J. Biol. Chem.-1992-Sadoshima-10551-60

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THE OURNALF BIOLOGICALHEMISTRYQ 1992by The American Society for Biochemistry and Molecular Biology. Inc. Vol.267, No. 15, Issue ofMay 25 , pp. 10551-10560,1992Printed in U.S.A.

Molecular Characterizationof the Stretch-induced AdaptationofCultured Cardiac CellsAN IN VITRO MODEL OF LOAD-INDUCED CARDIAC HYPERTROPHY*

(Received for publication, December 31, 1991)

Jun-ichi Sadoshima$ll**, Lothar Jahn$1$$,Toshiyuki TakahashiST, Thomas J. KulikQII ndSeigo Izumo$lQ#From the $Molecular Medicine Unit and CardiovascularDivision, Be th Israel Hospital, the Department of Cardiology,Childrens Hospital, and the Departments of lMed icine and IIPediatrics, H arvard M edical School, Boston, Massachusetts 02215

Although it is a well-known fact that hemodynamicload is a major determinant of cardiac muscle mass andits phenotype, little is known as to how mechanicalload is converted nto ntracellular signals of generegulation. To address this uestion, we characterizedthe stretch-induced adaptation of cultured neonatalcardiocytesgrown on a stretchablesubstrate n aserum-free medium. S tatic stretch (20%)of the cellswas applied without cell injury. Stre tch caused hyper-trophy in myocytes and hyperplasia in non-myocytes.Stretch caused an induction of immediate-early genessuch as c-fos, c-jun, c-myc, JE, and Egr-1, ut notHsp70. Immunostaining showed that the stretch-in-duced Fos protein localized in the ucleus of both myo-cytesnd non-myocytes. Nuclear extracts fromstretched myocytes contained DNA binding activi ty tothe A P- 1 and Egr- consensus sequences. In myocytes,the induction of immediate-early genes was followedby expression of fetal genes uch as skeletal a-actin,atrial natriu retic factor, and &myosin heavy chain.DNA transfectionxperiments showed thathestretch-response element of the c-fos gene promoteris present within 366 base pairs of the 6flankingregion, whereas that of the atrial natriuretic factorand the&myosin heavy chain genes isprobably locatedoutside of 3412 and 628 ase pairs of the 5-flankingregion, respectively. These results demonstrate thatthe phenotype of stretched cardiocytes n this in vitromodel closely mimics that of hemodynamic load-in-duced hypertrophy in vivo. This model seems to be asuitable system with which to dissect the molecularmechanisms of load-induced hypertrophy of cardiacmuscle.

Hypertrophy, an increase in cell size without cell division,is a fundamental adaptive process employed by post-mitoticmuscle cells (1). It is a well-known fact that external load* This work was supported in part by grants from the WhitakerFoundation and the National Insti tute of Health. The costs of pub-lication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertise-ment in accordance with 18U.S.C. Section 1734 solely to indicatethi s fact.** Fellow of Human Frontier Science Program.$$ Supported by a Fellowship in Basic Science Research at theHarvard-Thorndike Laboratory.3s Established Investigator of the American Heart Association. Towhom correspondence should be addressed Molecular Medicine Unit,Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215.Te1.l617-735-2656;ax: 617-735-2913.

plays a critical role in determining muscle mass and tsphenotype in both cardiac and skeletal muscles in uiuo. How-ever, in intact animals it is extremely difficult to isolate andevaluate the role of external load in the regulation of musclegene expression. The lack of a well-characterized in vitromodel of load-induced cardiac hypertrophy has hamperedfurther mechanistic investigation in this area, and little isknown as to how mechanical load is initially converted intointracellular signals of gene regulation (reviewed in Refs. 2,3) .The firstdirect evidence that muscle cells are able tosense the external load, in he absence of neuronal orhormonal factors, came from a study by Vandenburgh andKaufman (4 ) who demonstrated that he ate of proteinsynthesis of cultured chick skeletal muscle cells grown onelastic substrate increased significantly in response to s taticstretch of the substrate. A similar phenomenon was observedin adult cardiocytes plated on silicone sheet (5). More re-cently, it has been shown that stretchof neonatal cardiocytesin culture caused induction of c-fos protooncogene and skele-tal a-actin (6, 7), a phenotype also observed in the myocar-dium in response to pressure overload in uiuo (8).Thesestudies suggest that stretching cardiocytes in vitro may be asuitable experimental ystem with which to address the ques-tion of how mechanical load is transduced into intracellularsignals regulating gene expression. However, since previousstudies have been limited to the nalysis of the rate f proteinand RNA synthesis and expression of c-fos and skeletal a -actin genes (5-7), the appropriateness of this system as amodel with which to dissect molecular mechanisms of load-induced hypertrophy can only be determined after furthercharacterization of the system.For example, it has not been demonstrated whether or notthe stretch-induced c-fosgene expression and increase inprotein synthesis in cultured cardiocytes is a result of cellinjury, rather than that of physiological stimulus. This is animportant question because the c-fos gene is known to beinduced by a variety of nonspecific stimuli, such as cell injuryor cell dispersion by trypsin reatment (9). Furthermore,repetitive stretch of skeletal myotubes in culture is known tobe associated with an initial cell injury (10). Thus, it s criticalto determine whether or not the cardiocyte stretch model isan injury repair model.Second, pressure overload in vivo causes hypertrophy ofmyocytes and proliferation of non-myocytes (11).Smce neo-natal myocytes still retain some capacity to divide (12) , it isimportant to determine whether this in uitro stretch modelrepresents hypertrophy or hyperplasia of neonatal myocytes.It is also important to determine whether stretch affects the10551


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10552 Stretch- inducedardiacyper trophy in V i t r ogrowth of non-muscle cells in vitro.Third, the mmediate-early (IE)' genes induced by pressureoverload in vivo are not limited to c-fos, but include a largenumber of genes such as c-jun, jun B, Egr-1, c-myc, C-Ha-ras, nur77, and a major heat shock protein, Hsp7O (8 andreviewed in Ref. 13). It is not known whether these genes arealso induced by in vitro stretch. It would be interesting toexamine this because there have been several examples sug-gesting tha t the profile of IE gene expression is stimulus-specific, and distinct combinatorial expression of IE genesmight confer phenotypic specificity in the cellular responseto different stimuli (14, 15).Fourth, most previous studies concerning IE gene expres-sion in cardiocytes have been done only at the mRNA leveland not at the rotein level. Recently, Roux et al. (16) reportedthat in the absence of serum, Fos protein could not be trans-located into nucleus and stayed inytoplasm in rat mbryonicfibroblast and mouse fibroblast cell lines. If the same is truefor cardiocytes, which are usually cultured in serum-free me-dia, it will have important implications for our understandingof the roles of c-fos in cardiac hypertrophy because it wouldsuggest that the c-fos gene product may not be necessary forcells to develop the hypertrophic phenotype. Thus, it is im-por tant to determine whether Fos protein is localized in thenucleus after the stretch stimulus, and whether there is aninducible DNA binding activity in thenuclear extracts to thetarget sequence of Fos/Jun proteins.Fifth, following induction of IE genes, expression of severalgenes are known to change in response to pressure overloadin the rat ventricle. These include induction of the P-MHC,ANF, skeletal a-actin, smooth a-actin, &tropomyosin and B-type creatine kinase, as well as down-regulation of the SRCa2+-ATPase reviewed in Refs. 1,13). A previous study hasshown that theexpression of skeletal a-actin is up-regulatedby in vitro stretch (7).However, skeletal a-actin gene expres-sion may not be the best marker for hypertrophy becauseinduction of this gene in vivo is usually transient (lasting 1week) and is often no longer up-regulated in well-establishedhypertrophy (8, 17, 18). In contrast, up-regulation of the 8-MHC and ANF genes persists in the chronic phase of hyper-trophy (8, 13, 19). Therefore, it would be important to deter-mine whether these "stable late markers" of hypertrophy arealso expressed in the in vitro stretch model.Finally, the skeletal a-actin gene contains multiple CC(A/T),GG motif CArG box) in he proximal promoter (20),which is essential for the basal as well as growth factor-inducible expression of this gene in cardiac myocytes (21).This element is very similar to the serum response element(SRE) of the c-fos geneand cross-competes for the binding ofthe serum response factor to SRE (22, 23). The factor thatbinds to the CArG box of the skeletal a-actin gene is appar-ently identical to serum response factor (24). It has beensuggested that the activation of the c-fos and skeletal a -actingenes by stretch may occur by a common mechanism(7). A t present, however, a link between IE gene expressionand the late phenotypic changes remains highly conjectural.Therefore, it would be interesting to examine whether otherpromoters that do not contain the CArG box or SRE, such asthe /3-MHC and the ANF genes (25,26), arealso up-regulatedby stretch.

