathe, R. W., Yokoyama, S., Fasano, 0..Goldfarb, M., Wigler, M., and Rosenberg. M.(1984) Mol. Cell. Biol. 5 , 1015-1021 cell line PC12 which differentiates inresponse to Ras(16, 17) K.U.,Baracchini, E., Ross, R. S., Harris, A. N., Henderson, S. and whose normal differentiation in responseto nerve growth 11. Knowlton. A,, Evans, S. M.,Glembotski,C.C.,andChien,K. R. (1991) J. Biol. factor canbe inhibited by microinjection of an inhibiting antiChem. 266,7759-7768 S.-Y. Chen and S. Powers, unpublished observations.

' M. Schneider, personal communication.

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