WO1992013066A1

WO1992013066A1 - Mammalian cardiac myocyte cell line

Info

Publication number: WO1992013066A1
Application number: PCT/US1992/000559
Authority: WO
Inventors: Gary L. Engelmann
Original assignee: Loyola University Of Chicago
Priority date: 1991-01-24
Filing date: 1992-01-23
Publication date: 1992-08-06

Abstract

This invention provides stable, proliferative cardiac myocyte cell lines. The mammalian cardiac myocyte cell lines are established by the viral transformation of fetal cardiac myocyte cells to provide growth factor independent cells which retain desired morphological, biochemical and molecular characteristics of fetal cardiac myocyte cells.

Description

Mammalian Cardiac Mvocvte Cell Line

Background of the Invention

The present invention relates generally to stable, proliferative mammalian cardiac myocyte cell lines and specifically to a stable, proliferative homogeneous population of transformed cardiac myocyte cells produced by retroviral infection of fetal cardiac myocytes where the transformed cells exhibit morphological, biochemical and molecular characteristics associated with cardiac cells.

The heart is one of the first recognizable organs to develop in mammalian embryos. The ventricular myocyte cells or cardiomyocyte cells are a particularly interesting type of cell for a number of reasons. First, the initial growth and maturation of cardiomyocyte cells is critical to the developing embryo because of the need to accommodate the metabolic demands of the growing fetus. Second, shortly after birth cardiomyocyte cells stop typical cell replication, undergo unique processes of binucleation and nuclear polyploidation and then enter a life-long stage of cellular hypertrophy of a finite population of ventricular muscle cells. Third, cardiovascular disfunctions may be a direct result of morphogenetic heart abnormalities occurring during critical stages of fetal development.

Embryonic and fetal heart development represent the most critical periods of cardiac morphogenesis and growth. Aberrations and malformations that occur during this period of development may have life threatening consequences. This is particularly true for cardiovascular malformations that affect the heart and its great vessels since their functional integrity is essential for both continuing embryonic growth and for sustaining extrauterine life. Although the embryonic myocardium is a contrastile mass generating systemic circulation and fetal-placental exchange, its increase in size and function during embryonic and fetal stages of growth represents a uniquely brief period of rapid cardiomyocyte proliferation and development, all the while maintaining the retention of contractile activity. The end result is that the heart of a full term fetus contains a near complete complement of ventricular muscle cells, i.e., the total number of muscle cells that comprise the heart, that become terminally post mitotic shortly after birth. As a consequence cardiomyocyte maturation and increases in tissue mass of the heart during postnatal development are primarily the result of hypertrophic myocyte growth. Therefore, embryonic and fetal heart growth is exquisitely regulated by mechanisms which remain to be determined and which mechanisms may result in the development of a malformed or a normal fetal heart.

Many congenital abnormalities of the heart result from an interaction of genetically programmed development, environmental or maternal influences, and the heart's own hormonal or growth factor milieu. Aberrations in any or all of these factors may ultimately influence the development of the heart in the fetus. The determination, differentiation and proliferative growth of in vivo cardiac myocyte cells is limited to embryonic and fetal stages of mammalian development. Therefore, analysis and characterization of the regulatory factors involved in heart development are essential to understanding both congenital cardiovascular abnormalities and the long-term implications associated with these factors. The long-term implications are particularly relevant because maturation and hypertrophic growth of the myocyte occurs primarily in the postnatal and adult organism after the myocyte has stopped proliferating and further repair and restoration of structure and function by formation of new myocytes is no longer possible. In sum, a greater understanding of the specific cellular factors regulating cardiomyocyte cell development, growth and maturation provides opportunities to effectively identify the presence of and then intervene to correct embryonic and fetal abnormalities which ultimately may be associated with specific heart abnormalities.

The opportunity to investigate and understand the growth and differentiation of cardiomyocyte cells requires stable, proliferative cardiomyocyte cells that retain the unique morphological, biochemical and molecular characteristics of fetal cardiomyocyte cells. Unfortunately, the useful investigatory life time of isolated fetal cardiomyocyte cells is very short and attempts to develop regenerating cardiomyocyte cell dines have been unsuccessful.

