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a1-Adrenergic Receptor Stimulation of Sarcomeric Actin IsogeneTranscription in Hypertrophy of Cultured Rat Heart Muscle CellsCarlin S. Long,** Charles P. OrdahlV and Paul C. Simpson*Cardiovascular Research Institute and the Departments of *Medicine and tAnatomy, University of California San Francisco,San Francisco, California 94143; and *Cardiology Division (11 -C), Veterans Administration Medical Center, San Francisco,California 94121

Abstract

During pressure-load hypertrophy of the adult heart in vivo,there is up-regulation of the mRNAencoding skeletal a-actin,the sarcomeric actin iso-mRNA characteristic of mature skele-tal muscle and the fetal/neonatal heart. Wehave shown pre-viously that during al-adrenergic receptor-stimulated hyper-trophy of cultured rat heart myocytes, the induction of skeletala-actin mRNAis greater than that of the mRNAencodingcardiac a-actin, the sarcomeric actin iso-mRNA characteristicof the adult heart. To determine if this actin iso-mRNA switchduring cardiac hypertrophy reflects changes in the transcrip-tional status of the myocyte nucleus, we quantified the rate oftranscription of actin mRNAsand total RNA, using an in vitrorun-on transcription assay with nuclei isolated from the cul-tured myocytes after stimulation with norepinephrine (NE).Transcription of skeletal a-actin was increased at 3 h after NE,reached a maximum 6.1-fold increase at 12 h, and returned tothe control level at 24 h. The EC50 for NE was 200 nM, andpharmacologic studies indicated a,-receptor specificity. Tran-scription of cardiac a-actin was also increased rapidly by NE(maximum 4.6-fold vs. control at 3 h). However, cardiac a-actin transcription had returned to the control level at 6 h,when NE-stimulated skeletal a-actin transcription was stillincreasing. Transcription of the cytoskeletal (f) actin gene wasnot changed significantly by NE treatment. Total RNAtran-scription was not increased until 6 h after NE (1.5-fold vs.control) and remained elevated through 24 h. Inhibition ofprotein synthesis did not attenuate NE-stimulated actin genetranscription. Thus the a1-adrenoceptor mediates a rapid,transient, and selective increase in transcription of the sarco-meric actin isogenes during cardiac myocyte hypertrophy.Skeletal a-actin, the fetal/neonatal isogene, is induced prefer-entially to cardiac a-actin, the adult isogene. The differentkinetics of actin isogene and total RNAtranscription and theindependence of transcription from protein synthesis suggestthat transcriptional induction via the a, receptor is complexand may involve preexisting regulatory factors. These results

A preliminary report of this work has been published in abstract form(1988. Circulation. 78[Suppl. II]:11-562).

Address reprint requests to Dr. Simpson, VA Medical Center(1 1-C), 4150 Clement Street, San Francisco, CA94121.

Receivedfor publication 30 September 1988 and in revisedform 12December 1988.

are the first to demonstrate that the a1-adrenergic receptor is amolecular mediator of transcriptional changes underlying anisogene switch that is known to be associated with cardiacmyocyte hypertrophy.

IntroductionSarcomeric (a) actin has two known isoforms, designated car-diac a-actin and skeletal a-actin because of their abundance inadult heart and adult skeletal muscle, respectively. Skeletala-actin is expressed in the fetal and neonatal heart, but it ismarkedly down-regulated in the adult heart of birds andmammals, including humans (1, 2). It has been found recentlythat the skeletal a-actin iso-mRNA is reexpressed in the adultmyocardium in response to pressure overload (3). Reexpres-sion of isogenes characteristic of earlier developmental stages isa distinctive feature of certain forms of myocardial hyper-trophy in vivo (4). Thus myocardial hypertrophy is not simplya quantitative change in protein mass, but also involves quali-tative changes in myocyte mRNAand protein content. Inother systems, such qualitative changes in gene expression re-flect an altered differentiation state known to accompanymany regenerative and neoplastic processes.

