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K R Chien

Publications and source records attributed to K R Chien.

At least 91 records · Page 5Linked to original sources
PubMed1996

Neurofilament M mRNA is expressed in conduction system myocytes of the developing and adult rabbit heart.

We previously demonstrated that conduction-system myocytes of the rabbit heart express cytoskeletal proteins immunologically related to neurofilaments. In order to determine more precisely the nature of these proteins, we screened an expression cDNA library, prepared from the sino-atrial node region of the rabbit heart, using a monoclonal antibody which reacts with the M subunit of neurofilaments. Sequence analysis of the isolated cDNA clones shows high homology with rat and human neurofilament M mRNAs. Northern blot analysis demonstrates hybridization with a transcript expressed in brain, with the size expected for neurofilament M mRNA. An mRNA species of the same size is also detectable in the Northern blot of the sino-atrial node region RNA. In situ hybridization documents that in the adult rabbit the transcript accumulates in neurons and is localized in myocytes of the sino-atrial and atrio-ventricular nodes and of the atrio-ventricular bundle and bundle branches, but not in working atrial and ventricular myocytes. Developmental analysis was undertaken in order to determine the distribution of the neurofilament M mRNA in the rabbit embryonic heart. In situ hybridization shows that neurofilament M mRNA is detectable in a few ventricular myocytes in proximity to the atrio-ventricular groove after 9.5 days of embryonic development and it is accompanied by the presence of the protein. At subsequent stages of development neurofilament M mRNA is detectable in a number of cardiac myocytes, which are mainly localized at the atrio-ventricular junction and in the ventricular subendocardium and presumably correspond to myocytes of the heart conduction system.

Amino Acid Sequence↗
PubMed1996

Cardiotrophin-1: a multifunctional cytokine that signals via LIF receptor-gp 130 dependent pathways.

In a search for novel factors that induce cardiac myocyte hypertrophy, cardiotrophin-1 (CT-1) was identified by coupling expression cloning with an embryonic stem cell-based model of cardiogenesis. CT-1 is a new member of the IL-6 family of cytokines that induce their biological effects through the shared signaling subunit, gp 130. The expression pattern of CT-1 and its range of activities in the hematopoietic, neuronal, and developmental assays suggest that CT-1 may play an important role in other organ systems, in addition to its actions in cardiac development and hypertrophy.

Amino Acid Sequence↗
PubMed1996

Strategies for studying cardiovascular phenotypes in genetically manipulated mice.

Unraveling the pathogenesis of complex cardiovascular diseases, such as hypertension, requires the development of in vivo animal model systems. Although large-animal models have long served as the gold standard, recent advances in transgenic and gene-targeting approaches, mouse genetics, and microsurgical technology are initiating a revolution that has led to the unexpected coupling of in vivo molecular physiology with genetically engineered mice. This article discusses the design of strategies to study complex cardiovascular phenotypes in genetically modified mice, including both transgenic and gene-targeted animals. At this time, a number of strategies are used to address specific molecular or physiological questions, and examples are briefly highlighted. In addition, a number of potential problems in the generation and use of transgenic mice in the study of cardiovascular biology are presented.

Animals↗
PubMed1996

Developmental changes in ionic channel activity in the embryonic murine heart.

We have isolated murine embryonic atrial and ventricular cells derived from timed-pregnant females at different periods and used patch-clamp procedures to investigate age- and chamber-specific expression of ionic channels in the developing fetal mouse. Our data indicate that L-type Ca2+ channels play a dominant role in excitation during early murine cardiac embryogenesis and that Na+ channel expression increases dramatically just before birth. K+ channel expression is particularly sensitive to changes during development. Neither atrial nor ventricular cells express a slowly activating component of delayed rectification (IKs) until just before birth, and inwardly rectifying channel activity, associated with determination of cellular resting potential, is not markedly apparent until late stages of embryogenesis. Instead, we find robust expression of the ATP-regulated K+ channel at early and late states of embryonic development, which may indicate a novel functional role for this channel during morphogenesis of the heart. These results have important implications for the physiology and development of the murine cardiac conduction system and will also serve as a baseline for future studies designed to investigate developmental changes of ion channel expression in the myocardium of both wild-type and genetically modified mice.

