THE JOURNAL 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, Departments of §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, Uniuersity of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854

We have investigated the role of the proto-oncogene HRas in cardiac cell growth and hypertrophy. By di- rect needle microinjection of activated Ras protein into primary neonatal rat ventricular cardiac myocytes, we find that, unlike many other cell types, Ras does not induce DNA synthesis in these cells. However, injection of activated Ras does induce expression of both the c- Fos and atrial natriuretic factor (ANF) genes. Expres- sion of both these genes is associated with the hyper- trophic response in ventricular myocytes suggesting that Ras is involved in the hypertrophic signalling pathway. Ras injection also causes morphological changes in the cells so that they increase in profile and show changes in the organization of the contractile apparatus. Further support for a role for Ras in the hypertrophic response was obtained from studies showing that activated Ras stimulates ANF promoter activity in transient transfection assays. We also show that a dominant interfering Ras mutant inhibits the hypertrophic stimulation of the ANF promoter by phenylephrine, indicating a role for Ras in the hyper- trophic effect of an a-adrenergic agonist.

In response to a wide variety of mechanical, hormonal, and pathological stimuli, the myocardium is able to adapt 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 pro- liferation, 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 myo- cardial 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 mus- cle cell phenotype and the concomitant up-regulation of con-

* This work was supported by grants from the National Institutes of Health and the American 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 this fact.

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.

tractile protein gene expression is mediated by MyoD and related helix-loop-helix factors (4, 5). The expression of Ras in skeletal muscle myoblasts leads to the rapid induction of cell proliferation and prevents differentiation into the mature muscle phenotype (6-8). Accordingly, we have designed a series of experiments to test the role of the HRas protein in the growth and hypertrophy of primary ventricular cardiac muscle cells. We find that active Ras protein does not induce proliferation in these cells, but rather activates several phe- notypic features of muscle cell hypertrophy. Furthermore, we demonstrate that a dominantly acting negative HRas mutant significantly inhibits the hypertrophic effect of the a-adre- nergic agonist phenylephrine, suggesting that a Ras-depend- ent pathway, at least in part, mediates hypertrophy following stimulation of a classical G protein-coupled receptor.

EXPERIMENTAL PROCEDURES

Cell Culture and Microinjection-Primary neonatal rat ventricular myocytes were isolated from tissue by collagenase digest.ion and plated in serum-containing media onto laminin-coated chamber slides. In- jections were carried out by standard methods using modifications 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, cells maintained in serum-containing media survive better than cells maintained and injected in serum-free media. After injection, the 2,3-butanedione monoxime was removed by wash- ing 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 from Escherichia coli as 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 was co-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-lucifer- ase)’ along with the plasmid being tested. After staining for luciferase, the cells which expressed this plasmid were scored for ANF expres- sion.

D N A Synthesis Assay-After injection, the thymidine analog BrdU (Amersham) was added to the media at a lOOOX dilution of the stock

The abbreviations used are: RSV, Rous sarcoma virus; ANF, atrial natriuretic factor; MLC-2, myosin light chain-2; BrdU, bro- modeoxyuridine; CMV, cytomegalovirus; PBS, phosphate-buffered saline.

2244

HRas-induced Markers of Cardiac Cell Hypertrophy 2245

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 a monoclonal antibody from Amersham which contains DNase (the DNase partially digests the chromatin in the cell allowing the antibody to gain access to the BrdU). 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 (generously provided 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 sheep antibody 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 anti- bodies 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 pg of 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 was used to make up the total DNA for the samples where Ras was not expressed. After incubation for 48 h in serum-free or phenylephrine- containing medium as required, cells were harvested, and 0-galacto- sidase and luciferase activities were measured using standard enzy- matic assays. Luciferase activities were normalized to their corre- sponding @-galactosidase activities to correct for variation in trans- fection efficiency.

Plasmids-Plasmids for the transfection experiments were as fol- lows. 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 Biotech- nology Inc.) was used. The ANF-luciferase plasmid contains a 3.3- kilobase 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 to the luciferase gene. In the injection experiments, the same Ras expression plasmids or the pSVL control plasmid were used along with, in all the injection experiments, a constitutively active RSV-luciferase plasmid which uses the RSV long term repeat promoter.

