RESUMO
Fetal cardiomyocytes isolated from transgenic mice carrying a fusion gene of the alpha-cardiac myosin heavy chain promoter with a beta-galactosidase reporter were examined for their ability to form stable intracardiac grafts. Embryonic day 15 transgenic cardiomyocytes delivered directly into the myocardium of syngeneic hosts formed stable grafts, as identified by nuclear beta-galactosidase activity. Grafted cardiomyocytes were observed as long as 2 months after implantation, the latest date assayed. Intracardiac graft formation did not induce overtly negative effects on the host myocardium and was not associated with chronic immune rejection. Electron microscopy revealed the presence of nascent intercalated disks connecting the engrafted fetal cardiomyocytes and the host myocardium. These results suggest that intracardiac grafting might provide a useful approach for myocardial repair, provided that the grafted cells can contribute to myocardial function.
Assuntos
Transplante de Células , Coração Fetal/citologia , Transplante de Tecido Fetal , Junções Intercelulares/ultraestrutura , Miocárdio/citologia , Animais , Sequência de Bases , Comunicação Celular , Diferenciação Celular , Núcleo Celular/metabolismo , DNA/biossíntese , Primers do DNA , Eletrocardiografia , Junções Comunicantes/fisiologia , Junções Comunicantes/ultraestrutura , Marcadores Genéticos , Coração/fisiologia , Junções Intercelulares/fisiologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Miocárdio/ultraestrutura , beta-Galactosidase/análiseRESUMO
We have assessed the ability of skeletal myoblasts to form long-term, differentiated grafts in ventricular myocardium. C2C12 myoblasts were grafted directly into the heart of syngeneic mice. Viable grafts were observed as long as 3 mo after implantation. Immunohistological analyses revealed the presence of differentiated myotubes that stably expressed the skeletal myosin heavy chain isoform. Thymidine uptake studies indicated that virtually all of the grafted skeletal myocytes were withdrawn from the cell cycle by 14 d after grafting. Graft myocytes exhibited ultrastructural characteristics typical of differentiated myotubes. Graft formation and the associated myocardial remodeling did not induce overt cardiac arrhythmia. This study indicates that the myocardium can serve as a stable platform for skeletal myoblast grafts. The long-term survival, differentiated phenotype, and absence of sustained proliferative activity observed in myoblast grafts raise the possibility that similar grafting approaches may be used to replace diseased myocardium. Furthermore, the genetic tractability of myoblasts could provide a useful means for the local delivery of recombinant molecules to the heart.
Assuntos
Transplante de Coração/patologia , Miocárdio/citologia , Animais , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Coração/fisiologia , Isoenzimas , L-Lactato Desidrogenase/metabolismo , Camundongos , Miocárdio/enzimologiaRESUMO
This study describes a simple approach to generate relatively pure cultures of cardiomyocytes from differentiating murine embryonic stem (ES) cells. A fusion gene consisting of the alpha-cardiac myosin heavy chain promoter and a cDNA encoding aminoglycoside phosphotransferase was stably transfected into pluripotent ES cells. The resulting cell lines were differentiated in vitro and subjected to G418 selection. Immunocytological and ultrastructural analyses demonstrated that the selected cardiomyocyte cultures (> 99% pure) were highly differentiated. G418 selected cardiomyocytes were tested for their ability to form grafts in the hearts of adult dystrophic mice. The fate of the engrafted cells was monitored by antidystrophin immunohistology, as well as by PCR analysis with primers specific for the myosin heavy chain-aminoglycoside phosphotransferase transgene. Both analyses revealed the presence of ES-derived cardiomyocyte grafts for as long as 7 wk after implantation, the latest time point analyzed. These studies indicate that a simple genetic manipulation can be used to select essentially pure cultures of cardiomyocytes from differentiating ES cells. Moreover, the resulting cardiomyocytes are suitable for the formation of intracardiac grafts. This selection approach should be applicable to all ES-derived cell lineages.