Adenosine Triphosphate↗
PubMed1996

Developmental changes in beta-adrenergic modulation of L-type Ca2+ channels in embryonic mouse heart.

In the adult mammalian myocardium, cellular Ca2+ entry is regulated by the sympathetic nervous system. L-type Ca2+ channel currents are markedly increased by beta-adrenergic (beta-A) agonists, which contribute to changes in pacing and contractile activity of the heart. In the developing mammalian heart, the regulation of Ca2+ entry by this enzyme cascade has not been clearly established, because changes in receptor density and coupling to downstream elements of the signaling cascade are known to occur during embryogenesis. In this study, we systematically investigated the regulation of L-type Ca2+ channel currents during development of the murine embryonic heart. We used conventional whole-cell and perforated-patch-clamp procedures to study modulation of L- type Ca2+ channel currents and to assay functional activity of distinct steps in the beta-A signaling cascade in murine embryonic myocytes at different stages of gestation. Our data indicate that the L-type Ca2+ channels in early-stage (day-11 to -13) myocytes are unresponsive to either isoproterenol or cAMP. L-type Ca2+ channels in late-stage (day-17 to -19) murine myocytes, however, exhibit responses to isoproterenol and cAMP similar to responses in adult cells, providing evidence that the beta-A cascade becomes functionally active during this period of embryonic development. We found that L-type Ca2+ channel activity in early-stage cells is increased by cell dialysis with the catalytic subunit of cAMP-dependent protein kinase (cA-PK) and that dialysis of early-stage cells with the holoenzyme of cA-PK restores functional responses to forskolin and cAMP, but not to isoproterenol. Our results provide strong evidence that a key factor in the early-stage insensitivity of L-type Ca2+ channels to cAMP is the absence, or low expression level, of the holoenzyme of cA-PK but that in addition, another element in the signaling cascade upstream from adenylate cyclase is expressed at a nonfunctional level or is uncoupled from the cascade and thus contributes to L-type Ca2+ channel insensitivity to beta-A agonists in early stages of the developing murine heart.

Adrenergic beta-Agonists↗
PubMed1996

Cardiotrophin-1 displays early expression in the murine heart tube and promotes cardiac myocyte survival.

We have recently isolated a novel cytokine, cardiotrophin-1 (CT-1), from an in vitro embryonic stem cell system of cardiogenesis that can activate embryonic markers in neonatal rat cardiac myocytes. CT-1 is a new member of the interleukin 6 (IL-6)/leukemia inhibitory factor (LIF) cytokines, which activate downstream signals via gp130-dependent pathways. To define the developmental pattern of expression of CT-1 during murine embryogenesis, we have developed antibodies directed against a CT-1 fusion protein. As assessed by immunolocalization, CT-1 is predominantly expressed in the early mouse embryonic heart tube (E8.5-10.5). In the heart, CT-1 is primarily expressed in myocardial cells, and not in endocardial cushion or outflow tract tissues. After E12.5, CT-1 expression is found in other tissues, including skeletal, liver and dorsal root ganglia. Given the effects of a related family member (ciliary neurotrophic factor, CNTF) on neuronal cell survival, we studied the ability of CT-1 to promote cardiac myocyte survival and proliferation in vitro. Both CT-1 and LIF, which share the same receptors, dramatically promote neonatal cardiac myocyte survival, while IL-6 and CNTF are without effect. A cell proliferation assay documents that CT-1 provokes an approximate 2-fold increase in embryonic cardiac myocyte proliferation. Thus, CT-1 may play an autocrine role during cardiac chamber growth and morphogenesis by promoting the survival and proliferation of immature myocytes, most likely via gp130-dependent signaling pathways.