The abbreviations used are: IE, immediate-early gene; ANF, atria lnatriuretic factor; BrdU, bromodeoxyuridine; CAT, chloramphenicolacetyltransferase; FCS, fetal calf serum; MHC, myosin heavy chain;PBS, phosphate-buffered saline; kb, kilobase(s); bp, base pair(s);SR,serum response; SRE, serum response element; RSV, Rous sarcomavirus; PMSF, phenylmethylsulfonyl fluoride; HEPES, 4-(2-hydroxy-ethyl)-1-piperazineethanesulfoniccid.

These are some examples of questions that have not beenresolved by previous studies. In order to address these ques-tions, we have performed a systematical analysis of the phe-notype of ra t neonatal cardiocytes undergoing stretch in vitro.The results demonstrate that the phenotype of this in vitromodel closely mimics that of load-induced cardiac hypertro-phy in intact animals, but there are a few important differ-ences. Using this model, we have determined whether thetransfected promoters of the c-fos, ANF, and /3-MHC genesare responsive to stre tch in entricular cardiac myocytes.

EXPERIMENTALPROCEDURESMaterials-Collagenase Type IV was purchased from Sigma. Sili-cone sheet (0.005- or 0.01-inch thick) was Silastic sheet from DowCorning. Rat tail collagen was from Biomedical Technologies. Theair brush used for collagen coating was from Badger Air-Brush. Allother culture reagents were purchased from GIBCO. All radiochemi-cals were obtained from Du Pont-New England Nuclear. Anti c-Fosantibody was from Medac Molecular Biology (Hamburg, Germany).Anti c-Jun antibody was from Oncogene Science. All other chemicalswere from Sigma.In Vitro Stretch Deuice-The stretch device we used was a modi-fication of the system originally developed by Vandenburgh andKaufman (4) for the embryonic skeletal muscle cells. Silicone sheet(50 X 73 mm') in a stretching frame was finely coated with rat tailcollagen dissolved in 0.02 N acetic acid (0.25 mg/ml) using an airbrush. The stretching frame was put in a notched Plexiglas supportfixed ontoa 150-mm culture dish (Fig. lA).Uniaxial strain wasapplied by stretching the silicone sheet in the frame by 10 or 20% inthe length along the Plexiglas support. Ten and 20% stretch of theframe gave mean stretch of10.6 and 20.0% of the silicone sheet,respectively, which was obtained from average of the substrate lengthat various points measured in control and stretched states. In theexperiments of [3H]phenylalanine ncorporation and I3H[thymidineuptake, we used smaller version of the silicone sheet (15 X 20 mm').In the present experiments, we used 20% stretch unless otherwiseindicated. To eliminate differences due to substrate materials, staticcontrol cells or serum-stimulated ells were also grown on the siliconesheet.Preparation of Myocyte-rich Culture (Hereafter CalledMyocyte

Culture)-A diagram of experimental protocol is shown in Fig. 1B.Primary culturesof cardiac myocytes were prepared using a variationof the method described by Simpson et al. (27) and Orlowski andLingrel (28). Hearts were removed from 1-day-old Wistar rats anes-thetized by ether underaseptic conditions. The ventricles wereminced into 1-3-mm3 fragments. Digestion was performed by five tosix 15-min periods of incubation at 37 "C with HEPES-buffered salinesolution (mM):20 HEPES-NaOH, pH 7.6, 130 NaCl, 3 KCl, 1NaH2P04,4 glucose, 3.3 p~ phenol red containing 0.1% collagenaseIV, 0.1% trypsin, 15 pg/ml DNase I, and 1.0% chicken serum at37 "C. At the end of each cycle, the supernatant was stored on iceafter addition of calf serum (10% v/v) to neutralize trypsin. Thedissociated cells were collected by centrifugation and resuspended inDulbecco's modified Eagle'smedium/F-12 (GIBCO) (l :l , v/v) supple-mented with 5% horse serum, 3 mM pyruvic acid, 100 p M ascorbicacid, 1 pg/ml insulin, 1 pg/ml transferr in, 10 ng/ml selenium, and100 pg/ml ampicillin. To selectively enrich for myocytes, dissociatedcells were preplated for 1 h, during which period the non-myocytesattached readily to the bottom of the culture dish. The resultantsuspension of cardiocytes was plated onto he collagen-coated siliconesheet at a density of 1 X l o 5 cell/cm'. Bromodeoxyuridine (BrdU)(0.1 mM) was added during the first 24-36 h to prevent proliferationof non-myocytes except for the cultures used for thymidine uptakemeasurement. The culture medium was hanged 24-36 h after eedingto a defined serum-free Dulbecco's modified Eagle's mediump-12medium which had the same composition as described above, exceptthat 5% horse serum and BrdU were not added. To minimize theeffect of remnants of serum, cultured cells were rinsed twice with theserum-free medium. In the serum-free culture medium, we did notobserve proliferation of non-myocytes even in the absence of BrdU.Using this method, we routinely obtained contractile myocardial cellcultures with -90-95% myocytes, as assessed by microscopic obser-vation of cell beating and by immunofluorescence staining with amonoclonal antibody against arcomeric myosin heavychain (M F 20)(29). All experiments were done 24-36 h afterchanging to theserum-


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Stretch-induced Cardiac Hyp ertrop hy in Vitro 10553A

20% Stretch

I

B Dispersion d ardiacventricles(myocvtoand ron-myocyte)tpreplalingon plastic dlsh in5% HSfor1hr \Adhecent calls " "adherent cslist inlO%HS fCf481072ht 2ps~agesinlO%HS" t platedonsiliccnesubstratein5% HS+ BrdU. for24 hplsted on silicone wbaralo t in5%HS+BrdUurl2hGrown toConnuence Mediumchmge to removed " p

