Growth and hypertrophy of the heart: towards an understanding of cardiac specific and inducible gene expression.
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Cardiomegaly↗
Biomedical subjects
K R Chien
Publications and source records attributed to K R Chien.
A novel, tissue-restricted zinc finger protein (HF-1b) binds to the cardiac regulatory element (HF-1b/MEF-2) in the rat myosin light-chain 2 gene.
The AT-rich element MEF-2 plays an important role in the maintenance of the muscle-specific expression of a number of cardiac and skeletal muscle genes. In the MLC-2 gene, an AT-rich element (HF-1b) which contains a consensus MEF-2 site is required for cardiac tissue-specific expression. The present study reports the isolation and characterization of a cDNA which encodes a novel C2H2 zinc finger (HF-1b) that binds in a sequence-specific manner to the HF-1b/MEF-2 site in the MLC-2 promoter. A number of independent criteria suggest that this HF-1b zinc finger protein is a component of the endogenous HF-1b/MEF-2 binding activity in cardiac muscle cells and that it can serve as a transcriptional activator of the MLC-2 promoter in transient assays. These studies suggest that, in addition to the previously reported RSRF proteins, structurally divergent transcriptional factors can bind to MEF-2-like sites in muscle promoters. These results underscore the complexity of the regulation of the muscle gene program via these AT-rich elements in cardiac and skeletal muscle.
Amino Acid Sequence↗
Transcriptional regulation during cardiac growth and development.
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Amino Acid Sequence↗
Targeting gene expression to specific cardiovascular cell types in transgenic mice.
Transgenic techniques, which allow the introduction of exogenous genes into the genome of experimental animals, promise to bridge the gap between the in vitro observations made by molecular and cellular biologists on cardiac and vascular cells in tissue culture and the physiology and pathology of the whole organ system. One such application of these techniques is tissue targeting: by genetic manipulation to direct expression of a protein--such as a signaling peptide, a growth factor receptor, or an oncogene involved in cell growth--to a tissue where it normally would not be expressed (or where expression is tightly controlled) by fusing it to the transcriptional control sequences of another gene normally expressed in that tissue. In the cardiovascular system, regulatory sequences for cardiomyocyte-specific proteins, vascular endothelium-specific proteins, and smooth muscle-specific proteins can be used to target heterologous genes to their respective tissues in transgenic animals. The effects that such perturbations have on organ physiology and intracellular and intercellular communication can be observed by applying established physiological and molecular approaches. In this review, we highlight some tissue-specific genes from cardiac and vascular cell types whose regulatory sequences may be used to target heterologous proteins; we discuss neutral "reporter" proteins and signal transduction components as paradigms for the application of this technique; and we briefly touch on the potentials and pitfalls of transgenic approaches to molecular physiology.
Animals↗
Expression of SV40 virus large T antigen by recombinant adenoviruses activates proliferation of corneal endothelium in vitro.
Infection with the Ad5-SVR4 virus was used to introduce the large T antigen encoding region of the SV40 virus into bovine and human corneal endothelial cells. Expression of large T antigen occurred in 40% of bovine corneal endothelial cells after a 24-h incubation time versus 12% after 8 h of incubation. By 48 h after infection, almost all (92.8%) bovine corneal endothelial cells expressed large T antigen. Bovine and human corneal endothelial cells which expressed large T antigen proliferated and the characteristic morphologic features of corneal endothelium were maintained. This method may enable growth of enough corneal endothelium to perform studies to elucidate the biochemical mechanisms involved in regulating endothelial cell function.
Adenoviridae↗
Transcriptional activation of the cardiac myosin light chain 2 and atrial natriuretic factor genes by protein kinase C in neonatal rat ventricular myocytes.
