Molecular mechanism of cardiac hypertrophy

Pressure overload induces cardiac hypertrophy and reexpression of contractile protein isogenes. To ascertain the molecular mechanism of these events, we examined the expression of cellular oncogenes and the early change in the translational activity of specific cardiac mRNA by two-dimensional gel electrophoresis of in vitro translational products. Pressure overload increased the expression levels of c-fos, c-myc, and c-Ha-ras genes. The relative predominance of 8 species out of over 400 translational products was increased by pressure overload while that of 2 translational products was decreased. We cloned four pressure-overload-responsive cDNA clones by differential dot blot hybridization. The expression pattern of each cDNA clone in the pressure-overloaded hearts was similar to that in fetal hearts. To examine whether mechanical stimuli directly induce specific gene expression in the heart, we cultured rat neonatal cardiocytes in elastic silicone dishes and stretched these adherent cells. Myocytes stretching stimulated amino acid uptake and expression of the c-fos gene, which was blocked by protein kinase C inhibitors. These results suggest that there are some early responsive genes in cardiac hypertrophy and that mechanical loading directly stimulates gene expression possibly via protein kinase C activation.     

大鼠心肌组织中原癌基因（c—myc,Ha—ras,c—fos）表达的随龄性改变

目的　通过研究原癌基因(c-myc，Ha-ras，c-fos)在衰老心肌中的表达，探讨增龄对心肌生长相关的原癌基因的影响。方法　20月龄及6月龄健康雄性SD大鼠，以Northern杂交分析c-myc，Ha-ras，c-fos基因表达。结果　Ha-ras和c-fos在6月龄、20月龄大鼠心室肌中均表达，老龄鼠c-fos的表达水平明显高于青龄鼠(0.523±0.07，0.185±0.05)(P＜0.05)；c-Ha-ras则无显著差异(0.65±0.07，0.63±0.06)(P＞0.05)；c-myc在6月龄和20月龄大鼠心室肌中均无表达。结论　原癌基因((c-myc，Ha-ras，c-fos等)随增龄在心肌组织中的表达各不相同，探讨老年期中各基因重新激活和高表达的意义，有可能为老年心脏病的发生和心肌老化的内在机制提供一个新的解释。     

Expression of cellular oncogenes during embryonic and fetal development of the mouse.

Cellular oncogenes are conserved with great fidelity across a broad span of evolution. This avid conservation suggests possible roles in critical physiologic functions. Little, however, is known about their activity in normal cellular processes. In this study, we examined the expression pattern of eight cellular oncogenes during embryonic and fetal development of the mouse. Five of these genes (c-myc, c-erb, c-Ha-ras, c-src, and c-sis) were expressed at appreciable levels, and four were modulated in a consistent manner during the course of prenatal development.     

Patterns of mRNA expression during early cell growth differ in kidney epithelial cells destined to undergo compensatory hypertrophy versus regenerative hyperplasia.

An increase in cell size and protein content is characteristic of cells undergoing hypertrophy and of replicating cells prior to DNA synthesis. Cell enlargement in the two situations could be regulated by similar early events with an interruption of the cell cycle occurring in hypertrophy, or the two processes could be uncoupled. In vivo models were used to compare hypertrophy induced by unilateral nephrectomy and hyperplasia induced by folic acid injection in rabbit renal cortical cells. Within 48 hr, cell volume increased in both groups but the number of cells in the cell cycle and DNA synthesis was increased only after folic acid. Patterns of mRNA expression of the following three groups of cell cycle-dependent genes were analyzed: (i) protooncogenes (c-fos, c-myc, and c-Ha-ras), (ii) structural protein genes (vimentin and beta-actin), and (iii) transport protein genes (Na+, K+-ATPase, ADP-ATP translocase, and calcyclin). mRNAs for all genes, except calcyclin and c-Ha-ras, were detected in controls. Folic acid generally induced rapid, transient increases in mRNA levels, but after unilateral nephrectomy, expression of most mRNAs showed a gradual, progressive increase. These data indicate that gene expression in the early stages of cell enlargement differs in cells destined to undergo proliferation vs. hypertrophy. The term "sustained message amplification" is proposed to describe the hypertrophied cell.     