Medium changeto serum free mediumtR i m wice with seturn free medium Rmtwicewith serum free mediumMiurn change o m u m reemedium+t fCf24h t for24hNon-myocyto-rich fr8ction Yyocyto-rlch froctlont tStrotch oxp8rimonts Strotch oxporimonto

FIG. 1. Scheme of stretch culture dish (A ) and protocol ofcell preparation procedures (myocytes and non-myocytes)( B ) .A, Scheme of s tretch culture dish details of the device weredescribed in the text. Stretch was applied by gently pulling the frameto the direction indicated by the arrow. It took 2-3 s for the stretchto be accomplished. Morphology and beating of cardiocytes could beobserved through the silicone membrane by microscopy. a, 53 mm; b,75 mm. B , diagram of the cell preparation procedures (myocytes andnon-myocytes). *BrdU was omitted in [3H]thymidine uptake experi-ment. **BrdU was not used. ?The serum-free medium did not containBrdU. HS, horse serum.free medium. Lactate dehydrogenase and creatine phosphokinasewere measured by an IL Monac Auto Analyzer (Inst rumental Labo-ratory).Preparation of Non-myocyte-rich Culture (Hereafter Called Non-myocyte Culture)-Highly enriched cultures of non-myocytes wereprepared by two passages of cells adhered to the culture dish duringthe preplating procedure (Fig. 1B). Until the second passage, cellswere maintained in the same culture medium as above except that10% horse serum was used and BrdU was not used. After the secondpassage, the same serum-free medium as above was used.Isolation and Analysis of RNA-Total cellular RNA was isolatedfrom cardiocytes dissolved in 4 M guanidium thiocyanate, followed bycentrifugation through 5.7 M cesium chloride solution (30). Aliquotsof total cellular RNA were size-fractionated by 1%agarose gel elec-trophoresis, transferred to nitrocellulose membranes, and hybridizedwith cDNA probes labeled with [32P]dCTP 3000 Ci/mmol) by ran-dom priming (30). The following probes were used for Northern blotanalysis in this study: 1) c-fos: 2.1-kb EcoRI fragment of the mousec-fos cDNA clone pGEMfos3 (a gift from J. G . Belasco and M. E.Greenberg, Harvard Medical School); 2) glyceraldehyde-3-phosphatedehydrogenase: a 1.3-kb PstI fragment of the rat glyceraldehyde-3-phosphate dehydrogenase cDNA (31); 3) c-jun: a 2.1-kb fragment ofthe mouse c-jun cDNA (32); 4) Egr-1 (zif/268): a 2.8-kb fragment ofthe mouse zif/268 (= Egr-1) cDNA (33); 5) c-myc: the mouse cDNAclone pG2 myc5 (gift from C. Stiles, Dana-Farber Cancer Institute);6) JE: the mouse cDNA clone pJE (34); 7) Hsp70 the rat cDNAclone pDPF (35); 8) skeletal a-actin:asynthetic oligonucleotidecomplementary to he first 57 nucleotides of the 3"untranslated

region of the mouse skeletal a-actin cDNA (36); 9) ANF syntheticoligonucleotides (84-nucleotides long) complementary to the entirecoding sequence of the rat ANF (37). The filters were washed underappropriate conditions of stringency. The relative amounts of specificmRNA were quantitated by laser densitometry of the correspondingautoradiograms in the linear response range of the x-ray films. Thehybridization signals of specific mRNA were normalized to those ofglyceraldehyde-3-phosphate dehydrogenase mRNA to correct for dif-ferences in loading and/or transfer. The levels of glyceraldehyde-3-phosphate dehydrogenase mRNA were not significantly affected bystretch (not shown). To quantitate the relative levels of the a- and8-MHC mRNAs, S1 nuclease mapping was performed using the 3'end PstI fragment of therat &MHC cDNA clone pCMHC5 asdescribed (38).Incorporation of [3H]Phenylalanine-As an index of protein syn-thesis, [3H]phenylalanine incorporation was measured as described(39). Cells weregrown on 15 X 20-mm2 collagen-coated siliconemembranes. After incubation in serum-free medium for24 h, the cellswere stretched or stimulated with 20% fetal calf serum (FCS) forvarious periods of time with [3H]phenylalanine (10 pCi/ml) andunlabeled phenylalanine (0.36 mM) in the medium. For controls, cellswere harvested at comparable times without stretch. The cells werewashed with phosphate-buffered saline (PBS), and 10% trichloro-acetic acid was added at 4 "C for 60 min to precipitate protein. Theprecipitate was washed three times with 95% ethanol, then resus-pended in 0.15 N NaOH. Aliquots were counted by a scintillationcounter. The results were expressed as counts/minute/microgam oftotal protein, or counts/minute/dish. Protein concentration was de-termined by the method of Lowry (40), using bovine serum albuminas standard.Incorporation of r3H]Thymidine and Cell Count-Cells were grownon a 15 X 20-mm2collagen-coated silicone sheet without BrdU (Fig.1B). Even in this condition, we generally observed less than 10%non-myocytes in myocyte culture preparations, as measured by cellbeating and immunostaining with MF 20. This is probably due tohigh plating density (1 X 10' cells/cm2) and the use of horse serum(5%)which contains very low amounts of growth factors. After a 24-h culture in the serum-free medium, cells were stimulated by stretchor by addition of 20% FCS. After 18h, [3H]thymidine 5 pCi/ml) wasadded for 6 h. Cells were then washed with PBS and harvested with10% trichloroacetic acid. Trichloroacetic acid-precipitable countswere measured as above. For cell counting, each dish was rinsed threetimes with PBS. Cells were detached with 1ml of trypsin-EDTA anddissociated by trituration. Cell counts were performed using a hema-cytometer. Each sample was counted three times.Protein Content-Protein contents were measured using the meth-ods of McDermott et al. (41). Each dish was rinsed three times withPBS. The cell layer was scraped with 1 ml of 1 X standard sodiumcitrate containing 0.25% (w/v) sodium dodecyl sulfate and frozen at-20 "C. Prior to use the extracts were thawed and vortexed exten-sively. Total cell protein was assayed by the method of Lowry et al.(40). The results were normalized by DNA content, which was meas-ured fluorometrically in aliquots of each extract by the method ofLabarca and Paigen (42) using calf thymus DNA as a standard.P h m i d Constructs and DNA Transfection-The mouse c-fos CAT(chloramphenicol acetyltransferase) construct was a generous gift ofDr. M. Gilman (Cold Spring Harbor Laboratory) (43, 44). The ratANF CAT constructs were from Dr. C. E. Seidman (Brigham andWomen's Hospital, Boston) (45). The rabbit OMHC CAT constructswere gifts from Dr. N. Shimizu (Beth Israel Hospital, Boston) (46)and he at BMHC constructs from Dr. V. Mahdavi (Children'sHospital, Boston) (25). Transfection was performed by the calciumphosphate precipitationmethod (30). 15pg of one of these DNAs and5 pg of the RSV 8-galactosidase expression plasmid were transfected/dish. Glycerol shock was performed 8 h after transfection. 24 h aftertransfection, the cells were stretched for the time indicated andharvested. Cell extracts were made by repeat freeze/thaw cycles, andthe proteinconcentration of the extracts was measured. Equalamounts of protein extracts (100 pg) were incubated with [3H]chlor-amphenicol and butyryl-CoA. The butylated chloramphenicol wasseparated by a repeated phase extraction using xylene (47). Aliquotswere counted by scintillation counter. To correct for transfectionefficiency, @-galactosidaseactivity of cell extracts was measured asdescribed (30). The activity of the RSV promoter, normalized by theluciferase activity of the cotransfected cytomegalovirus-luciferaseconstruct , was not influenced significantly by stretch (1.16& 0.1-fold,stretch uersus control).Extraction of Nuclear Protein-Nuclear proteins were extracted