Wehave shown previously that the skeletal a-actin mRNAis reinduced during a,-adrenoceptor-mediated hypertrophy ofrat cardiac myocytes in culture (5). The control myocytes inthis culture system, although derived from the neonatal heart,express predominantly cardiac a-actin, the adult sarcomericactin isoform. Thus the culture system reproduces the sarco-meric actin iso-mRNA switching seen in overload-inducedhypertrophy of the mature heart in vivo.

It is unknown whether iso-mRNA switching during cardiachypertrophy in vivo and in culture simply reflects alteredmRNAturnover rates, or whether there are more fundamentalchanges in the transcriptional status of the myocyte nucleus.Recently, it has been shown that a I-adrenergic receptor stimu-lation leads to an increase in overall transcription and the rateof transcription of the gene encoding myosin light chain-2, aprotein occurring as a single isoform (6).

The goal of the present series of experiments was to deter-mine if the a,-adrenergic receptor-induced actin iso-mRNAswitch reflects fundamental changes in the transcriptional pro-gram of the myocyte nucleus. We used an in vitro run-ontranscription assay with nuclei isolated from cultured cardiacmyocytes after stimulation with norepinephrine (NE).' The

The Journal of Clinical Investigation, Inc.Volume 83, March 1989, 1078-1082

1078 C. S. Long, C. P. Ordahl, and P. C Simpson

1. Abbreviation used in this paper: NE, norepinephrine.

results show that a1-adrenergic stimulation mediates a rapidand preferential increase in transcription of the skeletal a-actingene as compared with total RNA. Moreover, the alteration intranscription of the skeletal a-actin gene is quantitatively andtemporally distinct from that of the cardiac a-actin gene.These results are the first to demonstrate that the aI-adrenergicreceptor is a molecular mediator of transcriptional changesunderlying an isogene switch that is known to be associatedwith cardiac myocyte hypertrophy.

MethodsEnzymes were obtained from Boerhinger Mannheim Diagnostics(Houston, TX) and New England Biolabs (Beverly, MA) and usedaccording to the manufacturers' instructions. RNAsin was from Pro-mega Biotec, (Madison, WI). [a-32P]UTP and [a-32P]GTP (400 Ci/mmol) were obtained from Amersham Corp. (Arlington Heights, IL).a-Amanitin and cycloheximide were from Sigma Chemical Co. (St.Louis, MO). DNAse I (RNAse-free) was obtained from Cooper Bio-medical Inc. (Malvern, PA). Sources of culture materials and adrener-gic agents were as noted previously (7).

Cell culture system. Primary cultures of neonatal rat heart musclecells were prepared as described previously (8). In brief, cells wereobtained from the hearts of l-d-old Sprague-Dawley rats by trypsini-zation and plated in MEM(Hanks' salts) with 5%calf serum. After 12h in culture, cells were transferred to a serum-free medium supple-mented with transferrin and insulin (each 10 ,g/ml). Cells were main-tained in 100-mm culture dishes at a density of 100-150 cells per mm2.Contaminating nonmuscle cells were kept at < 10% by preplating andby the addition of bromodeoxyuridine (0.1 mM) through day 2 ofculture. Under these control conditions, there is no change in cell sizeor number and no beating of the myocytes (7-9).

On day 4 of culture, freshly made adrenergic agents (agonists andantagonists) or their vehicle, ascorbic acid (100 1AM final), were addedto a final concentration of 2 uM (except as noted for dose-responsemeasurements). Cycloheximide, 2.8 ug/ml (10 MM), was present insome experiments, a concentration which inhibits protein synthesis by> 95% (7). Cells were then harvested for nuclear isolation after 1-24 hof treatment.

