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1075 Expression of Cellular Oncogenes in the Myocardium During the Developmental Stage and Pressure-Overloaded Hypertrophy of the Rat Heart Issei Komuro, Masahiko Kurabayashi, Fumimaro Takaku, and Yoshio Yazaki Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 Protooncogenes have been revealed to participate in normal cell proliferation as well as in cell transformation. Since cardiac myocytes are terminally differentiated, they cannot divide except in the fetal period. To determine the role of cellular oncogenes in the growth of the heart, the expression pattern of eight cellular oncogenes during the developmental stage and pressure-overloaded hypertrophy of the rat hearts were examined in vivo. Northern blot analysis was performed with eight oncogene probes (myc, fos, Ha-ras, src, erbA, erbB, sis, myb). Pressure overload increased the levels of cellular (c-) fos, c-myc, and c-Ha-ras. An increase of c-fos and c-myc was detected at 30 minutes and 2 hours, respectively; the levels peaked at 8 hours, and they returned to baseline by 48 hours after aortic constriction. However, the level of c-Ha-ras showed a gradual increase. During the course of development, the expression of c-myc was detectable only in the embryonic stage, whereas the expression of c-fos was not detected in the fetal period, was increased after birth, and peaked in 200day-old adults. The expression of c-Ha-ras was almost the same throughout the development. Cellular oncogenes were expressed in the heart in response to pressure overload and in a stage-specific manner. These results suggest that cellular oncogenes may participate in the normal developmental process and hypertrophy of hearts and that the cellular hypertrophy induced by pressure overload may share a similar mechanistic pathway with cell proliferation. (Circulation Research 1988;62:1075-1079) V arious types of malignancies can be induced by the RNA tumor viruses, and these retroviruses are known to carry specific genes, which are designated viral oncogenes.1 Cellular oncogenes (c-onc), homologous to retroviral oncogenes, have been hypothesized to play a role in growth control. Recent evidence that the products of several protooncogenes are either growth factors2 or growth factor receptors3 strongly suggested this hypothesis. Much information has accumulated that pathological expression of c-onc is related to cell transformation in vitro and malignant disease in vivo; however, very little is known about the role of c-onc in the physiology of normal cells. Cardiac myocytes can divide only in the fetal period, and soon after birth, they lose their ability to replicate DNA. After birth, hearts grow by an increase in cell size (hypertrophy), not in cell number (proliferation). Recently, the induction of cardiac myocyte hypertrophy has been reported to be associated with enhanced expression of the c-myc gene by exposure to From The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan. Supported by a grant-in-aid for scientific research and developmental scientific research from the Ministry of Education, Science and Culture; a grant for cardiomyopathy from the Ministry of Health and Welfare; and a grant from the research program on cell calcium signals in the cardiovascular system, Japan. Address for correspondence: Issei Komuro, The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Received September 3, 1987; accepted January 6, 1988. a,-adrenergic agonists in the culture cells.4 In the present study, we examined the expression of c-onc genes in the rat heart during the developmental stage and during hypertrophy caused by pressure overload. Materials and Methods Animals and Surgical Procedures To produce pressure-overloaded cardiac hypertrophy, male Wistar rats, which were 40 days old and weighed 180-200 g, were anesthetized with diethyl ether, and the upper part of the abdominal aorta was constricted with a hemoclip. Ninety-one of the 110 operated rats survived the procedures and were killed at predetermined times after the operation (0.5, 2 , 4 , 8, 12, 24, 48, and 72 hours). Sham-operated animals underwent an identical procedure except for placement of the hemoclip and were killed at different time intervals postoperatively. The mean aortic pressure in the aortic-constricted animals increased 46 ± 5 mm Hg compared with the sham-operated controls (n = 3). To investigate the developmental change, hearts of 12-, 15-, and 18-day-old embryos, 5-day-old neonates, and 40- and 200-day-old adults were examined. RNA Preparation Hearts were excised, and the atria, great vessels, and right ventricular free walls were removed. The left ventricles were rinsed with cold saline and quickly frozen in liquid nitrogen. Total cellular RNA was extracted from the myocardium by the lichium urea method.5 Poly-A + RNA was enriched by oligo(dT)cellulose chromatography. 