人主动脉硫酸乙酰肝素蛋白聚糖对胎儿主动脉平滑肌细胞c-fos和c-myc基因表达的影响

本室以往的研究发现硫酸乙酰肝素蛋白聚糖对培养的胎儿主动脉平滑肌细胞的增殖有明显的抑制作用。本实验采用Ｎｏｒｔｈｅｒｎｂｌｏｔ的方法观察硫酸乙酰肝素蛋白聚糖对胎儿主动脉平滑肌细胞的ｃ－ｆｏｓ，ｃ－ｍｙｃ原癌基因的表达的影响，发现前者能抑制后者的表达。因此硫酸乙酰肝素蛋白聚糖抑制平滑肌细胞增殖的途径之一可能为抑制原癌基因的表达。     

人主动脉硫酸乙酰肝素蛋白聚糖对胎儿主动脉平滑肌细胞c-fos和c-myc基因表达的影响

本室以往的研究发现硫酸乙酰肝素蛋白聚糖对培养的胎儿主动脉平滑肌细胞的增殖有明显的抑制作用。本实验采用Ｎｏｒｔｈｅｒｎｂｌｏｔ方法观察硫酸乙酰肝素蛋白聚糖对胎儿主动脉平滑肌细胞的ｃ－ｆｏｓ，ｃ－ｍｙｃ原癌基因的表达的影响，发现前者能抑制后者的表达。因此硫酸乙酰肝素蛋白聚糖抑制平滑肌细胞增殖的途径之一可能为抑制原癌基因的表达。     

Acute and Chronic Adaptation to Hemodynamic Overload and Ischemia in the Aged Heart

Congestive heart failure occurs more frequently in older individuals. This higher incidence of heart failure may be caused by the diminished capacity of aged hearts to adapt to increased hemodynamic overload and ischemia which are the most important triggers for heart failure in the aged. In the immediate early phase after the imposition of ascending aortic banding, the mRNA expression of proto-oncogenes (c-fos, c-jun and c-myc) was diminished in aged rat hearts compared with young adult hearts. In the later phase, the pattern of expression of contractile protein genes in aged hearts differed quantitatively from that in adult hearts. The hypertrophic responses to the imposition of not only pressure-overload but also volume-overload were also diminished at the organ and cellular levels. In addition, this diminution was observed both in the left and right ventricles. Against ischemic insults, aged hearts responded with a diminished expression of proto-oncogenes and heat shock proteins. Thus, aged hearts are characterized by poor adaptation to hemodynamic overload and by a poor self-protective mechanism against cell death through necrosis and apoptosis. Of interest, more severe hemodynamic overload elevated the diminished responses to a level similar to that in adult hearts, suggesting that the threshold for the heart to respond to hemodynamic overload or ischemia is elevated in aged hearts. In addition, even in aged hearts ischemic preconditioning upregulated the diminished gene expression in a gene-dependent manner. Thus, the capacity for adaptation to hemodynamic overload and ischemia is diminished in aged hearts, but aged hearts preserve the ability to respond to these under some conditions.     

CREG, a new regulator of ERK1/2 in cardiac hypertrophy

OBJECTIVES: The cellular repressor of E1A-stimulated genes (CREG), a mannose-6-phosphate-containing secreted glycoprotein, enhances differentiation and inhibits proliferation. In this study, our aim was to understand the role of CREG in cardiac hypertrophy. METHODS: Two models of cardiac hypertrophy were used: the in vivo pressure-overloaded rat cardiac hypertrophy and the in vitro stretched neonatal rat cardiomyocyte hypertrophy. CREG's function in cardiac cells was investigated after over-expression or antisense inhibition of CREG. RESULTS: We found reduced CREG expression in rat hearts after the in vivo overload, as shown by Northern blot analysis. CREG over-expression inhibited cardiac cell growth, as demonstrated by reduced protein content, cell area and ERK1/2 level in cultured neonatal rat cardiomyocytes, and by the reduced proliferation of cultured neonatal rat cardiac fibroblasts. Additionally, over-expression of CREG dampened the stretched cardiomyocyte hypertrophy through ERK1/2. On the other hand, the opposite effects were observed when CREG expression was decreased using antisense. This modulation of CREG expression resulted in no changes in the cardiomyocyte expressions of the hypertrophic or apoptotic signaling molecules such as protein kinase C (PKC) epsilon, PKC beta1, PKC alpha, PKC beta2, PKC delta, JNK1/2, p38, p53, Bax, Bcl2 and Fas. CONCLUSIONS: CREG was found to inhibit cardiac cell growth as a novel regulator of ERK1/2 and might participate in the development of cardiac hypertrophy under pressure overload. The insight that CREG inhibited the growth in rat hearts in vivo and in cardiac cells in vitro provides new clues for further investigation of the mechanism of heart remodeling.     