Nuclear run-on assay. Nuclei were isolated by a modification (10)of the procedure of Mulvihill and Palmiter (1 1). Cells were washed incold phosphate-buffered saline and scraped into homogenizationbuffer (300 mMultrapure sucrose, 10 mMTris [pH 8.0], 2.5 mMMgacetate, 0.5 mMdithiothreitol, and 0.25% Triton X-100). After celldisruption in a glass ball homogenizer, nuclei were pelleted at 2,000rpm (750 g) at 4VC, and resuspended in a buffer containing 50 mMTris (pH 8.0), 5 mMMgCl2, 0.1 mMEDTA, and 40% sterile glycerol.Resuspended nuclei were used immediately or after storage at -70°C.Storage at -70°C had no effect on transcriptional activity. Recovery ofnuclei averaged 75% of cell numbers and was the same for NE-treatedand control cultures. Nuclei were also isolated from freshly excisedadult and neonatal rat hearts by a similar protocol (10).

Run-on transcription assays were performed as described pre-viously (10). Five million nuclei from control or treated cells wereincubated at 30°C in a transcription reaction containing (final in 300Al): 28% glycerol (vol/vol), 150 mMKCI, 40 mMTris (pH 8.0), 6 mMMgCl2, 2 mMdithiothreitol, 1.0 mMeach ATP, CTP, GTP, 0.1 mMEDTA, 25 URNAsin/ml, and 300 MCi [a-32P]UTP. Reaction time was15 min, and incorporation of [32P]UTP into purified RNAwas linearover 30 min (data not shown). a-Amanitin (1 ,g/ml) was included insome reactions to test specificity for RNApolymerase II and to deter-mine the contribution of polymerases I and III (insensitive to a-aman-itin at this dose) to total transcription in control and treated cultures(12). Reactions were terminated by the addition of 125 ,g/ml ofRNAse-free DNAse I for an additional 10 min. Proteinase K (200mg/ml) was then added for I h at 37°C. The 32P-labeled run-off RNAproduced was purified by sequential phenol/chloroform extractions,precipitation with ice-cold 20% TCA, and two final ethanol precipita-

tions. In order to increase the 32P-RNA specific activity, several exper-iments were carried out using 300 MCi of [a-32P]GTP in addition to the[a-32P]UTP.

Purified 32P-labeled RNArun-on product was counted as a index oftotal transcriptional activity. Then 10-15 X 106 cpm of RNAproducedby equal numbers of nuclei (5 X 106) from control and treated cells washybridized to a panel of actin DNAprobes. In some experiments, equalcounts of 32P-labeled RNA from the different groups were used forhybridization; results were comparable to those in which equal num-bers of nuclei were used. The hybridization panel consisted of (a) pAC15.2, a 3.7-kb skeletal a-actin genomic fragment; (b) pc1 8, a 440-bpcardiac a-actin cDNA fragment containing 163 bp of the 3' untrans-lated region; (c) pAC 18. 1, a 4.4-kb ,-actin genomic fragment; and (d)as control, plasmid vector pBR322 DNA. 5 Mg of each plasmid probewas linearized and dotted onto nitrocellulose as described by Kafatoset al. (13). Hybridization of increasing amounts of 32P-RNA run-onproduct resulted in a corresponding increase in specific counts bound,verifying that the DNAprobes were in excess (data not shown). Filterswere prehybridized for 14-24 h at 520C in 30% deionized, freshlyrecrystallized formamide, 500 mMNaCl, 300 jg/ml salmon spermDNA, 50 mMHepes (pH 7.0), 0.4% SDS, and 2 mMEDTA. Afterhybridization for 72 h, blots were washed under stringent conditionsby the method of Spindler et al. (14). The washing conditions pre-vented cross-hybridization between the actin probes, as described pre-viously (15). Specificity of the probes was confirmed in two ways: (a)run-on experiments with nuclei isolated from neonatal and adult ratheart and skeletal muscle; and (b) hybridizations with control [32p]-and [3H]cRNAs transcribed in vitro from coding sequences for eachactin isoform inserted into pGEM4vectors (data not shown). Toquantify hybridization, labeled dots were excised and counted in aliquid scintillation counter to < 5%error. Gene-specific hybridization,in parts per million (ppm), was calculated by dividing the back-ground-corrected counts per minute hybridized to each probe by thetotal counts per minute included in the hybridization reaction andcorrecting for efficiency of hybridization. Background was determinedby hybridization to vector pBR322 DNA. Hybridization efficiency(average 30%under these conditions) was determined by hybridizationof known amounts of labeled [3H]cRNA synthesized from a cDNAinsert for cardiac a-actin cloned into a pGEM4plasmid.