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10554 Stretch-inducedardiacypertrophy in Vitrousing a variation of the method of Dignam et al. (48). Cells were lyseddirectly on the culture dish in.6 ml of cold lysis buffer (0.6% NonidetP-40,0.15 M NaCl, 10 mM Tris , pH .9,and1mM EDTA), transferredinto a 2-ml Eppendorf tube, and incubated for 5 min on ice. Thenuclei were pelleted (1250 X g,4 "C, 5 min), and nuclear proteinswere extracted from the pelleted nuclei in an equal volume of coldextraction buffer (500mM HEPES, pH 7.9, 0.5 mM EDTA, 0.75 mMMgC12,500 mM KCl, 1 mM dithiothreitol, 0.1 mM phenylmethylsul-fonyl fluoride (PMSF), 2 pg/ml leupeptin, 5 pg/ml aprotinin, and12.5% glycerol) on ice for 20 min, and cellular debris was removed bycentrifugation for 5 min a t 4 "C and 1250 X g. The supernatantcontaining nuclear proteins was dialyzed against buffer containing 10mM Tris-HC1, pH 7.9, 1mM EDTA, 5 mMMgC12, 10 mM KCl, 10%glycerol, 1 mM dithiothreitol, 0.1 mM PMSF. The ent ire procedurewas carried out a t 4 "C. The samples were stored at -85 "C.DNA Mobility Shift Assay-A double-stranded synthetic oligonu-cleotide containing the consensus sequence of AP-l (49) or Egr-1 (50)was labeled by [y3* P]ATP and olynucleotide kinase and was puri-fied by a 20% polyacrylamide gel. Samples of 15 pl containing 5 pg ofnuclear extract were incubated with 3 pg of poly(d1-dC) and 1 ng ofprobe (20,000 cpm) in the presence or absence of competitor oligo-nucleotides or antibodies for 20 min at room temperature. Beforeadding probe antibodies were preincubated with nuclear extract for15 min at room temperature. Binding reactions were electrophoratedon a 4% non-denaturing polyacrylamide gel and visualized by auto-radiography.Immunohistochemistry-Cells grown on silicone membranes werepermeablized with methanol (-20 "C) for 5 min followed by a shortdip in acetone (-20 "C). After air drying the cells were incubatedwith primary antibodies for 30 min in a humidified chamber, washedthree times in PBS, pH 7.4, and exposed to the econdary antibodiesfor 30 min. After another three washes in PBS, the cells were rinsedin deionized water, dehydrated in absolute ethanol, and mounted inmowiol (Polysciences, Warrington,PA).For double label experi-ments, both primary and secondary antibodies were applied (double-concentrated) simultaneously. Rabbit serum 456 against c-Fos wasfrom Medac. Monoclonal antibody MF 20 against sarcomeric myosinwas a generous gift of Dr. David Bader (Cornel1 University) (29).Secondary antibodies were fluorescein isothiocyanate-conjugated rTexas Red-coupled goat antibodies to immunoglobulins of rabbit ormouse (Jackson, West Grove, PA).Statistics-Data are given as mean f S.E. Statistical analysis wasperformed using analysis of variance and unpaired Student's t tes t,as appropriate. Significance was accepted a t p < 0.05 level.

RESULTSSubstrate Stretch Causes Cell Deformation withoutCell In-jury-We first examined whether stretching the ilicone sub-strate actually results in cell stretch or rather causes a "slip-page" of the cells rom the substrate. Fig. 2a shows ratneonatal cardiac myocytes plated sparsely in this experimentfor precise cell edge detection) observed using differentialinterference microscopy prior to stretch. A longitudinalstretch of the silicone substrate by 20% resulted in a compa-

FIG.2. Stretch of the silicone substrate resulted in an equiv-alent strain in culture d cardiac cells. Cellswere plated on acollagen-coated silicone sheet a i low density. The silicone substratewas stretched in the direction indicated by the long arrow in b by20%. Pictures of the same field taken before (a) , during ( b ) , nd after(c) release of the stretch are shown. In this example, the cells hadbeen stretched for 60 min before letting them to relax. The cells arehighlighted by short arrows. Note the increase in t he cell length withstretch ( b ) .The mean increase in cell length with s tretch was 20 f1%. Theelongation of the cells by st retch was reversible ( c ) and didnot cause detachment of the cells.

rable (20 f 1% )ncrease in cell length (or width) in thedirection of stretch, regardless of the orientation of the cells(Fig. 2b) . The cells returned to their prestretch length uponthe release of stretch (Fig. 2c). A similar result was observedwhen confluent cells were stretched by 20% (data not shown).In order to examine whether stretch causes cell injury, wemeasured release of lactate dehydrogenase or creatine kinasein the culture medium ollowing a 20% staticstretch ofconfluent cells. As shown in Fig. 3, there was no increase inrelease of lactate dehydrogenase or creatine kinase by stretch,either in the myocyte or in the non-myocyte culture. Thus,static stretch of the substrate membrane resulted in a com-parable strain of adherent cells without causing significantcell injury.Stre tch Causes True Hypertr ophy f Cardiac Myoc ytes-Wenext examined whether stretch causes hypertrophy or hyper-plasia of cardiac myocytes. Stretch of the cardiac myocytescaused an increase in protein synthesis in a time-dependentfashion, as measured by [3H]phenylalanine incorporation(Fig.a),n agreement with previous studies (5,7). n skeletalmuscle cells, tretch in serum-free medium causes an increasein protein synthesis, but the protein content of the cells didnot increase due to a concomitant increase in protein degra-dation (51). Serum factors were necessary for keletal musclecells to develop true hypertrophy in response to stretch (51).Whether stretch in serum-free medium causes true hypertro-phy of cardiac myocytes has not been addressed in previousstudies (5-7). Therefore, we measured the protein contentand rate of DNA synthesis of cardiac myocyte culture inresponse to stretch. As shown in Fig. 4B, stretch caused asignificant increase in protein content in serum-free medium.In contrast, stretch did not significantly increase the rate ofDNA synthesis, as measured by [3H]thymidineuptake during24 h (1.10-fold, n = 4 , Fig. 4 C ) . As a positive control, cellswere stimulated by 20% FCS,whichcaused a significantincrease in [3H]thymidineuptake (3.78-fold,n = 4,p < 0.001uersus control), indicating that these neonatal cells are capa-ble of DNA synthesis upon mitogenic timulation. The effectsof stretch are very similar to those of pressure overload onheart muscle cells in vivo, that is, increased load causes anincrease in protein synthesis and protein content without anincrease in DNA synthesis (11).