Assuntos
Engenharia Genética/métodos , Miocárdio/citologia , Seleção Genética , Células-Tronco/citologia , Transplante de Tecidos/métodos , Animais , Sequência de Bases , Biomarcadores , Diferenciação Celular/genética , Gentamicinas/farmacologia , Canamicina Quinase , Camundongos , Dados de Sequência Molecular , Miocárdio/ultraestrutura , Miosinas/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Regiões Promotoras GenéticasRESUMO
Intracardiac grafts comprised of genetically modified skeletal myoblasts were assessed for their ability to effect long-term delivery of recombinant transforming growth factor-beta (TGF-beta) to the heart. C2C12 myoblasts were stably transfected with a construct comprised of an inducible metallothionein promoter fused to a modified TGF-beta 1 cDNA. When cultured in medium supplemented with zinc sulfate, cells carrying this transgene constitutively secrete active TGF-beta 1. These genetically modified myoblasts were used to produce intracardiac grafts in syngeneic C3Heb/FeJ hosts. Viable grafts were observed as long as three months after implantation, and immunohistological analyses of mice maintained on water supplemented with zinc sulfate revealed the presence of grafted cells which stably expressed TGF-beta 1. Regions of apparent neovascularization, as evidenced by tritiated thymidine incorporation into vascular endothelial cells, were observed in the myocardium which bordered grafts expressing TGF-beta 1. The extent of vascular endothelial cell DNA synthesis could be modulated by altering dietary zinc. Similar effects on the vascular endothelial cells were not seen in mice with grafts comprised of nontransfected cells. This study indicates that genetically modified skeletal myoblast grafts can be used to effect the local, long-term delivery of recombinant molecules to the heart.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Terapia Genética/métodos , Coração/efeitos dos fármacos , Músculo Esquelético/transplante , Fator de Crescimento Transformador beta/farmacologia , Animais , Procedimentos Cirúrgicos Cardíacos , Sistemas de Liberação de Medicamentos , Tratamento Farmacológico/métodos , Metalotioneína/biossíntese , Metalotioneína/genética , Camundongos , Camundongos Endogâmicos C3H , Neovascularização Patológica/induzido quimicamente , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Transplante de Tecidos , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genéticaRESUMO
D-type cyclin/cyclin-dependent kinase (CDK) complexes regulate transit through the restriction point of the cell cycle, and thus are required for the initiation of DNA synthesis. Transgenic mice which overexpress cyclin D1 in the heart were produced to determine if D-type cyclin deregulation would alter myocardial development. Cyclin D1 overexpression resulted in a concomitant increase in CDK4 levels in the adult myocardium, as well as modest increases in proliferating cell nuclear antigen and CDK2 levels. Flow cytometric and morphologic analyses of dispersed cell preparations indicated that the adult transgenic cardiomyocytes had abnormal patterns of multinucleation. Histochemical analyses confirmed a marked increase in number of cardiomyocyte nuclei in sections prepared from the transgenic mice as compared with those from control animals. Tritiated thymidine incorporation analyses revealed sustained cardiomyocyte DNA synthesis in adult transgenic hearts.
Assuntos
Ciclinas/genética , Replicação do DNA/genética , Células Gigantes/patologia , Miocárdio/patologia , Proteínas Oncogênicas/genética , Animais , Ciclina D1 , Citometria de Fluxo , Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Camundongos Transgênicos , Miocárdio/ultraestruturaRESUMO
This report documents the formation of stable fetal cardiomyocyte grafts in the myocardium of dystrophic dogs. Preliminary experiments established that the dystrophin gene product could be used to follow the fate of engrafted cardiomyocytes in dystrophic mdx mice. Importantly, ultrastructural analyses revealed the presence of intercalated discs consisting of fascia adherens, desmosomes, and gap junctions at the donor-host cardiomyocyte border. To determine if isolated cardiomyocytes could form stable intracardiac grafts in a larger species, preparations of dissociated fetal canine cardiomyocytes were delivered into the hearts of adult CXMD (canine X-linked muscular dystrophy) dogs. CXMD dogs, like Duchenne muscular dystrophy patients and mdx mice, fail to express dystrophin in both cardiac and skeletal muscle. Engrafted fetal cardiomyocytes, identified by virtue of dystrophin immunoreactivity, were observed to be tightly juxtaposed with host cardiomyocytes as long as 10 wk after engraftment, the latest date analyzed. Confocal laser scanning microscopy revealed the presence of connexin43, a major constituent of the gap junction, at the donor-host cardiomyocyte border. The presence of intracardiac grafts was not associated with arrhythmogenesis in the CXMD model. These results indicate that fetal cardiomyocyte grafting can successfully augment cardiomyocyte number in larger animals.