DNAsynthesis. Cells were incubated with [3H]thymidine (5 ,Ci/ml, 20 Ci/mmol) from the time of plating through culture day 6.Cultures were taken for autoradiography, and the proportion of myo-cytes with labeled nuclei was determined (8). In each of two experi-ments, 1,000 cells in randomly selected microscopic fields of two tothree dishes were counted.

Statistics. Gene-specific transcription is presented as mean±stan-dard error (SE) ppmhybridized per 5 X 106 nuclei per 1 5-min reaction.NE-treated values were compared to concurrent controls using theStudent's t test. NE-treated/control ratios of mean 32p incorporationinto total purified run-off RNAwere analyzed for their deviation fromunity by calculation of confidence limits (16).

ResultsNeonatal rat heart muscle cell cultures were prepared andmaintained in serum-free culture for 3 d. On the fourth dayafter plating, cells were treated with either NEor vehicle (con-trol) for 1-24 h. Myocyte nuclei were isolated and incubatedwith labeled RNA precursors to allow previously initiatedRNAtranscripts to elongate (run-on). The labeled RNApro-duced was purified, counted, and hybridized to gene-specificDNAfragments immobilized on nitrocellulose. The amountof gene-specific hybridization is an index of the polymerase IIdensity on that gene at the time of nuclear isolation. Therefore,differences in hybridization to a particular gene between con-

trol and treated cells are indicative of changes in the rate ofinitiation of transcription of that gene (17).

a,-Stimulated Actin Gene Transcription 1079

In nuclei from control cultured myocytes, skeletal a-actingene transcription was 2.5±0.5 ppm, whereas cardiac a-actintranscription was 6.0±1.6 ppm (n = 26, P < 0.05). The re-duced level of transcription of the skeletal a-actin gene is con-sistent with the reduced level of skeletal a-actin mRNAincultured neonatal cardiocytes (5). Transcription of skeletal a-actin and cardiac a-actin by control myocytes did not varysignificantly over 24 h treatment with vehicle (Fig. 1 A).

Treatment of the cultured cells with 2 AMNEstimulated atransient increase in transcription of both the skeletal a-actinand cardiac a-actin genes (Figs. 1 A and 2). After 3 h, tran-scription of the skeletal a-actin gene was 3.2-fold higher inNE-treated cells than in control cells (P < 0.05). Skeletal a-actin gene transcription reached a maximum 6. 1-fold increasevs. control at 12 h (P' < 0.01) and returned to the control levelat 24 h, despite the continued presence of NE(Fig. 1 A). Tran-scription of the cardiac a-actin gene was stimulated more rap-idly than that of skeletal a-actin, with a' 3.7-fold increase at 1 h

A. mRNATranscription o ControlIO + Cycloheximide

skeletal a-actln

I8:

* NE* + Cycloheximide

cardiac a-actln

25 -

1 3 6 12 24

Time after NE (h)

mRNA TranscriptionNN

co eI-Gcm =. =(A (a)

0

3

w

z

12-

24-

48

& ."Ibk .. O.~~~~ ~~~~~~~~.. .,

.~~~~~~~~~~~~:1~~~~~ _M~~~~~~~~X,w *L1 2-

plus a-amanitinFigure 2. NE increases specific hybridization to actin probes. Cellswere treated with 2 ,uM NE for the times indicated, and nuclei wereharvested for the run-on transcription assay. The figure is an autora-diograph of run-on product hybridized to the probes indicated(WACT, (-actin; sACT, skeletal a-actin; cACT, cardiac a-actin). Notethe increase in cardiac a-actin hybridization by run-on product fromnuclei harvested at 3 h and the increase in skeletal a-actin hybridiza-tion after both 3 and 12 h of NEtreatment. There is no detectablechange in ,B-actin hybridization. Inclusion of a-amanitin (1 Mg/mI) inthe reaction done with nuclei harvested at 12 h abolishes all specifichybridization.