Time (h) Time (h)FIG. 3. Effect of stretch on release of lactate dehydrogenase(LDH) and creatine kinase (CPK) nto culture media n my-ocyte (left) nd non-myocyte right)culture. 8 X lo6 cells wereplated on a 53 X 75-mm2 silicone membrane. After 48 h in culture,culture medium was changed to serum-free medium (5.0 ml), andmedium samples (5 0 pl) were taken at th eime points ndicated, withand without 20% stretch of the silicone membrane. Release of lactatedehydrogenase and creatine kinase was less than 0.02% of the totalcell lactate dehydrogenase and creatine kinase obtained from celllysate. There was no significant difference in release of lactate de-hydrogenase or creatinekinase in stretched versus nonstretchedcultures, except th at at 2 h, a arger amount of lactate dehydrogenase

release was observed in nonstretchednon-myocyte culture. The phys-iological significance of th is observation is not clear. Each data pointrepresents the mean from three samples.


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Stretch-induced CardiacA

0 16 32 48TIME (HRB)

CC O N T R O LT R E T C H S E R U M

*+

C O N T R O L S T R E T C H SER U M

FIG.4. Effect of stretchandserumstimulationon ["HIphenylalanine incorporation ( A ) ,proteincontent ( B ) ,and["Hlthymidine uptake C ) n cardiac myocytes. A , ["Hjphenyl-alanine incorporat ion. Myocyte cul tures inserum-free mediumwerepreparedas n Fig. IH. A t each imepoint , hecp m of .'H wasnormal ized to meanof nonstretched control . Datawere also normal-ized to protein content f the d i sh ( to adjus t or the small variahi l i tyof the cel l counts hetween dishes) , al though we observed the sameresul ts without this normalizat . ion. Each data point represents mean+ S.E. rom four to s ix samples ( * p< 0.05, ** p < 0.01 uer.su.s control) .H , protein content . Cardiac myocy tes were st imulate d for 48 h. Da t awere normalized by m ean of n onst imulated control . Data were alsonormalized hy DNAcontent of the dish to normal ize he mal lvariabi l ity of the cell counts between dishes. Each data representsm e a n + S.E. from 9 to 10 samples (* p < 0.05, *' p < 0.01 uersuscontrol , +p < 0.05 ucrsus s t re tch) . C [ "Hl thymidine uptake . Cardiacmyocytes were st imulated for 24 h. For serum s t imula t ion , mediumwith 20% fetal calf serum was used. D a t a are expressed as relat ivecpm/dish normal ized to the mean cpmf control cel ls in each ex per-iment . Data a re mean + S.E. from four to five samp les (**p < 0.001ucrsus cont ro l ) .

Stretch CausesHyperplasia of Non-myocytes-Effects ofstretch on protein synthesis and the rate of DN A synthesiswere also examined in non-myocyte cultures obtained by twopassages of the prepla ting dish. Microscopical observationshowed no beating cells, and immunostaining with a mono-clonal antibody against sarcomeric myosin (M F 20) showedtha t thi s raction generally contained less than 15%of M F 20positive cells. 24 h of stretch of the non-myocyte culturecaused a small but stati stically significant ncrease (1.14 f0.01-fold, n = 6,p


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10556 Stretch-induced Cardiac Hypertrophynitrowas examined byNorthern blot analysis. These included theleucine-zipper class (c-fos, c-jun) (53), the helix-loop-helixfamily (c-myc) 54), the zinc fingerclass (Egr-l/Zif-268) (50),and a cytokine-like gene, JE (34). Stretch of myocytes pro-duced a rapid and transient induction of all IE genes exam-ined c-fos, c-jun, and J E (Fig. 6A), as well a s c-myc and Egr-1/Zif-268 (Fig. 6B). Although the expression of c-fos, c-jun,JE, and Egr-1 showed a peak a t around 30 min, that of c-mycrevealed a little ater peak around 1 h.Fig.6C shows aquantitative analysis of multiple Northern blots by laserdensitometry. Mean expression of c-fos, c-jun, and J E mRNAswere increased by 7.7-, 3.2-, and 3.0-fold, respectively. Theinduction of IE genes by stretch was also observed in primarynon-myocyte fraction (Fig. 7 and data not shown), althoughfor reasons not clear the levels of expression of IE genes innon-myocyte culture weremore variable (sometimes evenhigher) than those of myocyte culture (data not shown).It is known that heat shock proteins are induced by avariety of stimuli including high temperature, hypoxia, oxi-dative stress, and heavy metals (55). Heat shock proteins areinvolved in rotecting cells under adverse conditions by mech-anisms not yet fully understood.The stress protein Hsp7O isone of the principal constituents of the mammalian heat shockresponse and can be promptly up-regulated at the ranscrip-tional level in the absence of new protein synthesis, as in IEgenes (55 ) . A marked expression of Hsp7O mRNA by acutepressure overload in vivo has been demonstrated previously(8). nterestingly, however, stretching myocytes in vitro failedto elicit the heat shock response, although 1h of incubationof myocytes a t 42 "C strongly induced expression of Hsp70,particularly the larger transcript (inducible form) (Fig. 8).Unlike other IE genes, Hsp70 was not significantly inducedby serum stimulation either.Localization of Fos Protein in the Nucleus-The c-fos andc-jun genes encode uclear proteins Fos and Jun,espectively,which are known as transcriptional regulators (9). Fos andJun form a heterodimer and bind to AP-1 sites, present inthe promoter region of many genes (52).Recently Roux et al.(16) reported that in the absence of serum, the Fos proteincould not be translocated into nucleus and stayed in cytoplasm

A B

-c-fos

-c-jun

- JE-28s-18s

in rat embryonic fibroblasts and mouse fibroblast cell lines.Therefore, we examined whether or not stretch of cardiocytes(myocytes and non-myocytes)grown in serum-free conditionsactually leads to translocation of Fos protein to the nucleusafter its synthesis in the cytoplasm. Immunostaining of non-stretched cardiocytes with an antibody against Fos showedno specific signals (Fig. 9a). In contrast, staining of cardi-ocytes stretched for 60 min showed specific signals localizedin the nucleus (Fig. 9, b and c ) . Thus, in cardiocytes stretchinduces translocation of Fos to the nucleus in the absence ofserum. In order to confirm that both myocytes and non-myocytes express Fos protein in response to stretch, doubleimmunofluorescence staining was performed using anti-Fosand anti-sarcomeric myosin (MF 20) antibodies. Both myosinpositive cells (arrows) and myosin negative cellsarrowheads)(Fig. 9d) expressed Fos protein in response to stretch (Fig.9d'). Induction of Fos protein was a transient response be-cause little Fos signals were detectable 2 h after stretch (datanot shown).Induction of Nuclear DNA Binding Activities-To confirmthat stretch induces DNA binding activities to the AP-1element, we performed a DNA gel mobility shift assay. Nu-clear extracts were prepared from control and stretched myo-cytes and were incubated with a 32P-labeled onsensus AP-1oligonucleotide (49).The nuclear extract obtained from con-trol myocytes showed no significant binding (Fig. lOA, lane1). In contrast, tha t obtained from myocytes stretched for 1h showed significant binding ( l a n e 4 ) which can be competedby an excess of the unlabeled AP-1 oligonucleotide ( l a n e 5 )but not by unrelated sequences ( l a n e 6). Furthermore, prein-cubation of the nuclear extracts with antibodies against Fosand Junmarkedly diminished the DNA binding activity ( l a n e7), but antibodies against myosin had no effect l a n e 8). Theresidual DNA binding activity in lane 7 probably representsother members of AP-1 (such as fra, jun-B, jun-D, etc.). Whenthe Egr-1 consensus oligonucleotide was useds a probe (Fig.1OB) (50), detectable binding activity was observed n theextracts from control myocytes ( l a n e 2) , consistent with adetectable level of expression of Egr-1 mRNA in control state(Fig. 6B). On the other hand, there was a marked increase in