RESULTS AND DISCUSSION

Ras Does Not Activate Cardiac Muscle Cell Proliferation- Since 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 anti- gen) can exert a mitogenic effect on cardiac myocytes (14, 15), we initially investigated whether cardiac muscle cells would undergo a proliferative response following the microin- jection of purified, recombinant oncogenic Ras protein. Pri- mary 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

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 de- tected by indirect immunofluorescence using a mouse mono- clonal 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 fixed 24 h after 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 identi- fied 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 the injected 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 prolif- eration in ventricular myocytes. In a set of separate experi- ments, we were unable to detect DNA synthesis in Ras- injected 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 an inability of these cells to incorporate this analog into DNA.

Ras Activates Morphological and Genetic Markers of Hyper- trophy-Since the Ras oncogene protein did not induce pro- liferation 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), and lymph- oid (19) cells are differentiated in response 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 the ventricle of the embry- onic heart, but is down-regulated in neonatal and adult ven- tricular cells. Reactivation of ventricular ANF expression is dependent on hypertrophic stimuli and has become 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 serum- free media and then 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-in- duced hypertrophy and fixed 1 h (for analysis of c-Fos expres- sion) 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 co- injected marker, while c-Fos was detected with a rabbit poly- clonal anti-Fos antibody and ANF expression was assayed with a mouse monoclonal anti-ANF antibody. The expressed

2246 HRas-induced Markers of Cardiac Cell Hypertrophy

proteins were detected by staining with biotin-labeled anti- rabbit or anti-mouse followed 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 Val- 12 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

FIG. 1. Val-12 Has induces DNA synthesis in fibroblasts but not in cardiac myocytes. Quiescent C3H 10T1/2 fibroblasts (panels A and H ) or cardiac myocytes (panels C and D) were injected with Val-12 Ras plus a rabbit IgG and incubated in media containing RrdU. Twenty-four h later, the cells were fixed and analyzed by indirect immunofluorescence to detect the injected cells (A and C) and incorporated BrdU ( B and D), indicating that the Ras protein induces DNA synthesis in the fibroblasts but not in the cardiac myocytes. The magnification is 400X, the bar represents 25 pm.

FIG. 2. Val-12 Has induces c-Fos and ANF expression in cardiac myocytes. Cardiac cells were starved for 24 h in serum-free media before injection with either a marker antibody alone (panels A, R, E, and F ) or with Val-12 Ras plus the marker antibody (panels C, D, G, and H). Slides were fixed after 1 h (A, B, C, and D) or 30 h ( E , F, G, and H) and stained with an antibody against Fos or ANF. Each pair of images shows the same field stained for injected cells (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 proteins. Magnifications are 200X (panels A through D) or 400X (panels E through H), the bars represent 25 gm.

to Ras. Panels E, F, G, and H show the results obtained when ANF expression is monitored. Once again, while the control injections (panel E ) have not induced expression (panel F ) , injection of Ras protein (panel G) has resulted in 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 hy- pertrophy. By comparing Fig. 2, E and G, it appears that the Ras-injected cells display a larger cell profile and that the co- injected antibody which associates with dense protein in the cytoplasm reveals more highly organized myofibrils compared with the control injections. This is similar to previous studies examining the effect of bona fide hypertrophic stimuli on myocyte size and assembly of contractile proteins into orga- nized units (3, 20).

To extend these observations, injection experiments were carried out to analyze both ANF expression and MLC-2 organization in the same cells. Cells were injected as before, incubated in serum-free media for 30 h, and stained 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 phenyl- ephrine, while panels I and J show uninjected, untreated cells. By comparing the pattern of ANF expression and MLC-2 arrangement, as well as the size of the cells, it is clear that the Ras-injected cell has acquired morphological and genetic markers consistent with the phenylephrine-treated cells which have entered the hypertrophic pathway. It is also clear that the control injected cells maintain the phenotype of the uninjected, untreated cells, indicating that injection alone has not induced these phenotypic changes. In order to quantita- tively characterize these changes, injected cells from four separate experiments were 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-adrenergic agonist, phenyleph- rine. The increase in area of about 50% which we observe after Ras injection compares favorably with other agents which induce hypertrophy in these cells, for example, a growth factor secreted by nonmyocytes increases the area by about 30% (22). We also assayed the effect of microinjecting Ras expression plasmids on ANF expression. Since efficient expression of injected plasmids requires nuclear injection which is less efficient than cytoplasmic injection, we therefore used as a control a co-injected constitutively active reporter plasmid (RSV-luciferase) which was used to score for suc- cessful nuclear injection. After staining for both luciferase and ANF, the effect of injection of either wild type Ras or Val-12 Ras expression plasmids was measured. Table I1 shows that while the wild type expression plasmid had little effect on ANF expression, the Val-12 mutant induced ANF expres- sion similarly to that which we observed with the protein.