B. Total Transcription

2 -

p vs control:## -C 0.01# -c0.05

-aO

0

Q 11

1 3 6 12 24

Time after NE (h)

Figure 1. Time course of NE-stimulated actin mRNAand total RNAtranscription. Cultured myocytes were treated with 2 MMNEor ve-hicle (control). At the times indicated, nuclei were isolated, andequal numbers of nuclei in the two groups were assayed for (A) gene-specific transcription and (B) total transcription, as described inMethods. (A) Mean±SEppm run-on product hybridized to probesfor (left) skeletal a-actin and (right) cardiac a-actin. For the timepoints at 1, 3, 6, 12, and 24 h, the number of separate experimentswas 3, 4, 5, 10, and 4, respectively, with the exception of the 12-hcardiac a-actin value where n = 8. Cycloheximide was included at2.8 ,ug/ml in several 3-h experiments with NE (n = 7) and vehicle (n= 4). (B) NE-treated/control ratio for total 32p incorporation into pu-rified run-on RNAat each time point. Each value is the mean±SEfor the number of experiments indicated in A.

after NE(P < 0.01) and a maximum 4.6-fold increase at 3 h (P< 0.01). However, NE-stimulated cardiac a-actin gene tran-scription was much more transient than that of the skeletala-actin gene, returning to the control level before skeletal a-actin gene transcription reached its maximum increase (Fig. 1A). fl-actin gene transcription was unchanged by NE, exceptfor a small and statistically insignificant increase at 3 h afterNE (1.6-fold vs. control, P> 0.05). Inclusion of a-amanitin inthe transcription reaction abolished specific hybridization toall actin probes, indicating specificity for RNApolymerase II(Fig. 2).

The total 32p incorporation into purified run-on RNAbycontrol nuclei during the 15-min in vitro reaction was10.7±1.1 X 106 cpm per 5 X 106 nuclei (n = 26) and did notvary significantly over the times studied. Total incorporationwas not changed by 2 AMNEat 1 or 3 h (Fig. 1 B), althoughtranscription of the skeletal a-actin and cardiac a-actin geneswas increased at these times (Fig. 1 A). At 6 h of NEtreatment,NE-stimulated total transcription was increased 1.5±0.2-foldvs. control (n = 5, P< 0.05) and remained at this elevated levelfor 24 h (Fig. 1 B). Inclusion of a-amanitin in the in vitroreaction inhibited total transcription by 45-50%. Both a-

1080 C. S. Long, C. P. Ordahl, and P. C. Simpson

In. .t.

11 P.- l:

I 0

amanitin-sensitive and a-amanitin-resistant portions of totaltranscription were increased significantly by NEat 6 and 12 h(data not shown). At 24 h, the a-amanitin-resistant portion oftotal transcription (i.e., polymerases I and III activity) wasincreased in NE-treated cells (1.7±0.1 -fold vs. control, n = 4, P< 0.01). However, at the same time (24 h), a-amanitin-sensi-tive transcription (i.e., polymerase II activity) was not in-creased significantly by NE (1.3±0.3-fold vs. control, n = 4, P= NS).

NEstimulation of skeletal a-actin transcription at 12 h wasdose-dependent, with an EC50 of 200 nM (Fig. 3). The a,-adrenergic antagonist terazosin (2 MM)reduced NE-stimulatedskeletal a-actin transcription at 12 h to control levels (2.1 ± 1.0ppm for NEplus terazosin, vs. 2.2±3.7 ppm for control, n = 3paired experiments, P = NS). Further, the f3-adrenergic recep-tor agonist isoproterenol (2 yM) did not alter incorporationinto skeletal a-actin transcripts at 12 h (1.6±1.7 ppm for iso-proterenol, vs. 2.2+3.7 ppm for control, n = 3, P = NS).Similar a1-adrenergic receptor specificity was seen for cardiaca-actin in cells treated with NE for 3 h (data not shown).