Thus, a need exists for a suitable proliferative cardiomyocyte cell line which maybe used in improved analytical procedures for cardiovascular pharmacological agents, in methods for detecting cardiac abnormalities and which allows research into the nature of the developing mammalian heart.

Summary of the Invention

The present invention provides a method for obtaining and maintaining proliferative cardiomyocyte cells which retain the desired morphological, biochemical and molecular characteristics of embryonic or fetal cardiomyocyte cells. Preferably, a transformed mammalian cardiomyocyte cell of the present invention provides a stable, growth factor independent cardiomyocyte cell which retains the cellular capability of expressing the proteins encoded by cardiac embryonic or fetal genes including, but not limited to, Troponin C, Troponin Z, Myosin heavy chain-beta, alpha-cardiac Actin and Early Growth Response Gene-I. Three particularly preferred cardiomyocyte cell lines of this invention are on deposit with the American Type Culture Collection (ATCC) in Rockville, Maryland and may be identified by the deposit numbers assigned to CLEM deposited under Accession No. 10675 on January 24, 1991, ELEM deposited under Accession No. 10673 on January 24, 1991 and BWEM deposited under Accession No. 10674 on January 24, 1991.

The present invention also provides a process for producing a homogeneous population of stable, growth factor independent cardiomyocyte cells by employing the steps of transfecting mammalian fetal cardiomyocyte cells with a recombinant retrovirus to establish growth factor independent proliferation in the cardiomyocyte cells, identifying the transformed cells from untransformed cells by serial passage under selective growth conditions, and isolating a homogeneous population of stable, growth factor independent cardiomyocyte cells. Specific retroviruses which are particularly useful to practice this invention include recombinant viruses containing the v-myc or the v-H-ras oncogenes. Two viruses, Maloney urine leukemia virus containing the v-myc oncogene and Harvey sarcoma virus containing the v-H-ras oncogene, are especially preferred when used in conjunction with the helper viruses, lOA-1 virus or 4070 amphotrophic virus, respectively.

Further, the present invention provides an analytical method to identify or characterize a specific pharmacological agent or family of agents (such as alpha-, or beta-adrenergic blockers, possible channel blockers of the potassium or calcium family, or digitalis-like compounds) which have potential therapeutic specificity to the cardiovascular system and particularly to the heart muscle. The present method includes the steps of contacting a stable proliferative mammalian cardiomyocyte cell, a cellular component (such as the cell membrane) , or a cellular product thereof with an aliquot a specific cardiovascular agent, allowing the agent and the cell, cellular component or cellular product thereof to interact and generating a detectable response wherein the response signifies the interaction and/or therapeutic activity of the specific cardiovascular agent. Preferably, the detectable response is generated by a specific change in receptor occupancy, post-receptor responsiveness, or a specific change in the expression of a gene product to produce encoded proteins by the cardiac cell as the result of contact with the specific cardiovascular pharmacological agent.

Further, the present invention provides additional applications by providing for the production and isolation of cellular derived products having broad applications to cells and organ systems in addition to the cardiovascular system. These products include but are not limited to, products atrial naturietic factor, nerve growth factor, digitalis-like compounds, endothelin, and growth factor compounds specific for ventricular myocyte cells or for other types of cells.

Brief Description of the Drawings Figures 1A-1E are photographic reproductions of agarose gels which compare the total extracted RNA of cell lines of the present invention with the RNAs of a primary cell culture of fetal and neonatal cardiomyocytes. Figure 2 is a photographic reproduction of a polyacrylamide gel which compares the cellular constituents of cell lines of the present invention with the cellular constituents of a primary culture of fetal cardiomyocytes. Figures 3A and 3B are photographic reproductions of agarose gels which compare enzymatically digested DNA of cell lines of the present invention with similarly digested DNA of a rat heart endothelial cell line.

Detailed Description The present invention provides a proliferative, immortal mammalian cardiomyocyte cell where the cardiomyocyte cell is transformed by retroviral infection so as to be growth factor independent but the cell still retains important morphological, biochemical and molecular characteristics of uninfected cardiomyocyte cell. The transfection process provides a cell line that satisfies a need in the art for a stable, proliferative growth factor independent cell line which may be used to investigate a variety of unresolved questions that have been difficult to study because of the lack of an immortal cardiomyocyte cell.