Assuntos
Transplante de Células , Transplante de Tecido Fetal , Proteínas de Membrana , Distrofia Muscular Animal/terapia , Miocárdio/citologia , Animais , Proteínas do Citoesqueleto/análise , Cães , Distrofina/análise , Distrofina/genética , Terapia Genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Miocárdio/ultraestrutura , UtrofinaRESUMO
Thymoma is the most common tumor of the anterior-superior mediastinum. We have identified a line of transgenic mice which spontaneously and heritably develop thymomas at a very high penetrance. The available data suggest that thymoma formation in these mice results as a consequence of transgene insertional mutagenesis. Immune histologic analyses indicate that the thymomas are of epithelial cell origin. Survival studies indicate that tumor progression is more aggressive in females as compared to males (73.9 vs 41.7% mortality at 20 weeks of age, respectively). Fluorescent in situ hybridizations have localized the transgene integration site to the F2-G region of mouse chromosome 2. Translocation encompassing the syntenic region in humans has been implicated in lympho-epithelial thymoma. These animals may constitute a useful resource for the identification of gene(s) which participate in thymoma progression, as well as a model system for screening anti-thymoma therapeutic agents.
Assuntos
Timoma/etiologia , Neoplasias do Timo/etiologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Feminino , Complexo Principal de Histocompatibilidade , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Transgênicos , Mutação , Fator de Crescimento Transformador beta/genéticaRESUMO
Cardiomyocytes in the adult mammal retain little or none of their developmental capacity for hyperplastic growth. As a consequence of this differentiated, nonproliferative phenotype, cardiomyocyte loss due to injury or disease is irreversible. Therapeutic intervention in end-stage diseased hearts is currently limited to cardiac transplantation. An increase in cardiomyocyte number in diseased hearts could improve function. Augmentation of the cardiomyocyte population may be achievable by the expression of regulatory proteins in the myocardium, or by intracardiac grafting of exogenous cardiomyocytes.
Assuntos
Coração/fisiologia , Miocárdio/citologia , Regeneração , Animais , Diferenciação Celular , Divisão Celular , Expressão Gênica , Cardiopatias/terapia , Humanos , Hiperplasia , Mamíferos , Miocárdio/metabolismoAssuntos
Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Endotelinas/farmacologia , Mitógenos/farmacologia , Miocárdio/citologia , Animais , Bombesina/farmacologia , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados , DNA/biossíntese , Endotélio Vascular/fisiologia , Mitose , Ploidias , Salamandridae , Tretinoína/farmacologiaRESUMO
Cardiomyocyte DNA synthesis was examined in normal and injured adult mouse hearts. In preliminary studied DNA synthesis was monitored by [3H]thymidine incorporation, followed by autoradiographic analysis of dispersed cell preparations. No synthetic cells were identified when 20,000 ventricular cardiomyocytes from normal adult hearts were examined. A high throughput assay was developed to establish the actual labeling index for the adult mouse heart. The assay utilized [3H]thymidine incorporation in transgenic mice which expressed a nuclear-localized beta-galactosidase (beta-Gal) reporter gene exclusively in cardiac myocytes. Cardiomyocyte DNA synthesis was evidenced by colocalization of beta-Gal activity and silver grains in autoradiograms of histological sections. Examination of 180,000 ventricular cardiomyocyte nuclei from normal adult transgenic mice identified a single synthetic nucleus, suggesting a maximum labeling index of 0.0005%. Cardiomyocyte DNA synthesis was next examined in hearts injured by focal cauterization of the left ventricular free wall. Only three synthetic nuclei were identified when 36,000 cardiomyocyte nuclei in the perinecrotic zone of the injured heart were examined. No additional synthetic nuclei were identified when 180,000 nuclei in regions distal to the necrotic zone were examined. These data confirm that cardiomyocyte DNA synthesis in the adult mouse heart is extremely rare and provide baseline data for analyses in genetically modified animals.