C

p io.w* i ontrolE# Stretch 30'-c-myc

-2if-268(Egr-11-28s- 18s

c-fos("=I 1 c-jun JE(".7) ( k 7 )FIG. 6. Stretch- induced expressionof mmedi a te early genes in cardiac myocytes as determi ned byNorthern blot analyses .A , expression of c-fos, c-jun, and JE genes. B, expression of c-rnyc and Zif 26 8 (Egr-1)genes. Myocyte cultures in a serum-free medium were prepared as in Fig. 1B.Myocytes were stretched for the timeindicated above. 15 pg of total RNA were loaded in each lane. Ethidium bromide staining of 18 S and 28 S RN Ashowed that equal amounts of RNA was loaded in each lane. Gene expression 60 min after addition of 20% fetalcalf serum to the medium is also shown as a positive control. C, quantitative analysis of the stretch-inducedexpression of c-fos, c-jun, and JE gene s. Each hybridiza tion signal was quantitated by laser densitome try and isexpressed as th e ratio of the experimental value to that score obtained from nonstretched samples, which was set

as 1.0.For each analysis, the hybridization signal was normalized to the signal obtained with a glyceraldehyde-3-phosphate dehydrogenase probe as an internal control. The magnitude of induction of J E gene was more variablethan that of other IE genes. Results represent the mean + S.E. of 7-11 independent experiments.


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S t r e t c h - i n d u c e da r d i a cy p e r t r o p h yni t r o 10557C.-St r e t ch E

: - f o s

FIG. 7. Stretch-induced expression of the c-fos gene n thenon-myocyte as determined by Northern blot analysis. Non-myocyte cultures in a serum-free medium were prepared a s in Fig.1H . Non-myocytes were stretched for the imes ndicated. c-fosexpression 30 min after addition of 20 % fetal calf serum is also shownas apositivecontrol. Hybridization withaglyceraldehyde-3-phos-phate dehydrogenase probe showed equivalent amounts of signal ineach lane (not shown).

2 8 s -

1 Hsp 70 (inducible)Hsp 70 (constitutive)1 8 s -FIG.8. Lack of stretch-induced expression of Hsp7O geneas determined by Northernblot analysis. Cardiac myocytes werestretched for the times ndicated. As a positive control, Hsp7O expres-sion by 1 h of incubation at 42 C is also shown. Although 1 h ofincubation at 42 C caused a marked induction of both inducible andconstitutive transcripts, stretch for 1 or 2 h, or 1 h of treatme nt with20% fetal calf serum failed to induce Hsp70 mRNA significantly.Hybridization with a glyceraldehyde-3-phosphate ehydrogenaseprobe showed equivalent amounts of signal in each lane (not shown).

th e binding ac t iv ity to the Egr-1 target sequence in extractsfrom the stretched ells ( lane 4 ) ,which was specifically com-peted by excess unlabeled EGR -1 oligonucleotide (la ne 5 ) b u tnot by unrelated sequences (lane 6) . Similar esults wereobserved when myocytes were stim ulated by 20% F CS (lanes7 a n d 8) .Induction of Fetal Genes-To exam ine whether stretch nthi s model is accompanied by the late phenotyp ic changesthat are cha racte ristic in in uiuo models of hypertrophy (8,13, 19), we examined expression of several fetal genes by along term s tretch. For this exp eriment, a physiologic dose ofTa 1nM) was add ed to theedium to accelerate maturationof neonatal cardiac myocytes in uitro (56) because neo natalventricular cells are known to exp ress 3-MHC, ANF , skeletala-actin. and other fetal genes (8, 19, 56). Innonstretchedcells, A NF and skeleta l cy-actin geneshowed low levels ofexpression n hisculturecond ition. However, 12-48 h ofstret ch caused a progressive accum ulation of AN F an d kele-ta l cy-actin mR NAs (Fig. 1 lA ). S1 mapp ing analysis revealedth at 48 h of stretch also induced expression of the @ M HCmRNA (Fig. 11B).T hus , hepatt ern of thephenotypicchanges induced by stretch was very simi lar to tha t f in vivohypertroph y produced by hem odyna mic stress (8, 19). Fur-therm ore, genes whose expression is not depe nden t of CArGbox or SR E eleme nts (ANF an d @-M HC ) 25, 45) were alsoinducible by in uitro stretc h.Transfection Experiments-The above results suggest th atthi s in uitro model closely mimics th e phen otyp e of in uiuohypertroph y in expression of IE genes and late fetal genes.Therefore, we examined whether the promo tersf c-fos, AN F,

FIG. 9. Immunofluorescent staining of cells with anti-Fosand anti-myosinantibodies. Cardiac myocytes were platedonsilicone membrane in serum-free conditions as in ig. 1B.After a 60-minstretch, he cells were fixed and reatedas described underExperimental Procedures. a, stain ing of control (nonstretched) ellswith the anti-Fos antibody. Note that the bright spots are artifact asthey correspond to non-cellular materials in the phase contrast mi-croscopy image (not shown). b , staining by the anti-Fos antibody ofcells stretched for 60 min (low power view). Granular nuclear stainingwas observed in most of the cells in the field. c, the high power viewof b. c, phase contras t image of c. Arrows correspond to the samenuclei in c and c. d , immunostaining of stretched cells with the anti-sarcomeric myosin antibody using Texas Red as a secondary antibody.d, staining of thesame field by theanti-Fosantibody usingafluorescein isothiothiocyanate-labeled econdary antibody. Both my-osin positive cells (arrows) and myosin negative cells (arrowheads)in d had specific nuclear staining by the anti-Fos antibody ( d ) .FO Sstaining isweaker in dthan in , probably due to echnical difficultiesin performing double immunofluorescence on the silicone membrane.Bars indicate 20 pm.and &M HC enes contain stretch-responsive lements. Th e-356 c-fos CA T co nstru ct co ntains up to 56 base pairs (bp)ups tream of th e mouse c-fos promoter including two majorinducible elements , the SRE and the alcium/cAMP-respon-sive element (Ca+/CRE) (43, 44, 52). When this constructwas trans fect ed into myocytes, the re was 4-5-fold inductionof CAT activity after 2 h of stretc h (Fig. 12), in agreem entwith the previous reports (6) . TheANF-BglII-CAT constructcon tains 3412 bp of AN F 5-flanking sequence including AP-1 as well as a p utative Ca*+/C RE (45). It has been shownpreviously that this con stru ct is expressed in atrial cells butth at its expression is very low in neon atal ventricular cells(26) . When this ANF co nstruct was transfected to neonatalventricular myocytes, there was no induction of CAT activityby stretc h for 48 h (Fig. 12). Similarly, AN F A HindIII-CAT,producedfromANF -BglII-CAT by deletion of an nte rna l556-bp Hind111 fragm ent conta ining the atrial-specific ele-ment and AP-1 (45) ,ailed to show induction by stretch (datanot shown) . The @-MHC-1,2, and -3 co nstructs containupto 295, 348, and 628 bpupstream of the rabbit@-MHCprom oter, respectively (46 ), and the rat -673 @-M HCcon-struct con tains 73-bp 5-flanking sequence f the rat @-M HCgene 25). Allof these cons tructs have been shown to behighly active in skele tal muscle cells, altho ugh n culturedcardiac myocytes their levels of expression are low (25, 46,