As used in this patent, the following terms or phrases are intended to have the following meanings:

"Growth factor independent" means a cell or cell line, preferably a mammalian cell or cell line, which will unceasingly proliferate when cultured in the presence of a suitable growth medium under suitable conditions; minimally, such cells exhibit stable proliferation for from at least ten to preferably twenty serial passages,

"Morphological properties of embryonic cardiomyocyte cells" means transformed cells retaining biochemical and molecular characteristics of cardiomyocytes including the expression of polypeptides or polypeptide fragments by the transcription and subsequent translation of known cardiomyocyte genes provided that the properties of bi-or poly-nucleation and the property of spontaneous or responsitory contractile behavior are not necessary characteristics of the transformed cells, and

"Cardiac specific protein" means one or more proteins found in mammalian cardiac tissue and expressed by the translation and subsequent transcription of naturally occurring cardiomyocyte genes, including, but not limited to, such genes as tropinin C, tropinin I, myosin heavy chain-beta, alpha-cardiac actin or early growth response-I gene. To date no general procedures are available for predictably transforming a variety of cell types, particularly cardiomyocyte cells.

The determination and identification of the cellular causes of solid tumors or acute neoplasms have shown that certain viral oncogenes may transform certain normal, regulated cells into unregulated, proliferating cells. Specifically, certain retroviruses which carry transforming oncogenes have been found to induce specific diseases caused by a change from regulated to unregulated cell growth in infected animals. For example, it is known that avian retrovirus MC29 induces a myeloid neoplasm in chickens. Similarly, infection by the avian Rous sarcoma virus is known to be the cause of sarcomas in chickens. These well characterized and extensively studied avian transforming retroviruses provide a defined source of transforming retroviruses.

Recombinant DNA techniques have exploited the transforming capability of avian retroviruses by inserting the well characterized oncogenes of avian transforming retroviruses into the genome of some mammalian retroviruses. For example, modified mammalian retroviruses have been used to eliminate the growth factor dependency of several specific types of cells. Brightman et al., J. of Virology. 60:68-81 (1986). Fetal liver cells have been transformed from growth factor dependent to growth factor independent cells by infection with a recombinant retrovirus, Birchenall-Roberts et al., Oncogene. 4:731-735 (1989). Other cell lines which have also been transformed to growth factor independent cells include immune mast cells infected by Harvey sarcoma virus and murine NIH 3T3 fibroblast cells infected by a murine leukemia virus containing a recombinant modified avian oncogene, Rein et al. , Molecular and Cellular Biology. 5:2257-2264 (1985).

In the case of liver cells, the investigators were not directly attempting to establish a monocyte cell line. Rather, they were attempting to establish a fetal liver cell line because liver hepatocytes do not proliferate in the adult, and fetal tissue is the primary source of actively dividing cells. Because of the prevalence of the hematopoietic stem cell types as constituents of the fetal livers, these cell types were infected during the random nature of the immortalization process. Immortal myocyte cell lines are similar because fetal myocytes are also actively proliferative only during the time period of fetal development. Although the processes are related, the process of infectivity and subsequent cell line formation is a random process that generated only three cell lines from many millions of similarity treated cells.

In the case of liver cells, the investigators were not able to conclusively establish why the specific fetal liver cells used were transformed but felt that the undifferentiated nature of the fetal cells was an important factor. Specifically, the failure of earlier attempts to transform bone marrow cells or mesenchymal cells may have been a result of the type of cells used because it has been postulated that undifferentiated precursor cells might be expected to have the greatest proliferative capacity.

In addition, the transformation of mortal cells to immortal cells associated with viral oncogene infection may be cell type specific. For example, Rein et al., Mol. Cell. Biol.. 5:2257-2264 (1985) developed an immortal mast cell but other apparently infected cells such as erythroid progenitors did not develop the capability of long term growth.