Protooncogene induction and reprogramming of cardiac gene expression produced by pressure overload.

Hypertrophy, an increase in cell size without cell division, is a fundamental adaptive process employed by postmitotic cardiac and skeletal muscle cells. Cardiac myosins undergo an adult-to-fetal isoform transition in various models of hypertrophy. Using gene-specific cDNA probes, we show here that in the adult myocardium the mRNAs encoding the fetal (skeletal muscle type) isoforms of alpha-actin and sarcomeric tropomyosin are re-expressed within 2 days in response to pressure overload. In addition, atrial natriuretic factor mRNA, so far believed to be expressed primarily in the atria, was readily detectable in the ventricles of neonates and was induced to markedly high levels in pressure-overloaded adult ventricles. In contrast, cardiac hypertrophy produced by thyroid hormone excess was not associated with induction of the atrial natriuretic factor gene or fetal contractile protein isogenes. Furthermore, the c-fos and c-myc protooncogenes and a major heat shock protein gene (hsp70) are induced in the ventricular myocardium within 1 hr after imposition of pressure overload. These results suggest that induction of cellular protooncogenes and heat shock (stress) protein genes is an early response to pressure overload, whereas reinduction of the genes normally expressed only in perinatal life, such as fetal isoforms of contractile proteins and atrial natriuretic factor, is a later event. These two types of responses might represent the general pattern of growth induction to work overload by terminally differentiated cells that have lost the ability to undergo DNA replication.     

Adrenergic agents,but not triiodo-L-thyronine induce c-fos and c-myc expression in the rat heart

Summary We have examined the expression of two nuclear-acting oncogenes, c-fos and c-myc in the rat heart following administration of hormones implicated in the development of cardiac hypertrophy. A single injection of norepinephrine (2.5 g/kg to 2.5 mg/kg) led to transient increases in the levels of both c-fos and c-myc mRNA. The response was sequential: elevated levels of c-fos mRNA were first observed 15 min after treatment and peaked at 1 h whilst c-myc mRNA levels increased 30 min after treatment and peaked at 2h. The response of both cellular oncogenes to norepinephrine was reduced significantly by a blockade but (3 blockade was less effective. Administration of triiodo-L-thyronine (0.25 mg/kg), a level known to promote cardiac hypertrophy, did not produce elevated levels of c-fos or c-myc mRNA. In an initial study, it was possible to demonstrate induction of c-fos and c-myc in rat hearts perfused in vitro with medium containing 2x10^–7 M norepinephrine. These results provide support for the notion that c-fos and c-myc expression may play a transducing role in the development of adrenergic-mediated, but not thyroid hormone-mediated cardiac hypertrophy.     

Effects of fluid shear stress on expression of proto-oncogenes c-fos and c-myc in cultured human umbilical vein endothelial cells

The objective of our research was to reveal the effects of different shear stresses on the expression of proto-oncogenes c-fos and c-myc in cultured human umbilical vein endothelial cells (HUVEC). A parallel plate flow chamber was used to control the value and duration of shear stress (SS), and the expression of c-fos and c-myc protein was measured by immunocytochemistry methods and image analysis software. Some important conclusions were drawn. In the stationary state, c-fos protein levels were very low. The SS's of 4 dyn/cm(2) and 10 dyn/cm(2) induced rapid increases of c-fos protein levels, especially the SS of 10 dyn/cm(2). The levels peaked at 1.0 h. Then, c-fos protein levels began to decrease, after 2.5 h, they declined to almost basal levels. In the stationary state, the c-myc protein levels were also very low slowly increasing after the onset of shear stress. The effects of 4 dyn/cm(2) and 10 dyn/cm(2) SS on c-myc protein expression levels had no difference and apparently were less than the effect on c-fos protein levels. The c-myc protein levels peaked at 1.5 h. Subsequently, they fell to basal levels at 2.5 h. Increased expression of these proto-oncogenes mediated by shear stress may have important effects on the regulation of critical cell activities, such as proliferation and differentiation.     