Cycloheximide (2.8 jg/ml, 10 MM) included in the culturemedium inhibited protein synthesis by > 95% (7 and data notshown) but did not inhibit the NE-stimulated increase in skele-tal a-actin or cardiac a-actin transcription at 3 h (Fig. 1).

NEdid not stimulate DNAsynthesis, as measured by auto-radiography after incubation for 6 d in the presence of[3H]thymidine. There were 5.8±0.4% control myocytes withlabeled nuclei, vs. 4.0±1.0% of myocytes treated with 2 jiMNE(mean ± range, n = 2). Cell numbers were the same in thetwo groups (7, 9 and data not shown). Thus changes in tran-scription were not produced by altered template availability.There were < 10% nonmuscle cells in all cultures, and thesecells do not respond to NE (5, 7, 9).

Discussion

The critical new finding of this work is that stimulation of thea1-adrenergic receptor is coupled to preferential transcrip-tional induction of the skeletal a-actin gene, the fetal/neonetalsarcomeric actin isogene characteristic of pressure-load myo-cardial hypertrophy in vivo. The increases in transcription ofboth sarcomeric actin isogenes exceeded the increases in tran-scription of cytoskeletal (fl) actin and total RNA(Fig. 4), indi-cating that transcription of genes encoding contractile proteinsis increased selectively in cardiac myocyte hypertrophy. How-ever, the a,-mediated increase in transcription of the skeletala-actin gene was greater in magnitude and duration than theincrease in transcription of the cardiac a-actin gene (Fig. 4).The greater increase in skeletal a-actin mRNAtranscription is

0 0.02 0.2NE(IM)

Figure 3. Response of skeletal a-

actin transcription to NE concen-

tration. Cells were treated for 12 hwith the indicated concentrationsof NE, and nuclei were isolatedfor determination of skeletal a-

actin (sACT) transcription. Eachpoint is the ratio of NE-treated/control hybridization. Control

2 20 transcription was 2.3 ppm in thisexperiment.

11-

6 -

-6 cACT Gener2" 5 __Transcription

3 I

> I /

2.4-1 I

03

sACT {mRNA]

T Geneiscription

cACT ImRNA]

Cell Size [Total Protein]& Total Transcription

Tii12

ime after NE (h)24

Figure 4. Transcriptional regulation in a,-stimulated hypertrophy.The diagram summarizes the time course of activation of transcrip-tion and accumulation of specific mRNAsand total protein afterstimulation of cultured neonatal rat heart muscle cells with NE. Re-sults for cell protein and steady-state levels of actin iso-mRNAs aretaken from references 5, 7, and 8. (sACT, skeletal a-actin; cACT,cardiac a-actin).

consistent with the disporportionate increase in skeletal a-actin mRNAcontent observed previously in this model ofhypertrophy (Fig. 4; reference 5). Concomitant modificationsof mRNAstability cannot be excluded but do not appear to berequired to explain the observations. Therefore, this work pro-vides the first evidence that an isogene switch characteristic ofcardiac myocyte hypertrophy is due to an alteration in thetranscriptional program of the cardiac myocyte, and that thisprogram can be altered by stimulation of a specific cell surfacereceptor.

A second major finding in the current experiments was theresolution of distinct temporal stages in the transcriptionalresponse after a,-receptor stimulation (Fig. 4). Increased tran-scription of the skeletal a-actin and cardiac a-actin genes wasseparated in time, with delayed and prolonged activation ofskeletal a-actin mRNAtranscription. Asynchronous tran-scription of closely related genes has also been described in acyclic AMP-dependent system (18). Thus a single receptor canmediate transcriptional regulation of specific genes accordingto different time courses.