Animals↗
PubMed1996

An HF-1a/HF-1b/MEF-2 combinatorial element confers cardiac ventricular specificity and established an anterior-posterior gradient of expression.

The molecular determinants that direct gene expression to the ventricles of the heart are for the most part unknown. Additionally, little data is available on how the anterior/posterior axis of the heart tube is determined and whether the left and right atrial and ventricular chambers are assigned as part of this process. Utilizing myosin light chain-2 ventricular promoter/beta-galactosidase reporter transgenes, we have determined the minimal cis-acting sequences required for ventricular-specific gene expression. In multiple independent transgenic mouse lines, we found that both a 250 base pair myosin light chain-2 ventricular promoter fragment, as well as a dimerized 28 bp sub-element (HF-1) containing binding sites for HF1a and HF1b/MEF2 factors, directed ventricular-specific reporter expression from as early as the endogenous gene, at day 7.5-8.0 post coitum. While the endogenous gene is expressed uniformly throughout both ventricles, the transgenes were expressed in a right ventricular/conotruncal dominant fashion, suggesting that they contain only a subset of the elements which respond to positional information in the developing heart tube. Expression of the transgene was cell autonomous and its temporospatial characteristics not affected by mouse strain/methylation state of the genome. To determine whether ventricular-specific expression of the transgene was dependent upon regulatory genes required for correct ventricular differentiation, the 250 base pair transgene was bred into both retinoid X receptoralpha and Nkx2-5 null backgrounds. The transgene was expressed in both mutant backgrounds, despite the absence of endogenous myosin light chain-2 ventricular transcript in Nkx2-5 null embryos. Ventricular specification, as judged by transgene expression, appeared to occur normally in both mutants. Thus, the HF-1 element, directs chamber-specific transcription of a transgene reporter independently of retinoid X receptoralpha and Nkx2-5, and defines a minimal combinatorial pathway for ventricular chamber gene expression. The patterned expression of this transgene may provide a model system in which to investigate the cues that dictate anterior-posterior (right ventricle/left ventricle) gradients during mammalian heart development.

Animals↗
PubMed1996

The molecular genetics of retinoic acid receptors: cardiovascular and limb development.

The vitamin A metabolite retinoic acid (RA) is utilized as a signalling molecule in wide variety of developmental processes, defined by defects which occur after nutritional vitamin A deficiency or after exposure to excess vitamin A. We have initiated a genetic analysis of RA function through the establishment of lines of mice which carry germline mutations in the genes which encode retinoid receptors. Defects which result from developmental RA deficiency or excess have been recovered in embryos which are deficient in various combinations of retinoid receptors. In this chapter, our current understanding of the role of RA and retinoid receptors in cardiovascular and limb development are described, as for these our level of understanding is most advanced.

Animals↗
PubMed1995

Ventricular expression of a MLC-2v-ras fusion gene induces cardiac hypertrophy and selective diastolic dysfunction in transgenic mice.

p21ras has been implicated in the hypertrophic response of cultured cardiac myocytes to defined growth stimuli. To determine if activation of ras-dependent intracellular signaling pathways is sufficient to induce in vivo hypertrophy, transgenic mice were created that express oncogenic ras in the cardiac ventricular chamber. Mice homozygous for the transgene displayed morphological, physiological, and genetic markers of marked cardiac muscle hypertrophy. Miniaturized catheterization technology documented a selective prolongation of cardiac relaxation, similar to that seen in early human hypertrophic heart disease. An increase in left atrial mass, in the absence of transgene expression in that chamber, further supported physiologically abnormal left ventricular diastolic function. Histological analysis revealed myofibrillar disarray, indistinguishable from that in hypertrophic cardiomyopathy in man. These studies establish a ras-dependent pathway for hypertrophic heart disease and document the feasibility of mapping in vivo signaling pathways for cardiac hypertrophy and dysfunction by applying in vivo microphysiological assays to genetically manipulated mice. ras-dependent pathways may also be a rational target for developing new approaches to inhibit the genesis of hypertrophy in certain pathological settings.