In order to extend these observations with an independent experimental approach, we carried out a series of transient transfection experiments to test whether an ANF-luciferase fusion gene, which is activated by hypertrophic agonists (3, ll), would be trans-activated by expression of Val-12 Ras. The ANF-luciferase reporter plasmid was co-transfected with a plasmid which expresses the Val-12 Ras mutant. As a control for transfection efficiency, a CMV-p-galactosidase plasmid was included, and the luciferase levels were normal- ized to the p-galactosidase activity within each dish of cells.

HRas-induced Markers of Cardiac Cell Hypertrophy 2247

To confirm the validity of this approach in these cells, w e also performed experiments with an AP-1-dependent lucifer- ase construct which, as expected, was t ransact ivated by the mutant Ras. Fig. 4 shows the result of three separate trans- fection 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 the activated Ras induces this response.

HRas Is Required for Maximal 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 , i t does not necessarily follow from this that Ras is involved in the normal

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 maintained in the serum-free media. After incubation for 1 h, the cells were fixed and stained for both the injected antibody and Fos.

Iniection Cells exmessing Fos %

Uninjected 32 f 3 ( n = 95)" Val-I2 protein 30 f 3 ( n = 42)" Control urotein 38 ? 4 ( n = 35)"

" p < 0.01 by Student's t test.

FIG. 3. Val-12 Ras induces both ANF expression and mor- phological changes in cardiac myocytes in a manner s imilar 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 kept in 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, i t is clear that the Ras-injected cells appear like the phenylephrine- treated cells both in terms of morphology, MLC-2 arrangement, and ANF gene expression. Magnifications are 400X, the bar represents 25 m .

TABLE I1 Ras induces ANF expression and an increase in myocyte area

while dominant inhibitory Ras prevents phenylephrine-induced ANFexpression

Cells were injected with either Val-12 Ras protein plus marker antibody or with the marker antibody alone. As further controls, uninjected cells were either maintained in serum-free media or treated with 100 pM phenylephrine (PE). After incubation for 30 h, the cells were fixed and stained for injection and ANF 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 RSV- luciferase 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.

Injection Cells expressing ANF

%

Uninjected 14 ? 7 ( n = 423)' Val-12 Ras protein 85 f 2 ( n = 110)" Control protein 21 ? 9 ( n = 75)" Uninjected PE 84 f 5 ( n = 572)" Wild type Ras plasmid 26 +. 11 ( n = 38)" Val-12 Ras plasmid 62 f 8 ( n = 84)" Ala-15 Ras plasmid P E 18 +. 2 ( n = 92)" Control Dlasmid PE 77 f 7 ( n = 48)''

(arbitrary units) Mean area

100 +. 10 ( n = 50)" 167 f 13 ( n = 40)" 111 +. 6 (n = 25)" 155 f 12 ( n = 75)"

NDb ND ND ND

" p < 0.01 by Student's t test. ND, not determined.

8 luciferase activity ,

I ANF-luc exot 1 T

alone Val12 Ras

FIG. 4. Val-12 Ras transactivates the ANF promoter. Tran- sient transfections were carried out in primary cardiac myocytes with either an ANF-luciferase or an AP-1-luciferase plasmid alone or after co-tranfection with a Val-12 Ras expression plasmid. All the lucifer- ase activities shown are normalized relative to a co-transfected CMV- @-galactosidase plasmid. Each bar represents the mean plus or 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 exper- iments, it is clear the Val-12 Ras expression stimulates transcription from the ANF promoter.

i j / 20 .u n

SVL SVL PE Aim15 Rss PE

FIG. 5. Ala-15 Ras inhibits phenylephrine-induced activa- tion 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 WM phenylephrine ( P E ) as required, cells were harvested and the luciferase activity was normalized to @-galactosidase levels. 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.