A cultured myocardial cell model was used to examine the role of protein kinase C-dependent pathways in the transcriptional activation of two cardiac muscle genes [myosin light chain 2 (MLC-2) and atrial natriuretic factor (ANF)] during alpha-adrenergic receptor-mediated hypertrophy. Phorbol ester (phorbol 12-myristate 13-acetate) and the alpha-adrenergic agonist phenylephrine both activate protein kinase C (PKC) and induce 4- to 5-fold increases in the expression of MLC-2 and ANF promoter/luciferase reporter genes with little effect on Rous sarcoma virus/luciferase or minimal prolactin promoter/luciferase genes. To further assess the role of PKC in cardiac gene regulation, PKC expression vectors encoding constitutively activated PKC-alpha or PKC-beta, or a catalytically inactive PKC, were transiently cotransfected with the cardiac promoter/luciferase constructs. Cotransfection of either activated PKC-alpha or PKC-beta cDNA induces the expression of MLC-2 and ANF promoter/luciferase genes and of a reporter gene responsive to the transcription factor AP-1. The Rous sarcoma virus/luciferase and minimal prolactin promoter/luciferase genes are not concomitantly induced by cotransfectin with the PKC genes, indicating specificity of the transcriptional effect. The finding that activated PKC increases cardiac gene transcription suggests that activation of this enzyme may be a proximal signal for coregulation of two cardiac genes, MLC-2 and ANF, during the course of myocardial cell hypertrophy.
Amino Acid Sequence↗
A ubiquitous factor (HF-1a) and a distinct muscle factor (HF-1b/MEF-2) form an E-box-independent pathway for cardiac muscle gene expression.
Recent studies have identified a conserved 28-bp element (HF-1) within the rat cardiac MLC-2 gene which confers cardiac muscle-specific and inducible expression during myocardial cell hypertrophy. Utilizing a combination of independent experimental approaches, this study characterizes two cardiac nuclear factors which bind to HF-1, a ubiquitous factor (HF-1a), and an A + T-rich binding factor (HF-1b) which is preferentially expressed in differentiated cardiac and skeletal muscle cells. The HF-1a binding site is located in a core region of the 28-bp conserved element, immediately upstream from the A + T-rich HF-1b site, which is homologous to the MEF-2 site found in a number of muscle genes. By a number of separate criteria (gel mobility shift, competition, and mutagenesis studies), HF-1b and MEF-2 appear to be indistinguishable and thus are either identical or closely related muscle factors. Transient assays of luciferase reporter genes containing point mutations throughout the 28-bp HF-1 regulatory element document the importance of both the HF-1a and HF-1b sites in transient assays in ventricular muscle cells. In the native 250-bp MLC-2 promoter fragment, mutations in the single E box had little effect on cardiac muscle specificity, while point mutations in either the HF-1a or HF-1b binding site significantly reduced promoter activity, underscoring the importance of both the HF-1a and HF-1b sites in the transcriptional activation of this cardiac muscle gene. Thus, this study provides evidence that a novel, ubiquitous factor (HF-1a) and a muscle factor (HF-1b/MEF-2) can form a novel, E-box-independent pathway for muscle-specific expression in ventricular cardiac muscle cells.
Animals↗
Microinjection of antibodies and expression vectors into living myocardial cells. Development of a novel approach to identify candidate genes that regulate cardiac growth and hypertrophy.
BACKGROUND: Microinjection approaches in the cardiac cell context have allowed delivery of various calcium dyes and monitoring of short-term physiological responses. However, unlike other cell types, it has proved difficult to microinject myocardial cells without the concomitant loss of long-term cell viability. METHODS AND RESULTS: An analysis of experimental variables was conducted to adapt microinjection techniques to the neonatal rat ventricular cell context. Among the variables optimized were the selection of culture dishes, plating substrate, microinjection parameters, and a variety of maneuvers to inhibit myocyte hypercontracture, injury, and consequent death after micropuncture. With the modified technique, the percentage of injected cells that maintained long-term viability (48 hours) increased from less than 1% to 30%. Similarly, an increased efficiency of gene transfer and expression (measured as the percentage of injected cells that express the delivered gene) was obtained after either cytoplasmic or nuclear injection of a beta-galactosidase expression vector into cardiac myocytes. Microinjection of marker immunoglobulin G does not interfere with the induction of the hypertrophic response or the expression of a coinjected atrial natriuretic factor promoter-luciferase reporter fusion gene construct. CONCLUSIONS: To the best of our knowledge, this study provides the first description of the efficient microinjection of neonatal cardiac muscle cells with maintenance of long-term cell viability. The microinjection technique is now a viable approach to examine cause-and-effect relations between specific gene products and any defined feature or response of cardiac myocytes that can be assayed at a single-cell level.
Animals↗
Recent advances in the molecular biology of cardiac growth and hypertrophy: of mouse and man.
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Animals↗
Segregation of atrial-specific and inducible expression of an atrial natriuretic factor transgene in an in vivo murine model of cardiac hypertrophy.