Prognostic significance of the expression of c-fos,c-jun and c-erbB-1 oncogene products in human squamous cell lung carcinomas

The expression of the oncogenes c-fos, c-jun, c-myc, c-erbB-1 and c-erbB-2 at the protein level was analyzed in squamous cell lung carcinomas of 121 patients by means of immunohistochemistry. Patients with overexpression of proteins encoded by the oncogenes c-fos, c-jun and c-erbB-1 had significantly shorter survival times than these without overexpression of these oncogene products (c-fos:p=0.009; c-jun:p=0.029; c-erbB-1:p=0.018). No significant correlations were found between the expression of c-myc and c-erbB-2 products and the survival of the patients. In addition to the univariate analyses (Kaplan-Meier-estimates) multivariate analyses (Cox-regression-model) revealed that protein expression of the oncogenes c-fos, c-jun and c-erbB1 are significant prognostic factors in addition to staging.     

Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein.

The c-fos gene product Fos has been implicated in many cellular processes, including signal transduction, DNA synthesis, and resistance to antineoplastic agents. A fos ribozyme (catalytic RNA) was designed to evaluate the effects of suppressing Fos protein synthesis on expression of enzymes involved in DNA synthesis, DNA repair, and drug resistance. DNA encoding the fos ribozyme (fosRb) was cloned into the pMAMneo expression plasmid, and the resultant vector was transfected into A2780DDP cells resistant to the chemotherapeutic agent cisplatin. The parental drug-sensitive A2780S cells were transfected with the pMMV vector containing the c-fos gene. Morphological alterations were accompanied by significant changes in pharmacological sensitivity in both c-fos- and fosRb-transfected cells. pMAMneo fosRb transfectants revealed decreased c-fos gene expression, concomitant with reduced thymidylate (dTMP) synthase, DNA polymerase beta, topoisomerase I, and metallothionein IIA mRNAs. In contrast, c-myc expression was elevated after fos ribozyme action. Insertion of a mutant ribozyme, mainly capable of antisense activity, into A2780DDP cells resulted in smaller reductions in c-fos gene expression and in cisplatin resistance than the active ribozyme. These studies establish a role for c-fos in drug resistance and in mediating DNA synthesis and repair processes by modulating expression of genes such as dTMP synthase, DNA polymerase beta, and topoisomerase I. These studies also suggest the utility of ribozymes in the analysis of cellular gene expression.     

WHV/myc转基因小鼠肝癌发生过程中的基因异常

目的探测ＷＨＶ／ｍｙｃ转基因小鼠肝癌发生过程中有关基因在肝脏中的异常表达。方法用原位杂交方法检测ＷＨＶ／ｍｙｃ转基因小鼠肝肿瘤形成的不同阶段,ｃ－ｍｙｃ转染基因、胰岛素样生长因子Ⅱ（ＩＧＦ－Ⅱ）基因ｃ－ｊｕｎ、ｃ－ｆｏｓ和ｃ－Ｈ－ｒａｓ等基因的表达。结果ｃ－ｍｙｃ转染基因和ＩＧＦ－Ⅱ基因在转基因小鼠出生后早期的肝脏中异常表达,随后表达水平即降低至不能测出。上述两基因的表达在肝肿瘤形成期重新出现。ｃ－ｊｕｎ、ｃ－ｆｏｓ和ｃ－Ｈ－ｒａｓ基因在转基因小鼠肝癌发生前肝脏中的表达强度明显高于正常小鼠。结论ｃ－ｍｙｏｔ染基因和ＩＧＦ－Ⅱ基因在小鼠出生后早期和肿瘤形成期的异常表达对肝肿瘤的发生和瘤细胞转化表型的维持可能具有重要意义;在ｃ－ｍｙｃ转染基因表达的“沉默”期,一些癌基因在肝脏中的异常表达对日后肝脏肿瘤的形成可能也具有一定作用。     

Mechanisms of cardiac hypertrophy and injury--possible role of protein kinase C activation.