2248 HRas-induced Markers of Cardiac Cell Hypertrophy

Ras antibody (24) or by overexpression of another dominantly interfering Ras mutant (25). However, an important impli- cation of the current study is that a-adrenergic receptors, which are normally coupled to G,-dependent signalling path- ways, 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 and Ras interact during the activation of this important adaptive response.

FIG. 6. Ala- 15 Kas inhibits phenylephrine-induced activa- tion of endogenous ANF expression. Suclear microinjection of IiSV-luciferase plus the parental pSVL plasmid (panek 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 the control plasmid, inhibits endogenous ANF expression. Arrows indicate luciferase expressing (i .e. successfully injected with the expression plasmids) cells.

signaling pathways during cu-adrenergic-mediated hypertro- phy of cardiac muscle cells. In order to test this point more directly, additional transfection experiments were performed in which the effect of a different Ras mutant (Ala-15), which acts as a negative interfering mutant (23) and inhibits the growth-stimulatory activity of the wild type molecule in fibro- blasts was analyzed.2 Fig. 5 shows the results obtained. As expected ( l l ) , treatment of the cells with 100 PM phenyleph- rine caused a large transactivation of the ANF promoter. Co- transfection with the Ala-15 mutant significantly inhibits this effect. The inhibition which is observed is unlikely to be due to nonspecific promoter competition since the luciferase ac- tivity was again normalized to P-galactosidase activity from the CMV-P-galactosidase. Specific promoter competition be- tween the ANF promoter and the SV40 promoter is also excluded since the parental SV40 plasmid (SVL) was used in the control transfections. These data therefore suggest that the transcriptional activation of the ANF gene by a-adrener- gic agonists is mediated, at least in part, through the endog- enous wild type Ras protein. These results also show that the inhibition is not complete, which may indicate that phenyl- ephrine can activate ANF expression through different sig- nalling pathways, not all of which involve Ras. To further extend these observations to the endogenous ANF gene, mi- croinjection experiments were performed with the Ala-15 expression plasmid. As before, the plasmid was co-injected with an RSV-luciferase plasmid which was used to identify cells which had been successfully injected in the nucleus. Fig. 6 shows the result of this analysis indicating that cells which had been injected with the RSV-luciferase plus the Ala-15 expression plasmid did not show phenylephrine-induced ANF expression (panels C and D). Conversely, the control injec- tions with RSV-luciferase plus the parental SV40 expression plasmid (panels A and B ) did not show any inhibition of phenylephrine-induced expression of the endogenous ANF gene. As expected, uninjected cells in both fields expressed ANF as a result of the phenylephrine treatment. The data from these experiments are also summarized in Table 11.

The response of cardiac muscle cells to oncogenic Ras is reminiscent of the situation found in the pheochromocytoma cell line PC12 which differentiates in response to Ras (16, 17) and whose normal differentiation in response to nerve growth factor can be inhibited by microinjection of an inhibiting anti-

SUMMARY

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 a- adrenergic receptor-dependent signalling pathways require the wild type Ras protein to maximally activate the expression of a genetic marker of the hypertrophic response. Recent work from another group has shown that a different embryonic gene (skeletal a-actin) is also stimulated by Ras in these cells, thus supporting our conclusions." In atrial cells, the Ras protein is involved in the regulation of potassium channel interaction with muscarinic receptors (26), and it is therefore possible that we have detected different functions for the Ras protein in atrial and ventricular cells. It is interesting to note that the response to active Ras protein in cardiac muscle cells is quite different from that in skeletal muscle, in which Ras stimulates proliferation and inhibits the differentiated phe- notype (6, 7). It is not clear whether this difference reflects inherent differences between the two muscle types or whether it reflects different responses to Ras in fully differentiated (as the cardiac cells are) versus determined but not completely differentiated muscle cells (as the skeletal myoblasts are). Nevertheless, our results emphasize that the response to activation of a signalling pathway, in this case through Ras, is dependent upon the cellular context. In the future, it will be of interest to investigate the molecular mechanisms which mediate the different responses of skeletal and cardiac muscle cells to Ras. In addition, the cultured myocardial cell model provides a novel system to determine how Ras signalling pathways interact with those which are activated by classical G protein-coupled adrenergic receptors.

Acknowledgments-We are grateful to our colleagues in both the Chien and Feramisco laboratories for providing plasmids and for discussion during the course of this work. In particular, we are grateful to Art Alberts for help with the statistical analysis.

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