Assuntos
DNA/biossíntese , Traumatismos Cardíacos/metabolismo , Miocárdio/metabolismo , Animais , Autorradiografia , Cauterização , Núcleo Celular/metabolismo , Traumatismos Cardíacos/patologia , Ventrículos do Coração , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Transgênicos/genética , Miocárdio/patologia , Cadeias Pesadas de Miosina/genética , Valores de Referência , Prata , Timidina/metabolismo , beta-Galactosidase/genéticaRESUMO
The ability of cardiomyocytes to synthesize DNA in response to experimentally induced cardiac hypertrophy was assessed in adult mice. Isoproterenol delivered by osmotic minipump implantation in adult C3Heb/FeJ mice resulted in a 46% increase in heart weight and a 19.3% increase in cardiomyocyte area. No DNA synthesis, as assessed by autoradiographic analysis of isolated cardiomyocytes, was observed in control or hypertrophic hearts. A survey of 15 independent inbred strains of mice revealed that ventricular cardiomyocyte nuclear number ranged from 3 to 13% mononucleate, suggesting that cardiomyocyte terminal differentiation is influenced directly or indirectly by genetic background. To determine whether the capacity for reactive DNA synthesis was also subject to genetic regulation, cardiac hypertrophy was induced in the strains of mice comprising the extremes of the nuclear number survey. These data indicate that adult mouse atrial and ventricular cardiomyocytes do not synthesize DNA in response to isoproterenol-induced cardiac hypertrophy.
Assuntos
Cardiomiopatia Hipertrófica/metabolismo , DNA/biossíntese , Miocárdio/metabolismo , Animais , Cardiomiopatia Hipertrófica/induzido quimicamente , Cardiomiopatia Hipertrófica/patologia , Diferenciação Celular , Núcleo Celular/ultraestrutura , Ventrículos do Coração , Isoproterenol , Camundongos , Camundongos Endogâmicos C3H , Miocárdio/patologia , Ratos , Ratos EndogâmicosRESUMO
The adult newt cardiac ventricular myocyte has been successfully placed in cell culture and has been shown to undergo in vitro DNA synthesis. Although several growth factors have been reported to increase DNA synthesis in cardiac myocytes in vitro, PDGF has not been reported to do so, but has been shown to be active in other systems. Ventricles were removed from the adult red-spotted newt and were enzymatically and mechanically dissociated in a solution containing trypsin and collagenase. Cells were preplated on to plastic to remove non-myocytes. Myocytes were then plated onto laminin. Groups of myocytes were fed control medium and medium containing porcine PDGF. Myocytes were given 1 microCi/ml of tritiated thymidine 6 or 24 h before fixation. Control myocytes showed a peak DNA synthesis at 12-14 days in culture. One ng/ml of PDGF increased DNA synthesis significantly to 22% above control. Myocytes responded to PDGF with significantly increased DNA synthesis in about 12 h. PDGF did not induce earlier DNA synthesis, but increased synthesis at all days of culture tested. These results indicate that PDGF acts upon cardiac myocytes, increasing their DNA synthesis.
Assuntos
DNA/biossíntese , Coração/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Animais , Relação Dose-Resposta a Droga , Técnicas In Vitro , Cinética , Miocárdio/citologia , Miocárdio/metabolismo , Fator de Crescimento Derivado de Plaquetas/administração & dosagem , SalamandridaeRESUMO
To better understand the mechanisms governing the proliferation of cardiac myocytes it is important to identify the factors controlling this phenomenon, and to characterize their actions. DNA synthesis was quantified in vitro in ventricular myocytes from the adult red-spotted newt, Notophthalmus viridescens. Ventricles were enzymatically separated and plated onto laminin. Myocytes were fed modified L-15 medium with 10% fetal bovine serum, and were variously treated with transforming growth factor-beta, transforming growth factor-beta combined with platelet-derived growth factor, acidic fibroblast growth factor, basic fibroblast growth factor, 12-0-tetradecanoylphorbol-13-acetate, heparin, or conditioned medium from ventricular myocytes or non-myocytes (primarily endothelial cells). With their final feeding the cells were given 1 mu Ci/ml of tritiated thymidine, and 24 hours later were fixed and stained. Dishes were coated with photographic emulsion, exposed, and developed. The percent of cells with labeled nuclei was determined. Experimental media that significantly increased DNA synthesis included those containing acidic fibroblast growth factor (121% of control), basic fibroblast growth factor (119% of control), 12-0-tetradecanoylphorbol-13-acetate (233% of control) and conditioned medium from ventricular myocytes (230% of control) or non-myocytes (128% of control). Media significantly inhibiting DNA synthesis were those containing heparin (31% of control), transforming growth factor-beta (38% of control), non-myocyte conditioned medium and heparin (75% of control), or transforming growth factor-beta and platelet-derived growth factor (63% of control).