Animals↗
PubMed1995

Atrial-like phenotype is associated with embryonic ventricular failure in retinoid X receptor alpha -/- mice.

We have recently characterized a cardiac model of ventricular chamber defects in retinoid X receptor alpha (RXR alpha) homozygous mutant (-/-) gene-targeted mice. These mice display generalized edema, ventricular chamber hypoplasia, and muscular septal defects, and they die at embryonic day 15. To substantiate our hypothesis that the embryos are dying of cardiac pump failure, we have used digital bright-field and fluorescent video microscopy and in vivo microinjection of fluorescein-labeled albumin to analyze cardiac function. The affected embryos showed depressed ventricular function (average left ventricular area ejection fraction, 14%), ventricular septal defects, and various degrees of atrioventricular block not seen in the RXR alpha wild-type (+/+) and heterozygous (+/-) littermates (average left ventricular area ejection fraction, 50%). The molecular mechanisms involved in these ventricular defects were studied by evaluating expression of cardiac-specific genes known to be developmentally regulated. By in situ hybridization, aberrant, persistent expression of the atrial isoform of myosin light chain 2 was identified in the ventricles. We hypothesize that retinoic acid provides a critical signal mediated through the RXR alpha pathway that is required to allow progression of development of the ventricular region of the heart from its early atrial-like form to the thick-walled adult ventricle. The conduction system disturbances found in the RXR alpha -/- embryos may reflect a requirement of the developing conduction system for the RXR alpha signaling pathway, or it may be secondary to the failure of septal development.

Animals↗
PubMed1995

Retinoid-dependent pathways suppress myocardial cell hypertrophy.

Utilizing an in vitro model system of cardiac muscle cell hypertrophy, we have identified a retinoic acid (RA)-mediated pathway that suppresses the acquisition of specific features of the hypertrophic phenotype after exposure to the alpha-adrenergic receptor agonist phenylephrine. RA at physiological concentrations suppresses the increase in cell size and induction of a genetic marker for hypertrophy, the atrial natriuretic factor (ANF) gene. RA also suppresses endothelin 1 pathways for cardiac muscle cell hypertrophy, but it does not affect the increase in cell size and ANF expression induced by serum stimulation. A trans-activation analysis using a transient transfection assay reveals that neonatal rat ventricular myocardial cells express functional RA receptors of both the retinoic acid receptor and retinoid X receptor (RAR and RXR) subtypes. Using synthetic agonists of RA, which selectively bind to RXR or RAR, our data indicate that RAR/RXR heterodimers mediate suppression of alpha-adrenergic receptor-dependent hypertrophy. These results suggest the possibility that a pathway for suppression of hypertrophy may exist in vivo, which may have potential therapeutic value.

Animals↗
PubMed1995

Expression cloning of cardiotrophin 1, a cytokine that induces cardiac myocyte hypertrophy.

Heart failure continues to be a leading cause of mortality worldwide. A hallmark of this disease is dilated cardiac hypertrophy, which is accompanied by a reactivation of genes expressed in fetal heart development. Reasoning that fetal or embryonic growth factors may mediate the onset of cardiac hypertrophy, we have coupled expression cloning with an embryonic stem cell-based model of cardiogenesis to isolate a 21.5-kDa protein, cardiotrophin 1, that potently induces cardiac myocyte hypertrophy in vitro. Amino acid similarity data indicate that cardiotrophin 1 is a member of the leukemia inhibitory factor/ciliary neurotrophic factor/oncostatin M/interleukin 6/interleukin 11 family of cytokines. Several members of this family that are known to signal through the transmembrane protein gp130 stimulate cardiac myocyte hypertrophy, like cardiotrophin 1, suggesting that the gp130 signaling pathway may play a role in cardiac hypertrophy. A 1.4-kb cardiotrophin 1 mRNA is expressed in the heart and several other mouse tissues.