To examine the molecular mechanisms by which mechanical stimuli induced cardiac hypertrophy and injury, we cultured rat neonatal cardiocytes in deformable dishes and imposed an in vitro mechanical load by stretching the adherent cells. Myocyte stretching increased total cell RNA content and mRNA levels of c-fos. Marked accumulation of c-fos mRNA followed increases in intracellular Na+ and protein kinase C activation. The accumulation of c-fos mRNA by cardiocyte stretching was suppressed by protein kinase C inhibitors but not by stretch channel blockers. Moreover, myocyte stretching increased inositol phosphate levels, and activation of protein kinase C by phorbolesters stumulated the expression of c-fos. We also examined TGF beta expression in the heart. TGF beta is known to be stimulated by protein kinase C activation, and the mRNA level of TGF beta was increased in in vivo heart by pressure overload. Furthermore, collagen synthesis was stimulated by TGF beta in cultured fibroblasts from hearts. These findings suggest that hemodynamic overload may stimulate cardiac hypertrophy and induce cardiac injury (fibrosis) through protein kinase C activation.     

Sequential expression and cooperative interaction of c-Ha-ras and c-erbB genes in in vivo chemical carcinogenesis.

The level of expression of several cellular protooncogenes is examined at different stages of 7,12-dimethylbenzanthracene (DMBA)-induced tumor development in hamster buccal pouch epithelium (HBPE). Results presented demonstrate overexpression of c-Ha-ras gene at a very early stage of tumor development, and this elevated level of expression of the gene persists throughout the tumorigenesis process. The expression of the cellular protooncogene c-erbB, on the other hand, can be detected only after 8-10 weeks of DMBA treatment of the tissue and increases with the progression of the disease. The overexpression of c-erbB gene can be correlated with the stage of extensive proliferation and subsequent invasion of the HBPE cells into the underlying connective tissue. This sequential pattern of stage-specific expression of the two cellular protooncogenes can be observed in (i) treated tissues, (ii) stage-representative cultured cells, and (iii) NIH 3T3 transformants derived with DNA from HBPE cells. The low-level expression of c-myc and c-sis genes detected in control tissues remains unaffected, while c-fos gene activity cannot be detected at any stage of tumor development. The overexpression of c-Ha-ras gene alone in HBPE cells derived from tissues treated for 5 weeks (DM5) is not sufficient to induce tumors in athymic mice, whereas expression of c-Ha-ras and c-erbB genes at later stages of tumor development (DM10 and HCPC cells) induce histopathologically defined epithelial cell carcinoma in athymic mice within 2-3 weeks. The sequential overexpression of c-Ha-ras and c-erbB genes in a stage-specific manner and their cooperative interaction in the DMBA-induced in vivo oral carcinogenesis have been demonstrated.     

Effects of 17 beta-estradiol and growth factors on c-fos gene expression in endometrial epithelial cells in primary culture

Primary culture of guinea-pig endometrial cells was made quiescent by serum depletion. When added to quiescent cells, 17 beta-estradiol (E2) alone affected neither c-fos and c-myc gene expression, nor DNA synthesis and cell proliferation. Insulin or epidermal growth factor (EGF) only induced DNA synthesis. An association of both growth factors allowed significant cell proliferation without inducing c-fos or c-myc expression. A response including c-fos induction (maximal expression at 75 min), but not c-myc expression, DNA synthesis and a marked cell proliferation was only obtained when 17 beta-estradiol was associated with insulin plus EGF. In this case, cycloheximide raised the c-fos gene expression. These data suggest that in endometrial epithelial cells, E2 is mitogenic only when it acts in association with EGF plus insulin and the c-fos gene expression may not be correlated with cell proliferation.