1076 Circulation Research Vol 62, No 6, June 1988 Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 Hybridization Analyses Poly-A+ RNA (3 fig) was denatured at 60° C for 7 minutes, fractionated by electrophoresis through 1.2% agarose gels, and transferred to nylon membranes. The membranes were exposed to ultraviolet rays for 2.5 minutes, prehybridized, and hybridized at 42° C with 32 P-labeled oncogene probes: human c-myc, exon ID, EcoR I/Cla I6; v-fos, Bgl II/Pvu IT; v-Ha-ras, Bal I/Sal P; v-erbA, Pst I/Pst I; v-erbB, BamH I/EcoR P; v-sis, Pst I/Pst I10; v-myb, Sal I/Sal I"; and v-src, Pvu II/Pvu II. n The human c-myc probe was a generous gift of Dr. T. Sekiya, National Cancer Center, Tokyo. Other viral oncogenes were obtained from the Japanese Cancer Research Resources Bank, Tokyo. Prehybridization was performed in a solution containing 5 X SSPE (saline-sodium-phosphate-EDTA) buffer, 5 x Denhardt's solution, 1% sodium dodecyl sulfate (SDS), 10% dextran sulphate, and 100 /xg/ml heat-denatured salmon sperm DNA for 6-12 hours at 42° C. Hybridization was performed in the same solution with the addition of 5 X 103 cpm/ml 32P-labeled probe for 24-36 hours at 42° C. Probes were prepared with the random-priming procedures. Membranes were washed twice at 42° C c-fos with 2 X SSC (saline sodium citrate) buffer and 0.1% SDS, twice at 42° C with 0.2 x SSC and 0.1% SDS, air dried, and exposed to x-ray film (Kodak XAR-5) for 24 hours or 5 days with an intensifying screen at - 70° C. Relative amounts of c-onc expression were determined by a densitometric scanner. Results RNA Blot Hybridization Analysis of c-onc in Pressure-Overloaded Cardiac Hypertrophy Figure 1 shows Northern blot analysis of three oncogenes, c-fos, c-myc, and c-Ha-ras, which were expressed at appreciable levels in rat hearts because of pressure overload. Since in the normal adult hearts, these oncogenes were either slightly or not expressed, the sample that was pressure-overloaded for 8 hours is used in the figure. The expression of the other five conc were not detected in this study. Figure 2 shows the expression patterns of these three genes during pressure overload. Sham operation did not change the expression levels of these genes. The increase of the expression of c-fos-related sequences was recognized as early as 30 minutes after operation. Two peaks were c-myc c-Ha-ras Z8S- FIGURE 1. RNA blot hybridization analysis of three c-onc genes in rat hearts. Poty-A + RNA (3 fig) of the rat heart, which was pressure-overloaded for 8 hours, was separated on 1.2% agarose gel, blotted onto a nylon membrane, and hybridized to a random-priming probe (v-fos, c-myc, and v-Ha-ras). The autoradiograms were exposed for 24 hours with intensifying screen at — 7CP C. Size of rRNA is indicated as marker. 18S- k. Komuro et al Proto-oncogenes in the Growth of Hearts 1077 sham 0° 30' 2° 4° 8° 12° 24° 48 8 C- fosk c-myc c-Ha-ras Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 FIGURE 2. Expression of c-onc transcripts in rat hearts at various times after aortic constriction. Aorta of male Wistar rats was constricted with a hemoclip. Rats were killed at indicated times after operation, and RNA was extractedfrom hearts. Poly-A + RNA (3 pg) was separated on 1.2% agarose gel, blotted onto a nylon membrane, and hybridized to three oncogene probes. Autoradiograms were exposedfor 5 days with intensifying screen at - 70° C. Data on the sham-operated control taken 8 hours after operation were also demonstrated. observed, one at 30 minutes and one at 8 hours after operation. By densitometric scanning, the more prominent peak at 8 hours after operation showed an 8- to 10-fold increase over the level of the preoperative control (Figure 3). The level of the expression was gradually decreased thereafter to the baseline at 48 hours. The expression pattern of c-myc was similar to that of c-fos. Its expression was detectable by 2 hours, peaked at 8 hours, and decreased to baseline at 48 hours after operation. Comparison of relative levels of the expression by densitometric scanning of the autoradiograms showed an 8- to 10-fold increase in the c-myc-related message at 8 hours compared with 2 hours after operation. Some c-Ha-ras-related sequences were expressed in sham-operated hearts and increased gradually by pressure overload. Densitometric scanning revealed a threefold to fivefold increase of c-Ha-ras-related sequences at 48 hours, as compared with the preoperative controls (Figure 3). c-fos c-myc RNA Blot Hybridization Analysis of c-onc During Development of Rat Heart Three of the same genes were also detected. Sequences that were c-mycrelatedwere detectable in the fetal period but disappeared soon after birth. Relative levels of the expression determined by densitometric scanning of the autoradiograms were almost the same throughout the embryonic stage. In contrast, embryonic hearts showed no detectable c-fos