Assuntos
DNA/biossíntese , Miocárdio/metabolismo , Notophthalmus viridescens/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados , Fator 1 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Heparina/farmacologia , Miocárdio/citologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Fator de Crescimento Transformador beta/farmacologiaRESUMO
The proliferative capacity of embryonic stem (ES) cell-derived cardiomyocytes was assessed. Enriched preparations of cardiomyocytes were isolated by microdissection of the cardiogenic regions of cultured embryoid bodies. The identity of the isolated cells was established by immunocytology, and mitotic activity was monitored by [3H]thymidine incorporation and pulse-chase experiments. ES-derived cardiomyocytes were mitotically active and predominantly mononucleated at 11 days after cardiogenic induction. By 21 days postinduction, cardiomyocyte DNA synthesis was markedly decreased, with a concomitant increase in the percentage of multinucleated cells. Interestingly, the duration of active cardiomyocyte reduplication in the ES system appeared to be roughly similar to that observed during normal murine cardiogenesis. Given these observations, as well as the genetic tractability of ES cells, ES-derived cardiogenesis might provide a useful in vitro system with which to assess the molecular regulation of the cardiomyocyte cell cycle.
Assuntos
Núcleo Celular/ultraestrutura , DNA/biossíntese , Embrião de Mamíferos/citologia , Coração/embriologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Ciclo Celular , Diferenciação Celular , Células Cultivadas , Camundongos , Camundongos Endogâmicos C3HRESUMO
Expression of p107, a protein with homology to the retinoblastoma tumor suppressor (pRB), was monitored during murine development. Northern blot tissue surveys identified two transcripts of 4.9 and 2.4 kb that hybridized to a p107 cDNA clone. Expression of both transcripts was detected in fetal tissues, with particularly high levels in the liver and heart. In contrast, p107 transcripts were markedly decreased in most adult tissues examined. Molecular cloning analyses revealed that the 4.9- and 2.4-kb transcripts encoded proteins with deduced molecular masses of 119 and 68 kDa, respectively. Genetic mapping studies suggested that the two p107 transcripts arose by alternative splicing of a common precursor. The protein encoded by the 2.4-kb transcript lacks the spacer and B motif of the "pocket domain," a region of homology between p107 and pRB that is required for binding to cell cycle regulatory proteins. Structural modifications resulting from alternative splicing may thus confer functional diversity upon the 119- and 68-kDa proteins.
Assuntos
Processamento Alternativo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/genética , RNA Mensageiro/metabolismo , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Sequência de Bases , DNA Complementar/análise , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Proteína p107 Retinoblastoma-LikeRESUMO
The therapeutic recourse for end-stage heart disease is currently limited to cardiac transplantation. The ability to augment cardiomyocyte number in an end-stage heart might facilitate myocardial function. Augmentation of cardiomyocyte number may be achievable by the targeted expression of cell cycle regulatory genes to the myocardium. Alternatively, intracardiac grafting of exogenous cardiomyocytes might also provide a viable approach to augment cardiomyocyte number. Potential strategies for heart muscle regeneration via gene therapy and cellular transplantation are discussed.
Assuntos
Cardiopatias/fisiopatologia , Cardiopatias/terapia , Coração/fisiopatologia , Cicatrização , Animais , Divisão Celular , Transplante de Células , Humanos , Miocárdio/patologiaRESUMO
Cardiomyocyte DNA synthesis and binucleation indexes were determined during murine development. Cardiomyocyte DNA synthesis occurred in two temporally distinct phases. The first phase occurred during fetal life and was associated exclusively with cardiomyocyte proliferation. The second phase occurred during early neonatal life and was associated with binucleation. Collectively, these results suggest that cardiomyocyte reduplication ceases during late fetal life. Northern and Western blot analyses identified several candidate genes that were differentially expressed during the reduplicative and binucleation phases of cardiomyocyte growth.