These studies indicate it would not be predicted that embryonic cardiomyocyte cells would be successful candidates for immortalization associated with growth factor independent proliferation, a major reason being the potential variability of many factors which have to he satisfied to ultimately allow for growth factor independent proliferation. The retroviruses used to transform and immortalize fetal cardiomyocytes according to this invention and the use of retroviruses to infect and ultimately transform fetal cell lines are described by Birchenall-Roberts et al., Oncogene, 4:731-735 (1989) . Briefly, the procedure to transform the fetal cardiomyocyte cells involves infection of the cells with a known virus stock such as Maloney murine leukemia virus (M-MuLV) pseudotyped with lOA-1 helper virus or Harvey sarcoma virus pseudotyped with 4070 amphotrophic helper virus. After infection, fresh medium is added to the infected cell cultures and the cells are maintained by the replenishment of medium about once a week for the first five weeks and once every three days thereafter or as may be required.

According to one aspect of the present invention, fetal ventricles are obtained from day-16 gestation rat pups and cardiomyocyte cells are isolated by collagenase digestion procedures. A population of desired cells is isolated, placed in culture, and at the end of the culture period (about 48-96 hours) the cells may be identified and characterized by immunofluorescent microscopy utilizing antisera directed against specific cardiac proteins such as Troponin C, Troponin I and myosin. All desired cultures may then be initiated overnight with Ventrex PC-I serum-free medium on collagen coated culture dishes. Cultures are then preferably maintained under serum-free conditions using media mixture containing a 1:1:1 ratio of Ventrex PC- l:Dulbecco's MEM with glucose:Ham's F12 (growth medium) . This media is preferred because it maintains cardiomyocyte protein synthetic rates at similar levels to that found in vivo and minimizes the proliferative advantage of non-muscle cells in these cultures.

The initiated and isolated fetal cardiomyocytes are isolated and cultured in growth medium containing IGF-I and acidic-FGF for about 36-48 hours post-isolation to ensure that a majority of the cells are actively traversing the cell cycle. Infections are performed according to the procedures for fetal liver cells with either v-myc in a construct of Maloney murine leukemia virus (M-MuLV) pseudotyped with lOA-1 helper virus or v-ras in a construct of Harvey sarcoma virus pseudotyped with 10A-I helper virus. These retroviruses were received from F.W. Ruscetti, Laboratory of Molecular Immunoregulation, NCI, Frederick, Maryland. Preferably, replication-defective viral constructs are used to transform the myocyte cells because these replication defective recombinant viruses successfully transform primary cultures of fetal cells to yield monocyte cell lines.

For infection, the viruses are preferably added to about 1 x 10⁶ cells in about 1 ml of growth medium containing about 25 μg/ml POLYBRENE (Aldrich Chemical Company, Milwaukee, WI) . The virus stocks preferably have titers of about 2 x 10⁴ focus forming units/ml (as determined on fibroblast cell lines) . The viral infection typically proceeds (for less than about 1 hour) at 37°C because of the limited viability of the viruses in culture medium. After the infection period, the viral-containing growth medium is removed by aspiration and fresh growth medium is added.

Following infection, culture growth medium is typically changed about twice a week during the first two weeks of culture and about twice a week thereafter. All infected cells are maintained for 2-3 months and during this time the non-infected cardiomyocyte cell population is generally lost. Infection of the cells with either the helper virus or the defective retrovirus itself has been shown to produce no transformed cell lines or functional viruses. Established primary cultures of mortal, short-lived fetal cardiomyocytes or cell lines may be used as comparative cells and the steady state transcript levels of the various proliferation, differentiation and growth factor receptor genes may be readily characterized by known procedures such as Northern blot hybridizations. These established blots may then be compared to similar blots of cardiomyocyte cells of the present invention. For example, Northern blot hybridizations may be performed with total or poly-adenylated RNA after electrophoresis, capillary transfer, and ultraviolet cross-linking to a solid support such as nitrocellulose. These blot hybridizations provide quantitative analysis of gene expression levels using vacuum loaded, serially diluted, and denatured RNA samples.