Amino Acid Sequence↗
PubMed1995

Inhibition of a signaling pathway in cardiac muscle cells by active mitogen-activated protein kinase kinase.

Signaling via the Ras pathway involves sequential activation of Ras, Raf-1, mitogen-activated protein kinase kinase (MKK), and the extracellular signal-regulated (ERK) group of mitogen-activated protein (MAP) kinases. Expression from the c-Fos, atrial natriuretic factor (ANF), and myosin light chain-2 (MLC-2) promoters during phenylephrine-induced cardiac muscle cell hypertrophy requires activation of this pathway. Furthermore, constitutively active Ras or Raf-1 can mimic the action of phenylephrine in inducing expression from these promoters. In this study, we tested whether constitutively active MKK, the molecule immediately downstream of Raf, was sufficient to induce expression. Expression of constitutively active MKK induce ERK2 kinase activity and caused expression from the c-Fos promoter, but did not significantly activate expression of reporter genes under the control of either the ANF or MLC-2 promoters. Expression of CL100, a phosphatase that inactivates ERKs, prevented expression from all of the promoters. Taken together, these data suggest that ERK activation is required for expression from the Fos, ANF, and MLC-2 promoters but MKK and ERK activation is sufficient for expression only from the Fos promoter. Constitutively active MKK synergized with phenylephrine to increase expression from a c-Fos- or an AP1-driven reporter. However, active MKK inhibited phenylephrine- and Raf-1-induced expression from the ANF and MLC-2 promoters. A DNA sequence in the MLC-2 promoter that is a target for inhibition by active MKK, but not CL100, was mapped to a previously characterized DNA element (HF1) that is responsible for cardiac specificity. Thus, activation of cardiac gene expression during phenylephrine-induced hypertrophy requires ERK activation but constitutive activation by MKK can inhibit expression by targeting a DNA element that controls the cardiac specificity of gene expression.

Animals↗
PubMed1995

EFIA/YB-1 is a component of cardiac HF-1A binding activity and positively regulates transcription of the myosin light-chain 2v gene.

Transient assays in cultured ventricular muscle cells and studies in transgenic mice have identified two adjacent regulatory elements (HF-1a and HF-1b/MEF-2) as required to maintain ventricular chamber-specific expression of the myosin light-chain 2v (MLC-2v) gene. A rat neonatal heart cDNA library was screened with an HF-1a binding site, resulting in the isolation of EFIA, the rat homolog of human YB-1. Purified recombinant EFIA/YB-1 protein binds to the HF-1a site in a sequence-specific manner and contacts a subset of the HF-1a contact points made by the cardiac nuclear factor(s). The HF-1a sequence contains AGTGG, which is highly homologous to the inverted CCAAT core of the EFIA/YB-1 binding sites and is found to be essential for binding of the recombinant EFIA/YB-1. Antiserum against Xenopus YB-3 (100% identical in the DNA binding domain and 89% identical in overall amino acid sequence to rat EFIA) can specifically abolish a component of the endogenous HF-1a complex in the rat cardiac myocyte nuclear extracts. In cotransfection assays, EFIA/YB-1 increased 250-bp MLC-2v promoter activity by 3.4-fold specifically in the cardiac cell context and in an HF-1a site-dependent manner. EFIA/YB-1 complexes with an unknown protein in cardiac myocyte nuclear extracts to form the endogenous HF-1a binding activity. Immunocoprecipitation revealed that EFIA/YB-1 has a major associated protein of approximately 30 kDa (p30) in cardiac muscle cells. This study suggests that EFIA/YB-1, together with the partner p30, binds to the HF-1a site and, in conjunction with HF-1b/MEF-2, mediates ventricular chamber-specific expression of the MLC-2v gene.

Animals↗