Furthermore, the increase in actin isogene transcriptionwas rapid and transient in comparison with total RNAtran-scription, which increased more slowly and was not transient(Fig. 4). In particular, amanitin-insensitive transcription of(presumably) rRNA and tRNA by polymerases I and III, re-spectively, was still increased at 24 h, when transcription of theactin isogenes had returned to control levels. In a previousreport of altered transcription in a model for myocardial hy-pertrophy, the early and selective increase in contractile pro-tein gene transcription seen in the present study was not ob-served, perhaps because specific gene transcription was mea-

sured at 24 h only (6). Transient activation of mRNAtranscription has been seen in other systems, and attenuationof transcription has been attributed to synthesis of a transcrip-tional repressor (19, 20). Thus, rather than effecting a general,simultaneous increase in overall gene transcription, stimula-

a1-Stimulated Actin Gene Transcription 1081

6-

c 5-.2-.0

Q 2-

s. c -4~O

; 3-z-U

tion of the a1-adrenergic receptor leads to a distinctive tem-poral sequence of transcriptional activation. It will be interest-ing to see if conditions which induce myocardial hypertrophyin vivo also stimulate the same complex temporal sequence ofgene transcription as does stimulation of the a,-adrenergic re-ceptor.

A major implication of this work is that the a, receptortransduces a rapid signal or signals to specific genes in themyocyte nucleus. The results with cycloheximide suggest thatnew protein synthesis is not required for signal generation.Thus the transcriptional changes may result from modificationof preexisting molecules which interact with regulatory se-quences of the specific genes (19, 21-23). A protein kinaseC-mediated modification may be important in the presentcase, since protein kinase Cis activated through the al receptorin the cultured heart myocytes (24).

Finally, these results demonstrate a new dimension for therole of the a1-adrenergic receptor in cardiac myocytes. In ad-dition to its well-established role in mediating inotropic andchronotropic responses (25), we show here that the a1-adren-ergic receptor mediates changes in RNAtranscription whichunderly a gene switch accompanying heart muscle cell hyper-trophy. Regulation of transcription is independent of effectson contractility per se, since the myocytes in this culture sys-tem are quiescent with a, stimulation alone (7). It should nowbe possible to delineate the molecular mechanism(s) by whichthe a1-adrenergic receptor mediates these vastly different ef-fects.

Acknowledaments

Wethank Uri Nudel for the kind gift of the actin clones used in theseexperiments and Tom Cooper for his helpful advice in the technicalaspects of the work.

This study was supported by the U. S. Public Health Service(HL-35561, HL-31113) and the Veterans Administration ResearchService. Dr. Simpson is a Clinical Investigator of the Veterans Admin-istration. Dr. Long was supported by American Heart Association(California Affiliate) Fellowship 86-N25.

References

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2. Gunning, P., P. Ponte, H. Blau, and L. Kedes. 1983. Alpha-skel-etal and alpha-cardiac actin genes are co-expressed in adult humanskeletal muscle and heart. Mo. Cell. Bio. 3:1985-1995.

3. Schwartz, K. D. de la Bastie, P. Bouveret, P. Oliveiro, S. Alonso,and M. Buckingham. 1986. a-Skeletal muscle actin mRNAsaccumu-late in hypertrophied adult rat hearts. Circ. Res. 59:551-555.

4. Izumo, S., B. Nadal-Ginard, and V. Mahdavi. 1988. Protoonco-gene induction and reprogramming of cardiac gene expression pro-duced by pressure overload. Proc. Natl. Acad. Sci. USA. 85:339-343.

5. Bishopric, N. H., P. C. Simpson, and C. P. Ordahl. 1987. Induc-tion of the skeletal a-actin gene in a1-adrenoceptor-mediated hyper-trophy of rat cardiac myocytes. J. Clin. Invest. 80:1194-1199.

6. Lee, H. R., S. A. Henderson, R. Reynolds, P. Dunnmon, D.Yuan, and K. R. Chein. 1988. a1-Adrenergic stimulation of cardiac

gene transcription in neonatal rat myocardial cells: effects on myosinlight chain-2 gene expression. J. Biol. Chem. 263:7352-7358.

7. Simpson, P. 1985. Stimulation of hypertrophy of cultured neo-natal rat heart cells through an a1-adrenergic receptor and induction ofbeating through an a,- and #1-adrenergic receptor interaction: evi-dence for independent regulation of growth and beating. Circ. Res.56:884-894.