transcripts. An increase of the expression of the sequences related to the c-fos gene was detected at the neonatal period and a gradual increase continued and peaked in 200-day-old adults. The expression of c-Ha-ras-related sequences was detected throughout the developmental stage, and its level was not changed among three stages. Discussion Evidence has accumulated that normal cellular proliferation is controlled by the interaction of several c-Ha-ras % FIGURE 3. Relative amounts of c-onc expression in pressure-overloaded cardiac hypertrophy. Relative amounts of c-onc expression were determined by soft-laser density scanning of the RNA blot autoradiograms shown in Figure 2. Relative OD was plotted against time after aortic constriction, and values were expressed as percentage to the highest level of expression of a given c-onc. Mean ± SEM values from three separate experiments are shown. 100 so II'30" Z* 4' 8' I f ' 2 4 * 4 8 ' 7 2 ' V M " f 4' 8' 12*24'48^2*Y 30' ?' 4* 8' 12'24*48*72" 1078 Circulation Research Vol 62, No 6, June 1988 Neo. Fetus 12d Adult 40d 200(1 15d 18d c-fos c-myc Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 c-Ha-ras • • • • • FIGURE 4. Expression of c-onc transcripts in the heart during developmental stage. Hearts of 12-, 15-, and 18-day-old embryos (Fetus 12d, 15d, and 18d), 5-day-old neonates (Neo.), and 40- and 200-day-old adults (Adult 40d and 200d) were examined. Procedures were described in Figure 2. classes of proto-oncogenes and that changes in the expression of controlling elements of normal proliferation result in the uncontrolled cellular growth. In the present study, we have examined the relation between cardiac growth and proto-oncogene expression in rat hearts. Among the eight oncogenes investigated, three oncogenes were expressed at measurable levels. An increase of c-fos and c-myc was detected at 30 minutes and 2 hours, respectively, and the levels peaked at 8 hours and returned to baseline by 48 hours after aortic constriction. The expression of c-Ha-ras, however, was detected from the preoperative hearts and increased gradually by pressure overload. During the course of heart development, the expression of c-fos was detected after birth and increased gradually, whereas that of c-myc was detected only in the fetal stage. The level of c-Ha-ras was not changed throughout the development. The c-fos is the cellular homologue of the oncogene of two mouse osteosarcoma viruses, FBJ-MSV and FBR-MSV.13 All fos genes encode nuclear proteins and show a complex pattern of tissue-, cell type-, and stage-specific expression,14 suggesting a correlation with cellular differentiation and proliferation. The increase of c-fos was detected as early as 30 minutes, peaked at 8 hours, and returned to the uninduced level by 48 hours after aortic constriction. In the pressureoverloaded cardiac myocardium, not only is total protein content increased, but also different types of proteins are synthesized. For instance, the isozymic transition of myosin heavy chains was induced by 24 hours after aortic constriction (data not shown), and in the other contractile proteins, actin or tropomyosin, expression of fetal isoforms was reported in the hypertrophic, hearts. Therefore, the proteins, such as c-fos, that were increased in the early stage of pressure overload appear to play an important role in cardiac hypertrophy. During development, the level of c-fos expression was very low in the fetal periods and only gradually increased. Although the role of c-fos in the heart is unknown, its pattern of expression is distinctive and of interest. Further study is necessary to clarify the role of c-fos with regard to proliferation and differentiation processes during cardiac hypertrophy and aging. The oncogene c-myc also encodes nuclear protein, is expressed in relation to the cell cycle,15 and may play a role in cellular proliferation.16 Recently, the expression of c-myc was reported to be increased in cultured cardiac myocytes and in induced hypertrophy by ar adrenergic agents.4 In the present study, the c-myc gene was also expressed in pressure-overloaded hearts in vivo. The c-myc gene was expressed in the fetal heart, which is in the proliferative period. Cardiac myocytes are terminally differentiated and cannot divide after birth.17 The c-myc gene, which was expressed only in the fetal heart in the physiological state, was reintroduced in adult heart by pressure overload, suggesting that the cell hypertrophy induced by pressure overload may share a common mechanistic pathway with cell proliferation and that enhanced expression of the c-myc gene may be related to both cardiac cell division and cell hypertrophy. The Ha-ras genes encode 21-kDa proteins that appear to be involved in the control of cellular growth and differentiation,18 and mutations affecting ras and Komuro et al Proto-oncogenes in the Growth of Hearts overproduction of normal 21-kDa proteins can induce a transformed phenotype.19 Recently, 21-kDa proteins were reported to affect both the phosphatidylinositol4,5-bisphosphate breakdown pathway and the level of inositol-l,4,5-trisphosphate that would be elevated in ras-transformed cells.20 In cultured cardiac myocytes, a,-adrenergic agonists have been reported to induce hypertrophy through the phosphoinositide/protein kinase C pathway.4 In this study, mRNA encoding of the ras gene was highly expressed in the pressure-overloaded heart. These results and observations suggest that enhanced expression of the ras gene might be associated with cardiac hypertrophy. Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 In cardiac hypertrophy, c-fos and c-myc genes were expressed from as early as 30 minutes and 2 hours after pressure overload, respectively. Morkin and Ashford17 reported that there was little change in DNA synthesis in the interstitial cells of pressure-overloaded hypertrophic hearts until the 2nd postoperative day. Furthermore, the c-myc gene was recently demonstrated to be expressed in cultured cardiac myocytes in the induction of hypertrophy.4 Although oncogenes were reported to be expressed in fibroblasts or other nonmuscle cells in the proliferative period, together with these observations, it is possible to speculate that these cellular oncogenes were expressed in cardiac myocytes. But further investigation is needed to know the cellular origin of these changes and the relation between cellular oncogenes and cardiac hypertrophy. Acknowledgments We are indebted to Drs. Takao Sekiya and Yoshinori Murakami, National Cancer Center, Tokyo, for providing human c-myc probes. We would like to thank Miss Kazue Minamisako for her excellent technical assistance. 1. 2. 3. 4. References Bishop JM: Cellular oncogenes andretroviruses.Ann Rev Biochem 1983;52:301-354 Doolittle RF, Hunkapiller MW, Devare SG, Robbins KC, Aaronson SA, Antoniades HN: Simian sarcoma virus one gene, v-sis, is derived from the gene (or genes) encoding a plateletderived growth factor. Science 1983;221:275-277 Downward J, Yarden Y, Mayes E, Scrace G, Totty N, Stockwell P, Ulrich A, Schlessinger J, Waterfield MD: Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature 1984;307:521-527 Starksen NF, Simpson PC, Bishopric N, Coughlin SR, Lee WMF, Escobedo JA, Williams LT: Cardiac myocyte hyper- 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 1079 trophy is associated with c-myc protooncogene expression. 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J Virol 1980;36:575-585 Robbins KC, Devare SG, Aaronson SA: Molecular cloning of integrated simian sarcoma virus: Genome organization of infectious DNA clones. Proc Natl Acad Sci USA 1981;78:29182922 Bergmann DG, Souza LM, Baluda MA: Vertebrate DNAs contain nucleotide sequences related to the transforming gene of avian myeloblastosis virus. J Virol 1981;40:450^155 Delorbe WJ, Luciw PA, Goodman HM, Varmus HE, Bishop JM: Molecular cloning and characterization of avian sarcoma virus circular DNA molecules. J Virol 1980;36:5O-61 Curran T, Teich NM: Candidate product of the FBJ murine osteosarcoma virus oncogene: Characterization of a 55,000dalton phosphoprotein. / Virol 1982;42:114-122 Muller R, Muller D, Guilbert L: Differential expression of c-fos in hematopoietic cells: Correlation with differentiation of monomyelocytic cells in vitro. EMBO J 1984;3:1887-1890 Campici J, Gray HE, Pardee AB, Dean M, Sonenshein GE: Cell-cycle control of c-myc but not c-ras expression is lost following chemical transformation. Cell 1984;36:241-247 Makino R, Hayashi K, Sugimura T: c-myc Transcript is induced in rat liver at a very early stage of regeneration or by cycloheximide treatment. Nature 1984;310:697-698 Morkin E, Ashford T: Myocardial DNA synthesis in experimental cardiac hypertrophy. Am J Physiol 1968;215:14O91413 Mulcahy LS, Smith MR, Stacey DW: Requirement for ras proto-oncogene function during serum-stimulated growth of NIH 3T3 cells. Nature 1985;313:241-243 Chang EH, Furth ME, Scolnick EM, Lowy DR: Tumorigenic transformation of mammalian cells induced by a normal human gene homologous to the oncogene of Harvey murine sarcoma virus. Nature 1982;297:479^t83 Fleischman LF, Chahwala SB, Cantley L: Ras-transformed cells: Altered levels of phosphatidylinositoM,5-bisphosphate and catabolites. Science 1986;231:407^10 KEY WORDS • cardiac hypertrophy • cellular oncogenes cardiac development Expression of cellular oncogenes in the myocardium during the developmental stage and pressure-overloaded hypertrophy of the rat heart. I Komuro, M Kurabayashi, F Takaku and Y Yazaki Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 Circ Res. 1988;62:1075-1079 doi: 10.1161/01.RES.62.6.1075 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1988 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/62/6/1075 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/