Assuntos
Animais Recém-Nascidos/fisiologia , Núcleo Celular/ultraestrutura , DNA/biossíntese , Coração Fetal/metabolismo , Coração/fisiologia , Miocárdio/metabolismo , Animais , Animais Recém-Nascidos/metabolismo , Western Blotting , Ciclo Celular/fisiologia , Expressão Gênica , Genes Reguladores , Camundongos , Camundongos Endogâmicos C3H , Miocárdio/citologia , Fatores de TempoRESUMO
The long-term viability of cardiomyocyte grafts in the adult myocardium was tested. AT-1 cardiomyocytes, a differentiated tumor line derived from transgenic mice expressing an atrial natriuretic factor-simian virus 40 T antigen fusion gene, were grafted directly into the myocardium of syngeneic animals. Viable grafts were detected as long as 4 mo postimplantation. Thymidine uptake studies suggested that the grafted cardiomyocytes retained mitotic activity. The presence of AT-1 cardiomyocyte grafts and the associated myocardial remodeling were not accompanied by overt cardiac arrhythmia. Electron microscopic analyses showed that the majority of the grafts were juxtaposed directly to the host myocardium and were not encapsulated. This study indicates that the myocardium can serve as a stable platform for cells that have been manipulated in vitro and suggests that cardiomyocyte grafts may provide a useful means for the local delivery of recombinant molecules to the heart. The long-term survival of the AT-1 cardiomyocytes in the heart also raises the possibility that similar grafting approaches may be used to replace diseased myocardium.
Assuntos
Coração/fisiologia , Miocárdio/citologia , Transplante de Tecidos , Animais , Antígenos Transformantes de Poliomavirus/genética , Fator Natriurético Atrial/genética , Linhagem Celular , Sobrevivência Celular , Eletrocardiografia , Estudos de Viabilidade , Sobrevivência de Enxerto , Técnicas Imunológicas , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Miocárdio/ultraestrutura , Fatores de TempoRESUMO
Previous studies have identified several host proteins (p53, p107, and p193), which form prominent complexes with SV40 T antigen in transformed cardiomyocytes. Expression of p53 and p107 was monitored during normal and pathologic growth in nontransformed murine myocardium. Both genes were expressed at relatively high levels in embryonic cardiomyocytes. Transcript levels decreased markedly during the process of cardiomyocyte terminal differentiation and were very low or undetectable in adult animals. In contrast, retinoblastoma transcripts were observed at low levels throughout myocardial development. None of the tumor suppressor genes examined were transcriptionally activated during acute myocardial overload or isoproterenol-induced myocardial hypertrophy. The potential role of tumor suppressor gene product expression in myocardial development and pathology is discussed.
Assuntos
Cardiomegalia/metabolismo , Expressão Gênica , Genes Supressores de Tumor , Miocárdio/metabolismo , Animais , Antígenos Transformantes de Poliomavirus/biossíntese , Northern Blotting , Western Blotting , Peso Corporal , Cardiomegalia/induzido quimicamente , Linhagem Celular Transformada , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Genes Supressores de Tumor/efeitos dos fármacos , Células HeLa , Coração/efeitos dos fármacos , Humanos , Isoproterenol/toxicidade , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Tamanho do Órgão , Valores de Referência , Proteína do Retinoblastoma/biossíntese , Vírus 40 dos Símios/genética , Transcrição Gênica , Proteína Supressora de Tumor p53/biossínteseRESUMO
Targeted expression of the SV40 large T-antigen oncoprotein (T-Ag) induces cardiomyocyte proliferation in the atria and ventricles of transgenic mice. Previous studies have identified the p53 tumor suppressor, p107 (a homologue of the retinoblastoma tumor suppressor), and p193 (a novel BH3 only proapoptosis protein) as prominent TAg binding proteins in cardiomyocyte cell lines derived from these transgenic mice. To further explore the significance of these protein-protein interactions in the regulation of cardiomyocyte proliferation, a transgene comprising the human atrial natriuretic factor (ANF) promoter and sequences encoding a mutant T-Ag lacking the p53 binding domain was generated. Repeated micro-injection of this DNA gave rise to genetically mosaic animals with minimal transgene content, suggesting that widespread cardiac expression of mutant T-Ag was deleterious. This notion was supported by the observation that the transgene was selectively lost from the cardiac myocytes (but not the cardiac fibroblasts) in the mosaic animals. Crosses between the mosaic mice and animals expressing a cardiac restricted dominant negative p53 resulted in transgene transmission with ensuing overt cardiac tumorigenesis. Transfection of the mutant T-Ag in embryonic stem (ES) cell-derived cardiomyocytes resulted in wide-spread cell death with characteristics typical of apoptosis. Co-transfection with a dominant negative p53 transgene rescued mutant TAg-induced cell death in the ES-derived cardiomyocyte cultures, resulting in a marked proliferative response similar to that seen in vivo with the rescued transgenic mouse study. These results indicate that T-Ag expression in the absence of p53 functional abrogation results in cardiomyocyte death.