Specifically, typical hybridizations may be performed with the ³²P-labeled cDNA inserts or oligonucleotides such as: a-FGF cDNA, FGF receptor cDNA, IGF-I and II cDNAs, IGF-I and IGF-II receptor cDNAs, ornithine decarboxylase cDNA, thymidine kinase cDNA, early growth response gene-I cDNA; growth arrest specific gene (gas-I) cDNA, TGF-jSl and -β₂ cDNAs, and cDNAs, α-cardiac or /3-actin oligonucleotides, total myosin heavy chain cDNA, α- and /3-myosin heavy chain oligonucleotides, creatine kinase m and b cDNAs, troponin C cDNA, or troponin I CDNA. All cDNAs may be used as inserts after electrophoretic separation of restriction fragments in low melting agarose and labeled by random primer extension according to well known protocols. General hybridization conditions for cDNAs typically are: 50% formamide, 6X SSC, 0.1M Na₂PO_ή, 0.1% Na pyrophosphate, 0.1% SDS, IX Denharts solution, 100 μg/ml E.coli DNA and 10 mM vanadyl ribonucleoside complex heated to about 40-42°C for about 16-20 hours. Oligonucleotide probes are generally hybridized in 6X SSC, IX Denhardt's, 0.05% Na pyrophosphate, 0.1% SDS overnight, and 200 μg/ml yeast tRNA at 50-55°C.

Nitrocellulose filters probed with cDNAs are washed two times in 2X SSC with 0.1% SOS at 50°C for about thirty minutes and are finally washed in 0.1-1.0 X SCC with 0.1% SDS at about 55-60°C for 15 minutes. Nitrocellulose filters probed with oligonucleotides are washed in 6X SSC-0.1% SDS at room temperature for about 60 minutes, then for 30 minutes at 37°C and are finally washed at about 50-55°C for 30 minutes. All nitrocellulose filters may be exposed to XRA-5 film with an intensifier screen at -70°C for 1-4 days. For comparative purposes, primary cardiomyocytes or cell lines are biosynthetically labeled on days 1, 2, 3, 4 and 5 in culture in growth medium supplemented with ³H-leucine (100,000 dpm/nmol) . Cells are harvested by SDS lysis and lysates are divided for determination of myosin subunit content by SDS-PAGE and staining with ¹²⁵I-Coomassie brilliant blue and determination of DNA content. The remaining cell lysate is used for determination of leucine specific radioactivity in individual contractile protein subunits (MHC, MLC_lv, and MPLC_2v) by SDS-PAGE, acid hydrolysis of gel bands containing radiolabeled subunits, and double label radioassay of protein hydrolysates with 14C-dansyl chloride.

Cell line DNA synthetic response and resulting DNA content and cell numbers may be readily determined. Briefly, DNA labeling involves addition of 3H-thymidine (2 μCi/ml) for the final 4 hours of culture and subsequent perchloric acid precipitation of all macromolecules in the washed cell layer. The amount of DNA may be determined by known fluorometric methods. These studies are preferably done after about 24-96 hours of culture in the presence of reduced serum v-myc infected cell lines or 5-15% serum for the v-ras infected cell line.

The morphological properties of a cardiac myocyte cell of this invention are also important factors in the characterization of the cell line. To compare the morphological features of a primary culture and a cell line of this invention, both types of cell lines are established on Permanox culture dishes (Nunc) , fixed in 1% gluteraldehyde for one hour at 4°C and processed for electron microscopy. Ultrathin sections of the embedded cells may then be examined after sectioning "en face" to the original culture surface. Light microscopy examination of the cultured cell occurs after fixation of the cells by treatment for one hour with paraformaldehyde (4%) . The fixed material may be used for histochemical, immunofluorescent or in situ hybridization studies of peptides, proteins and gene transcripts, respectively. Histochemical analysis generally involved overnight incubation of a culture with primary antisera and subsequent peroxidase localization with AEC as the chromogen. Immunofluorescent studies utilize fixed, permeabilized monolayers that are incubated for 2 hours at 4°C with primary antisera and visualized after secondary, FITC or rhodamine conjugated antibody reaction using fluorescent microscopy. Utilization of the cardiomyocyte cell lines of the present invention provides a unique opportunity to address many cellular and molecular questions associated with heart development that are currently limited to nonhomogeneous primary cultures of fetal or neonatal cardiomyocytes. The cells of the present invention provide a meaningful "tool" to address these fundamental questions of the growth parameters and differentiated properties of these unique cell lines specific to the cardiovascular system. Embryonic and fetal heart development may now be studied at the cellular and molecular level using cells made available by the present invention.