8. Simpson, P., and S. Savion. 1982. Differentiation of rat myo-cytes in single cell cultures with and without proliferating nonmyocar-dial cells: cross-striations, ultrastructure, and chronotropic response toisoproterenol. Circ. Res. 50:101-116.

9. Simpson, P. 1983. Norepinephrine-stimulated hypertrophy ofcultured rat myocardial cells is an alpha,-adrenergic response. J. Clin.Invest. 72:732-738.

10. Long, C. S., and C. P. Ordahl. 1988. Transcriptional repressionof an embryo-specific muscle gene. Dev. Biol. 127:228-234.

11. Mulvihill, R. R., and R. D. Palmiter. 1977. Relationship ofnuclear estrogen receptor levels to induction of ovalbumin and conal-bumin mRNAin chick oviduct. J. Biol. Chem. 252:2060-2068.

12. Kedinger, C., M. Gniazowski, J. L. Mandel Jr., F. Gissinger,and P. Chambon. 1970. Alpha-amanitin: a specific inhibitor of one oftwo DNA-dependent RNApolymerase activities from calf thymus.Biochem. Biophys. Res. Commun. 38:165-171.

13. Kafatos, F. C., C. W. Jones, and A. Efstratiadis. 1979. Determi-nation of nucleic acid sequence homologies and relative concentra-tions by a dot hybridization procedure. Nucleic Acids Res. 7:1541-1552.

14. Spindler, S. R., S. H. Mellon, and J. D. Baxter. 1982. Growthhormone gene transcription is regulated by thyroid and glucocorticoidhormones in cultured rat pituitary tumor cells. J. Biol. Chem.257:11627-11632.

15. Mayer, Y., H. Czosnek, P. E. Zeelon, D. Yaffe, and U. Nudel.1984. Expression of the genes coding for the skeletal muscle and car-diac actins in the heart. Nucleic Acids Res. 12:1087-1 100.

16. Snedecor, G. W., and W. G. Cochran. 1967. StatisticalMethods. The Iowa State University Press, Ames, IA. 593 pp.

17. McKnight, G. S., and R. D. Palmiter. 1979. Transcriptionalregulation of the ovalbumin and conalbumin genes by steroid hor-mones in chick oviduct. J. Biol. Chem. 254:9050-9058.

18. Milsted, A., R. P. Cox, and J. H. Nilson. 1987. Cyclic AMPregulates transcription of the genes encoding human chorionic gonad-otropin with different kinetics. DNA(NY). 6:213-219.

19. Larner, A. C., A. Chaudhuri, and J. E. Darnell, Jr. 1986. Tran-scriptional induction by interferon: new protein(s) determine the ex-tent and length of the induction. J. Biol. Chem. 261:453-459.

20. Murdoch, G. H., E. Potter, A. K. Nicolaisen, R. M. Evans, andM. G. Rosenfeld. 1982. Epidermal growth factor rapidly stimulatesprolactin gene transcription. Nature (Lond.). 300:192-194.

21. Bohmann, D., T. J. Bos, A. Admon, T. Nishimura, P. K. Vogt,and R. Tijan. 1987. Human proto-oncogene c-jun encodes a DNAbinding protein with structural and functional properties of transcrip-tion factor AP-1. Science (Wash. DC). 238:1386-1392.

22. Dynan, W. S., and R. Tijan. 1985. Control of eukaryotic mes-senger RNAsynthesis by sequence-specific DNA-binding proteins.Nature (Lond.). 316:774-778.

23. Montminy, M. R., and L. M. Bilezikjian. 1987. Binding of anuclear protein to the cyclic AMPresponse element of the somatosta-tin gene. Nature (Lond.). 328:175-178.

24. Henrich, C. J., and P. C. Simpson. 1989. Differential acute andchronic response of protein kinase C in cultured neonatal rat heartmyocytes to a,-adrenergic and phorbol ester stimulation. J. Mol. Cell.Cardiol. In press.

25. Benfey, B. G. 1987. Function of myocardial a-adrenoceptors. J.Applied Cardiol. 2:49-70.

1082 C. S. Long, C. P. Ordahl, and P. C. Simpson