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10558 Stretch-inducedardiacypertrophy in VitroA B

control Sfretch Con l r o l Stretcherum

4 A P - 1

1 2 3 4 5 6 7 8

Stretch?m

4 - E G R . 1

FIG. 10. DN A gel m o b i l i t y s h i f t analysisof cardiac n u c l e a re x t r a c t s . A , stretching myocytes inducesanuclear actor whichspecifically binds to heAP -1 consensus sequence. Myocyte-richcultu res in a serum-free medium were prepared as inFig. 1B. Nuclearextracts ( N E ) were obtained from nonstretchedcontrol) ndstretche d cardiac myocytes (60 min ), and binding eactions werecarried out asdescribed in the textwith an end-labeled AP -1 consen-su s oligonucleotide. Th e specific complex formed by the factor indingto the P-1 probe is indicated y the arrow marked AP-I. Competitorsused are indicated above each lane. Lanes 1 a nd 4 , N E alone; lanes 2an d 5 , NE with 100-fold molar excess of cold AP-1 oligonucleotide;lane 6, NE with 100-fold molar excess of the thy roid hormone recep-tor-binding site of the a-my osin heavy chain ( M H C )oligonucleotide(61) used a s a nonspecific competitor; lanes 3 and 7, NE wi th 2 pl ofanti-Fos and Ju n antibodies; lane 8, NE wi th 2 pl of anti-MHCantibody. Antibodies were preincubated withN E a toom temperaturefor 15 min. B , stretch and serum stimulation significantly increasesa nuclea r factor which specifically binds to the Egr-1-binding sitesequence. The same NE as in were used. In addition, N E were alsoprepared from cardiac myocytes stimulated with 20% fetal calf serumfor 60 min. Binding reactions were carried o ut as described in thetex t with a n end-labeled oligonucleotide co ntaining thesequence 5 -CGCCCCCGC-3 (50). The specific complex formed by the factorbinding to the Egr-1 robe is indicated by the arrow marked EGR-I.Lane I, probe alone; lanes 2,4, an d 7, N E alone; lanes 3,5, and 8, N Ewith 100-fold excess of cold Egr-1 oligonucleotide; lane 6, NE with100-fold molar excess of th e a-M H Coligonucleotide (61).57). When hese p-MHC constructs were transfected intoneonatal cardiac myocytes, none of them showed significantinduction of CAT activity after 48 h of stretch (Fig. 12 anddata not shown).A trivial explanation for the apparent lack of stretch re-sponse of the ANF and the p-MHC promoters might be lowtransfection efficiency of cardiac myocytes, and a lack ofsufficient sensitivity of the CAT assay (as opposed to lucif-erase assay). We believe this is unlikely for the followingreasons. First, we were able to demonstrate that the c-fospromoter is stretch inducible, which serves as a positivecontrol of the system. Second, changing the method of trans-fection to electroporation or to lipofectin, which have beenreported to give a higher transfection efficiency in cardiocytes(26, 58), did not change our results (data not shown). Third,th e method of CAT assay we used is suitable to analyze weakpromoter activities because it is 25-fold more sensitive thanthe conventional method due to a much lower background(47). Fourth, the base-line levels of expression of the ANFand the p-MHC constructs we observed (see Fig. 12 legend)are quite comparable to those reported n the literature (aew% of RS V promoter) (25, 26, 57, 59). Taken together, it ishighly unlikely that we had failed to detecta significantinduction of the ANF and p-MHC promoters by stretch.

Sk. Aclln

P Po PolyIAla a 1 8S +

8-301ntCOO-

u-lsontFIG.11. S t r e t c h - i n d u c e d expression of f e t a l c a r d i a c g e ne s.A, induction of AN F and skeletal (Sk) -actin genes by stretch asdetermined by No rthern blot analyses. Myocyte cultures in a serum-free medium were prepared as in Fig. 1B. Myocytes were stretchedfor the tim es indicated. Hybridization was performed w ith 5 end-labeled specific oligonucleotide probes (36, 37). Hybridization withglyceraldehyde-3-phosphate dehydrogenase probe showed equivalentamount of signals in each lane (not shown). B , effects of long termstretch on a- and @ MH C gene expression a s determined by S1mapping analysis. S1 mapping was performed as previously as de-scribed (38). a M H C , a-myosin heavy chain; P, stI; w, amino acid;

nt,nucleotide.DISCUSSION

Our results demonstrate that the phenotype of stretchedcardiac myocytes in vitro highly resembles that of load-in-duced hypertrophy in vivo. More specifically, stretch causesan increase nproteinsynthesis andcontent without anincrease in DNA synthesis, a rapid and transient inductionof a variety of IE genes, followed byactivation of fetal genes,such as skeletal a-actin, /3-MHC, and ANF. Thus, thismodelseems to be a suitable system with which to dissect the signaltransduction pathway of load-induced hypertrophy.A very important question is whether the 20% stretch weused is a physiologically relevant stimulus. We confirmed thatthe cells did not slip from the subs tratewith this degree ofstretch , and there as no evidence of cell injury. In thewholeheart, a 20% increase in cell length would result in a aximumof 1.728 (U3)- fold ncrease in theend-diastolic volume. Thismagnitude of change in end-diastolic volume is well withintha t range observed in the intact heart 60).The second question concerns whetherstatic stretchof themembrane imposed a pure diastolic stress or a combinationof diastolic and systolic stress. In the ase of non-muscle cellsthere is no systolic component. On the other hand, myocytesin ourculture system actively contracted. Therefore, it islikely that stat ic stretch of myocytes resulted in an increasein both ystolic and diastolic stress. However, IE gene expres-sion was also observed in myocytes arrested with high K ortetrodotoxin in the culture medium, indicating that activetension generation is notnecessary for myocytes to sense thestretch stimulus.

* J. Sadoshima, T. Takahashi, L. Jahn, and S. Izumo, manuscriptsubmitted.