To study the mechanisms that activate expression of the atrial natriuretic factor (ANF) gene during pressure-induced hypertrophy, we have developed and characterized an in vivo murine model of myocardial cell hypertrophy. We employed microsurgical techniques to produce a stable 35- to 45-mmHg pressure gradient across the thoracic aorta of the mouse that is associated with rapid and transient expression of an immediate-early gene program (c-fos/c-jun/junB/Egr-1/nur-77), an increase in heart weight/body weight ratio, and up-regulation of the endogenous ANF gene. These responses that are identical to those in cultured cell and other in vivo models of hypertrophy. To determine whether tissue-specific and inducible expression of the ANF gene can be segregated, we used a transgenic mouse line in which 500 base pairs of the human ANF promoter region directs atrial-specific expression of the simian virus 40 large tumor antigen (T antigen), with no detectable expression in the ventricles. Thoracic aortic banding of these mice led to a 20-fold increase in the endogenous ANF mRNA in the ventricle but no detectable expression of the T-antigen marker gene. This result provides evidence that atrial-specific and inducible expression of the ANF gene can be segregated, suggesting that a distinct set of regulatory cis sequences may mediate the up-regulation of the ANF gene during in vivo pressure overload hypertrophy. This murine model demonstrates the utility of microsurgical techniques to study in vivo cardiac physiology in transgenic mice and should allow the application of genetic approaches to identify the mechanisms that activate ventricular expression of the ANF gene during in vivo hypertrophy.
Animals↗
Co-regulation of the atrial natriuretic factor and cardiac myosin light chain-2 genes during alpha-adrenergic stimulation of neonatal rat ventricular cells. Identification of cis sequences within an embryonic and a constitutive contractile protein gene which mediate inducible expression.
To study the mechanisms which mediate the transcriptional activation of cardiac genes during alpha adrenergic stimulation, the present study examined the regulated expression of three cardiac genes, a ventricular embryonic gene (atrial natriuretic factor, ANF), a constitutively expressed contractile protein gene (cardiac MLC-2), and a cardiac sodium channel gene. alpha 1-Adrenergic stimulation activates the expression and release of ANF from neonatal ventricular cells. As assessed by RNase protection analyses, treatment with alpha-adrenergic agonists increases the steady-state levels of ANF mRNA by greater than 15-fold. However, a rat cardiac sodium channel gene mRNA is not induced, indicating that alpha-adrenergic stimulation does not lead to an increase in the expression of all cardiac genes. Studies employing a series of rat ANF luciferase and rat MLC-2 luciferase fusion genes identify 315- and 92-base pair cis regulatory sequences within an embryonic gene (ANF) and a constitutively expressed contractile protein gene (MLC-2), respectively, which mediate alpha-adrenergic-inducible gene expression. Transfection of various ANF luciferase reporters into neonatal rat ventricular cells demonstrated that upstream sequences which mediate tissue-specific expression (-3003 to -638) can be segregated from those responsible for inducibility. The lack of inducibility of a cardiac Na+ channel gene, and the segregation of ANF gene sequences which mediate cardiac specific from those which mediate inducible expression, provides further insight into the relationship between muscle-specific and inducible expression during cardiac myocyte hypertrophy. Based on these results, a testable model is proposed for the induction of embryonic cardiac genes and constitutively expressed contractile protein genes and the noninducibility of a subset of cardiac genes during alpha-adrenergic stimulation of neonatal rat ventricular cells.
Animals↗
Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response.
Studies from both in vivo and in vitro model systems have provided an initial skeleton of the potential signaling pathways that might regulate cardiac genes during growth and hypertrophy. One of the first detectable changes in cardiac gene expression is the activation of a program of immediate early gene expression, which is distinct for the hypertrophic response, and is conserved in multiple models of both in vivo and in vitro hypertrophy. Diverse and distinct hormonal stimuli have been documented to activate several features of the hypertrophic response, including several autocrine and paracrine factors. Although the signaling mechanisms that link these factors with the activation of cardiac gene expression are unclear, recent studies suggest that the activation of protein kinase C may represent one of the most proximal common events in this signaling cascade. The activation of cardiac target genes induces a program of embryonic gene expression, including the atrial natriuretic factor (ANF) gene. The cis sequences that mediate cardiac-specific and inducible expression of an embryonic marker gene (ANF) can be segregated by studies in both cultured cell models and in vivo models of hypertrophy in transgenic mice, suggesting that specific sets of regulatory elements may exist for inducible expression of this class of cardiac gene responses. However, the induction of a constitutively expressed contractile protein gene (MLC-2) is mediated by a set of conserved elements that regulate both cardiac-specific and inducible expression. Finally, a subset of cardiac muscle genes appears to be noninducible during in vivo or in vitro hypertrophy in myocardial cells, demonstrating specificity of transcriptional activation during the hypertrophic process. The development of a bona fide in vivo pressure overload model of hypertrophy in a small animal model that can be genetically manipulated, such as the in vivo murine model recently described, should allow a rigorous analysis of the role of these specific signaling mechanisms in the activation of the responses of cardiac genes during the hypertrophic process in vivo.