Example

Cell Line Formation From Primary Cultures of Fetal Cardiomyocytes

Serum-free cultures of day-16 gestation fetal cardiomyocytes were prepared, stimulated to proliferate by exogenous IGF-I (20 ng/ml) and a-FGF (50 ng/ml) for 48 hours and then infected with the replicative defective retroviruses and their appropriate helper virus for one hour. The infecting retroviruses used and the resulting cells lines that were generated are identified in Table 1. POLYBRENE (20 mg/ml, Aldrich Chemical Company, Milwaukee, WI) was added to the media with the replicative defective viruses to facilitate the attachment and infection of the cells. After one hour, the infecting medium was removed and fresh medium containing 10% fetal calf serum was added. The infected cells continue to proliferate and were serially passed 3-5 times during which time several obvious changes occurred in both the infected and control cultures. Control cultures were soon overgrown with noncontractile, fibroblastic cells that were not retained for long-term analysis. The infected, proliferative cardiomyocyte cultures were found to be non-contractile, yet highly homogeneous in their appearance, particularly the v-myc transfected cells. All subsequent cell passages were done at large dilutions so as to yield highly homogeneous populations of cells that were further characterized. The results of the transfections were identified as the cardiomyocyte cell lines listed in Table 1.

Table 1

RETROVIRAL VECTORS CELL LINE PRODUCED M-MuLV containing v-myc BWEM Accession pseudotyped with 10A-I NO. 10674 helper virus

M-MuLV containing v-myc CLEM Accession pseudotyped with 4070 No. 10675 amphotrophic helper virus

Harvey Sarcoma with v-ras ELEM Accession pseudotyped with 4070 No. 10673 amphotrophic helper virus

The v-myc cell lines were found to be highly proliferative and were propagated in low serum (0.5-1.0% fetal calf serum), whereas the v-ras cell line required much higher concentrations

(10-20% fetal calf serum) to maintain the cells in a proliferative state.

Ultrastructural examinations of the three cell lines were compared to that of a primary culture of day 16 fetal cardiomyocytes. Using electron microscopic examination (about 7,250x), the morphology of the primary cultures showed cellular contractile apparatus which was generally well organized, yet areas of disorganized contractile filaments were also seen. In comparison, the morphology of all three cell lines (CLEM Accession No. 10675, BWEM Accession No. 10674, ELEM Accession No. 10673) showed a less differentiated, more embryonic type of muscle cell which was suggested by the poorly organized cytoskeletal myof ilaments located in their cytoplasm. The general morphology of the cell lines accurately reflects the noncontractile nature of the cells. Nevertheless, cytoplasmic evidence of some immature, sarcomere-like structures were often seen.

The cardiac lineages of these cell lines CLEM Accession No. 10675 (C) , BWEM Accession No. 10674 (A) and ELEM Accession No. 10673 (HE) , are illustrated in Figure 1. The expression of a series of genes that have a strong association with the myocardium are identifiable by examining the total RNA extracted from the three cell lines (labeled as A, C and HE) and comparing this total RNA obtained to the total RNA obtained from primary cultures of day-16 fetal (F) and 2-day neonatal cardiomyocytes (N) . Rat liver (RL or L) , total RNA was a negative control (double bars on left indicate 285 and 185 rRNA migration) . The expression of the Troponin C transcript (0.8 kb) is highly selective for cardiac muscle and is found in both primary cultures and the three cell lines. Another transcript (-3.7 kb) of interest and associated with the myocardium is Egr-1 (Early growth response-I gene) which is found in all three cell lines at levels which equal or exceed that of the primary cultures. The other identifiable myocardial lineage transcript is α-cardiac actin (α-CA, -1.8 kb) which is found at a slightly reduced level in the two vmyc cell lines compared to the levels observed in the primary cultures. There was little or no expression of α-CA in the ELEM Accession No. 10673 v-H-ras cell line. Significant cross-hybridization to jS-actin (-2.4 kb) by the α-CA oligonucleotide (56-mer) was found in all F & N myocyte RNA and cell line RNAs.