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Stretch-induced Cardiac Hyp ertrop hy in Vitro 10559

c-10s ANF P-MHCFIG. 12. Effect of stretch on the transfected -366 c-fos,-3412 ANF, and -628 @-MHC eporter constructs in primarycardiac myocytes. Primary ventricularmyocytes plated on siliconemembranes were cotransfected with 15 pg of eachCATreporterconstruct and5 pg of the RSV-&galactosidase expression plasmid ycalcium phosphateprecipitationmethod. 24 h after transfection,static stretchwasapplied to the dishes for 2 h (for the c-fos construct)or 48 h (for the ANF and B-MHC constructs). Cells were harvestedand CAT activity was determined by a modified phase extractionmethod as described (47).To normalize or transfection efficiency, -galactosidase activitywas measured as described (30). The activityf

th e RSV promoter was not influenced significantly by stretch (1.16& 0.1-fold, stretch versus control). To calculate the magnitude ofinduction by stretch, thenormalized CAT activity of stretched cellswas divided by that of nonstretched cells foreach construct. Resultsare expressed as mean f S.E. of three to seven independent experi-ments done in pairs (*p < 0.01 compared to nonstretchedcontrols).Th e mean base-line (nonstretched) CAT activities f each constructrelative to that of RSV-CAT (which has a very strong activity incardiocytes)were:pUC9CAT (a promoterlessnegativecontrol),0.27%;356 c-fos, 9.0%; 3412 ANF, 2.0%; -628 P-MHC,1.8%.Theactivities of the ANF and 0-MHC promoters were very similar tothose of comparable constructsreported in the literature (25, 26,57,59).IE genes have been proposed to function as mediators(third messengers) of long term cellular responses (9, 52).In some systems, downstream genes regulated by IE geneshave been identified. In the ra t fibroblast, c-fos expressionwas necessary for induction of stromelysin gene expressionby epidermal growth factor (62). However, in cardiac hyper-trophy, whether IE genes direct the subsequent phenotypicchanges remains to be elucidated. We demonstrated that thestretch-induced c-fos expression led to expression of Fos pro-tein in the nucleus in this model. DNA gel mobility shiftassay showed that stre tch nduced a DNA binding activity toth e AP-1 consensus sequence as well as to the Egr-1 targetsequence. Since many genes seem to contain the binding sitesfor AP-1 and Egr-1, these IE gene products may work as thethird messenger for some part of the hypertrophic response.On the other hand, it is very difficult to explain long term

changes in the expression of cardiac genes on thebasis of IEgene expression alone because it occurs only transiently inresponse to overload in vitro and in vivo.Among the IE genes which are known to be induced inresponse to external load in the intact heart nly Hsp7O wasnot induced in this n vitro stretch model. It has been reportedthat the uman Hsp70 gene is inducible by serum stimulationbut, unlike other IE genes, it takes 1 2 h before the inductionby serum to occur (63). Furthermore, &-regulatory sequencesrequired for heat shock response are different from thoserequired for serum induction (64,65). Thus, t is noturprisingthat Hsp7O and other IEgenes are regulated differentially incardiac myocytes. The reason for the lack of Hsp7O inductionby stretch in vitro is not clear yet. It is possible that pressureoverload in vivo may be accompanied by relative myocardialhypoxia due to an increase in oxygen demand and squeezing

of the microcoronary circulation or possibly by an increase inmyocardial temperature due to excess workload. On he otherhand, in our in vitro system, myocytes were well oxygenatedand a t constant temperature condition even in the stretchedstate. These differences may contribute to the ack of induc-tion of Hsp7O in in vitro stretch.In agreement with a previous report (6), we were able todemonstrate the stretch-induced activation of the c-fos genepromoter. However, we did not observe significant inductionof the promoter activities of the transfected ANF or the B-MHC genes by long term stretch despite activation of theendogenous genes by this procedure. In the case of ANF, theconstruct we used contained a 3.4-kb fragment from the 5-flanking sequence of the rat ANF genes which contains suf-ficient sequence to direct high levels of atrial-specific tran-scription in primary atrial cell cultures (26). However, theactivity of this construct in neonatal ventricular cells is verylow and not significantly inducible by glucocorticoids, potentinducers of the ANF promoter in atrial cells (26). These datasuggest that DNA sequences necessary for inducible ventric-ular expression of the ANF gene may be located outside ofthis 3.4-kb sequence. In support of this interpretation, Rock-man e t al. (66) demonstrated that , in a transgenic mouse linein which 500 base pairs of the human ANF promoter regiondirected the atrial specific expression of a marker gene, tho-racic aortic banding led to no detectable expression of themarker gene in the ventricle despite a 20-fold increase inendogenous ANF mRNA. This suggests that the DNA ele-ments necessary for the atrial-specific and pressure-inducible,ventricular expression of the ANF gene are distinct. The 3.4-kb fragment of the ANF promoter contains the AP-1consen-sus sequence which has been shown to bind to in vitro trans-lated c-foslc-jun heterodimers (45). However, the fact thatthe activity of the ANF construct could not be induced bystretch suggests that induction of c-fos and c-junalone is notsufficient to confer the stretch responsiveness to this pro-moter. In fact, an increase in ANF mRNA in response tooverload in vitro and in v ivo takes place when Fos protein isno longer detectable. These observations raise the question ofwhether the AP-l-binding sites of physiological significancein regulating the ANF promoter by hemodynamic overload.The P-MHC promoter constructs used in this study areknown to contain two muscle-specific positive elements andtheir expression is high in cultured skeletal myotubes (46).When transfected to cultured rat neonatal ventricular cells,however, their levels of expression are very low even in theabsence of thyroid hormone, a condition tha t causes a markedinduction of the endogenous B-MHC gene (38). Therefore, i tis possible that additional positive cis-elements may exist thatconfer a high level of expression in thecardiac myocytes. Thepresence of cardiac-specific elements, separate from theskeletal muscle positive elements, has been demonstrated inthe case of the chicken cardiac troponin T gene and themousecardiac troponin C gene (67, 68). It is possible that the p-MHC promoter may require such additional cardiac-specificelements in order to respond to stretch.Stretching primary non-myocyte caused a small but signif-icant increase in mitogenic activity. This result is consistentwith the observation in vivo tha t the cardiac interstitium,which comprises cardiac fibroblasts, endothelial cells, vascu-lar smooth muscle cells, macrophages, mast cells, and others,undergoes hyperplasia in response to pressure overload (11).These primary non-myocytes express IE genes in response tostretch in vitro, thus mimicking the early mitogenic responseto growth factors in a variety of cells (9). Our model may be


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10560 Stretch-induced Cardiac Hypertrophy in Vitrouseful to elucidate the mechanism of the interstit ial esponseto external load.It should be noted, however, that stretch responsivenessmay not be a general phenomenon because the stretch-in-duced IE gene expression was observed only in primary cul-tures but not in established cell lines such as NIH 3T3 andPC12 or in cultured pulmonary arterial smooth muscle oraortic endothelial cells that have been passaged multiple times(data notshown). These resultssuggest that the ignal trans-duction pathway for the stretch-induced response may utilizecomponents which are susceptible to down-regulation duringmultiple passages or in cell lines.In summary, the phenotype of the in vitromodel of theload-induced cardiac hypertrophy described here very closelymimics that of the load-induced response in vivo.his in vitromodel, which s amenable to molecular biological approaches,should allow us to elucidate further the precise mechanismsof gene regulation by mechanical stimuli in muscle cells andin other cell types.

Acknowledgments-We thank E. Wu for her technical assistance,R. D. Rosenberg and W. Grossman for their support and encourage-ment, C. E. Seidman, V. Mahdavi, M. Z. Gilman, M. Greenberg, B.Rollins, C. Stiles, M. Karin, N. Shimizu, and D. Nathans for plasmids,and D. Bader for MF 20 antibody.REFERENCES

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