Adaptation, Biological↗
A conserved 28-base-pair element (HF-1) in the rat cardiac myosin light-chain-2 gene confers cardiac-specific and alpha-adrenergic-inducible expression in cultured neonatal rat myocardial cells.
To study the transcriptional regulatory mechanisms which mediate cardiac-specific and inducible expression during myocardial cell hypertrophy, we have extensively characterized the rat cardiac myosin light-chain-2 (MLC-2) gene as a model system. The MLC-2 gene encodes a relatively abundant contractile protein in slow skeletal and cardiac muscle and is upregulated during in vivo cardiac hypertrophy and alpha-adrenergic-mediated hypertrophy of neonatal rat myocardial cells. In transient expression assays employing a series of MLC-2-luciferase constructs, recent studies have identified a 250-bp fragment which is sufficient for both cardiac-specific and alpha-adrenergic-inducible expression. Within this 250-bp fragment lie three regions (HF-1, HF-2, and HF-3), each greater than 10 bp in length, which are conserved between the chicken and rat cardiac MLC-2 genes, suggesting their potential role in the regulated expression of this contractile protein gene. As assessed by substitution mutations within each of the conserved regions, the present study demonstrates that HF-1 and HF-2 are important in both cardiac-specific and inducible expression, while HF-3 has no detectable role in the regulated expression of the MLC-2 gene in transient expression assays. HF-1 sequences confer both cardiac-specific and inducible expression to a neutral promoter-luciferase construct but have no significant effect in the skeletal muscle or nonmuscle cell contexts. Thus, these studies have identified a new cardiac-specific regulatory element (HF-1) which plays a role in both cardiac-specific and inducible expression during myocardial cell hypertrophy.
Animals↗
Heart-to-heart. New approaches for gene transfer in the myocardium.
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Animals↗
Endothelin induction of inositol phospholipid hydrolysis, sarcomere assembly, and cardiac gene expression in ventricular myocytes. A paracrine mechanism for myocardial cell hypertrophy.
The present study examined the effects of endothelin-1 on phosphoinositide hydrolysis, diacylglycerol formation, and the induction of myocardial cell hypertrophy utilizing a well characterized cultured neonatal rat myocardial cell model. In this system, a hypertrophic response can be assessed by increases in myocardial cell size, an increase in the assembly of an individual contractile protein (myosin light chain-2) into organized contractile units, accumulation of contractile proteins, the activation of a program of immediate early gene expression, and the induction of genes encoding contractile and embryonic proteins (Iwaki, K., Sukhatme, V., Shubeita, H.E., Chien, K.R., (1990) J. Biol. Chem. 265, 13809-13817). Utilizing these criteria, the present study documents that stimulation with endothelin-1 can produce myocardial cell hypertrophy, induce the expression and release of ANF in ventricular cells, and can activate the transcription of cardiac-specific genes. In addition, endothelin-1 stimulates phosphoinositide hydrolysis and the accumulation of diacylglycerol. It is proposed that endothelin-1 stimulation may represent an important paracrine mechanism for the in vivo regulation of cardiac growth and hypertrophy.
Animals↗
Alpha- and beta-adrenergic stimulation induces distinct patterns of immediate early gene expression in neonatal rat myocardial cells. fos/jun expression is associated with sarcomere assembly; Egr-1 induction is primarily an alpha 1-mediated response.