Expression of a -2.5 kb TG¥-β₁ transcript is very high in ELEM Accession No. 10673 v-ras cell line RNA. The expression of v-myc is at very high levels in only the CLEM Accession No. 10675 and BWEM Accession No. 10674 cell lines. High expression of v-ras is observed only in the ELEM Accession No. 10673 cell lines. Other genes of interest and relationship to the myocardium have been detected in the three cell lines, but at much reduced levels of expression, including jS-MHC, ANF, and Troponin I. Biosynthetic labeling of the cell lines indicated the production of many of the same cellular constituents seen in primary cultures of fetal cardiomyocytes. As illustrated in Fig. 2, sized, radiolabeled 14^c "rainbow" markers (Std. 200-21 kd) are shown as well as the most prominent bands of those proteins associated with the contractile apparatus, such as actin (42 kd) and a faint band at the myosin level (200 kd) in the CLEM Accession No. 10675 and BWEM Accession No. 10674 cell lines and are barely detected from the ELEM Accession No. 10673 cell line. The gel illustrated in Fig. 2 was loaded with equal amounts of radioactivity, not proteins, which may explain the low myosin signal in the cell lines. Nevertheless, this gel demonstrates that the cell lines are making many of the same proteins detected in the primary fetal cardiomyocytes.

Additional evidence of the integration of the oncogenes into the genomes of the cell lines of this invention is provided by Southern blots of Hind III genomic DNA digests which are illustrated in Figures 3A and 3B. The migration of DNA standards of varying sizes (kb) are shown. Only a single, unique integration site (approx. 1 kb) is evident in the v-H-ras infected ELEM Accession No. 10673 cell line, yet the probe used hybridized to C-ras in all four cell lines (-2.0 kb) , CLEM Accession No. 10675; BWEM Accession No. 10674; and rat heart endothelial cell line). Multiple sites (9.4 and ~15 kb) of v-myc integration were observed in both the CLEM Accession No. 10675 and BWEM Accession No. 10674 cell lines but no cross-hybridization to c-myc (usually found at -4.4 kb) was seen in either of the v-myc cell lines or in the ELEM Accession No. 10673 and rat endothelial cell lines. The foregoing detailed description and examples have been given for clearness of understanding only, and no unnecessary limitations of the appended claims should be understood therefrom, as modifications will be obvious to those skilled in the art.

Claims

Claims:

1. A cardiomyocyte cell characterized by stable, growth factor independent cell proliferation, expression of at least one cardiac specific protein, and retention of the morphological characteristics of embryonic cardiomyocyte cells.

2. The cardiomyocyte cell line of claim 1 wherein the cardiac specific proteins are selected from the group of proteins transcribed by the genes Tropinin C, Troponin I, Myosin heavy chain-beta, Alpha-Cardiac Actin and Early Growth Response-I Gene.

3. A cardiomyocyte cell of claim 1 wherein the cell is a mammalian cell.

4. A cardiomyocyte cell of claim 1 wherein the cell is CLEM Accession No. 10675 deposited on January 24, 1991 with the American Type Culture Collection, Rockville, Maryland.

5. A cardiomyocyte cell of claim 1 wherein the cell is BWEM Accession No. 10674 deposited on January 24, 1991 with the American Type Culture Collection, Rockville, Maryland.

6. A cardiomyocyte cell of claim 1 wherein the cell is ELEM Accession No. 10673 deposited on January 24, 1991 with the American Type Culture Collection, Rockville, Maryland.

7. The cardiomyocyte cell of claim 1 wherein the cell contains a nucleic acid sequence capable of expressing a cardiac protein.

8. A process for producing a homogeneous population of stable, growth factor independent cardiomyocyte cells comprising the steps of: i) transfecting a mammalian fetal cardiomyocyte cell with a retrovirus to establish growth factor independent proliferation in the cardiomyocyte cell, ii) identifying the transformed cells from untransformed cells by serial passage under selective growth conditions, and iii) isolating a homogeneous population of stable, growth factor independent cardiomyocyte cells.

9. A cardiomyocyte cell line produced by the process of claim 8.

10. A analytical method to identify a specific cardiovascular agent comprising the steps of: i) contacting an aliquot including the cardiovascular agent with a stable, proliferative cell produced by a cardiomyocyte cell line of claim 1 or a cellular product thereof, and ii) generating a detectable response wherein the response signifies the pharmacological activity of the cardiovascular agent.