The present study was designed to determine if alpha- and beta-adrenergic stimulation of neonatal rat myocardial cells might induce common and/or distinct members of the immediate early gene program and to assess whether their induction might correlate with the differential effects of these adrenergic agonists on cardiac gene expression, sarcomere assembly, and several features of myocardial cell hypertrophy. Alpha- and beta-adrenergic stimulation of neonatal rat myocardial cells both produce an increase in the assembly of an individual contractile protein (myosin light chain-2) into organized sarcomeric units and also rapidly induce mRNAs for the immediate early genes c-fos and c-jun, thereby suggesting a potential role for these protooncogenes in sarcomerogenesis. alpha-Adrenergic stimulation results in the co-induction of mRNAs encoding a zinc finger protein gene (Egr-1). However, beta-adrenergic stimulation does not produce a significant increase in the levels of Egr-1 mRNA, providing the first evidence in any cell system that c-fos and Egr-1 expression can regulated separately. Studies with norepinephrine in combination with various adrenergic receptor antagonists revealed that the induction of Egr-1 is primarily an alpha 1-mediated, pertussis toxin-insensitive response. These studies provide the first evidence for the induction of immediate early genes following adrenergic stimulation of myocardial cells and demonstrate alpha- and beta-adrenergic stimulation can rapidly activate the expression of common and distinct subsets of these transcriptional regulators. Since alpha- and beta-adrenergic agonists have differential effects on cardiac gene expression and on the acquisition of several features of myocardial cell hypertrophy, the induction of Egr-1 provides a potential mechanism for the induction of genes that are exclusively induced during alpha-adrenergic-mediated myocardial cell hypertrophy.
Animals↗
Deacylated lipopolysaccharide inhibits plasminogen activator inhibitor-1, prostacyclin, and prostaglandin E2 induction by lipopolysaccharide but not by tumor necrosis factor-alpha.
Bacterial LPS and TNF induce vascular endothelial cells to express a variety of response molecules. LPS that is partially deacylated (dLPS) by a human neutrophil enzyme blocks the ability of LPS, but not TNF, to augment one of these responses, the expression of endothelial cell surface molecules that promote neutrophil adherence (J. Exp. Med. 1987; 165:1393-1402). We show that dLPS can inhibit the ability of LPS, but not TNF, to elicit the expression of plasminogen activator inhibitor-1 (PAI-1), prostacyclin, and PGE2 by human umbilical vein endothelial cells. dLPS also prevented the accumulation of specific PAI-1 mRNA in response to LPS, but not to TNF. Neither the LPS- or TNF-induced expression of PAI-1 nor the dLPS inhibition of the LPS response was mediated by prostanoids. These results indicate that dLPS can specifically block a variety of endothelial cell responses to LPS and provide support for the hypotheses 1) that dLPS and LPS may interact with a common target molecule on or in endothelial cells, and 2) that dLPS, produced by enzymatic deacylation of LPS in vivo, could inhibit endothelial cell stimulation by LPS and thereby limit the host inflammatory response to invasive gram-negative bacteria.
Acylation↗
Phorbol esters induce immediate-early genes and activate cardiac gene transcription in neonatal rat myocardial cells.
The mechanisms which transduce intracellular signals for the accumulation of myofibrillar protein during the onset of myocardial cell hypertrophy are unknown. Although previous studies in skeletal muscle cells have suggested that the activation of protein kinase C induces de-differentiation, including the selective disassembly of myofibrils and inhibition of myofibrillar protein synthesis, the present study demonstrates that phorbol esters which activate protein kinase C lead to the accumulation of an individual contractile protein, myosin light chain-2 (MLC-2) and produce several features of myocardial cell hypertrophy. Utilizing immunoblotting and indirect immunocytofluorescence with MLC antisera, the present study demonstrates a several-fold increase in the content of MLC-2, and a marked increase in the assembly of MLC into organized contractile units in individual neonatal rat myocardial cells following treatment with phorbol 12-myristate 13-acetate (PMA). The concentration of PMA required to elicit this response and the lack of a response with an inactive phorbol ester is consistent with the activation of a protein kinase C dependent pathway. Furthermore, PMA treatment results in the rapid induction of a program of immediate-early gene expression (including the c-fos and c-jun proto-oncogenes, and an inducible zinc finger containing gene, egr-l), and activates cardiac gene transcription as assessed by nuclear run-on analyses. The results of the present study suggest the possibility that a protein kinase C dependent pathway may be involved in the up-regulation of myofibrillar protein content and the activation of cardiac gene transcription during growth and hypertrophy of neonatal rat myocardium, and that the induction of a program of immediate-early gene expression may be linked to this response.
Animals↗