Search PubMed⌕ Search

Biomedical subjects

K R Chien

Publications and source records attributed to K R Chien.

At least 109 records · Page 6Linked to original sources
PubMed1995

Cardiac muscle diseases in genetically engineered mice: evolution of molecular physiology.

Recent advances in molecular, cellular, and genetically based technologies now offer the possibility of generating genetically engineered mice that display physiological phenotypes with direct relevance to human pathophysiological states. The ability to create gene ablations, gene duplications, and gene modifications should allow the use of genetic approaches to map in vivo pathways responsible for complex physiological phenotypes. Recent work from our laboratory utilizing this approach to study cardiac muscle diseases in both the adult context (cardiac hypertrophy) and in the embryonic context (congenital ventricular defects) will be discussed, as well as the steps that led to the generation and characterization of these novel mouse model systems. A large body of work from independent laboratories now points to the inception of a new field of molecular physiology that will fuse mouse genetics and in vivo physiology using appropriate miniaturized physiological technology. Recent advances and prospects for future directions are summarized.

Animals↗
PubMed1995

Insulin-like growth factor-1 enhances ventricular hypertrophy and function during the onset of experimental cardiac failure.

To determine whether additional hypertrophy would be beneficial or maladaptive in cardiac failure, the effects of insulin-like growth factor (IGF-1) were investigated in rats with left ventricular (LV) dysfunction. In normal rats, 3 mg/kg per d of recombinant human IGF-1 for 14 d augmented LV wt (32%) and increased LV/body wt ratio (P < 0.01). 2 d after coronary occlusion, rats were randomized to IGF-1 (3 mg/kg per d) or placebo. After 2 wk, IGF-1-treated rats showed significant increases in LV wt (13%) and LV wt/tibial length ratio, but LV/body wt ratio was unchanged. By microangiography, compared with controls (n = 12) IGF-1-treated rats (n = 16) showed increased LV end-diastolic volume (19%) and stroke volume (31%) (both significant normalized to tibial length, but not to body wt). Average infarct size did not differ between groups. The LV ejection fraction (EF) was not significantly different between groups, but estimated cardiac output was higher in treated rats; there was a significant interaction for the EF between infarct size and treatment (P = 0.029) and a trend for EF to be higher in treated rats with large infarctions (EF 33.4 vs 25.1% in controls). Myocyte cross-sectional areas in noninfarcted LV zones tended to be larger in treated rats (232.1 vs 205.4 microns 2; P = 0.10), but there was no difference in capillary density and collagen content did not differ between groups. In conclusion, IGF-1 administration caused hypertrophy of the normal heart in vivo. When stimulated by IGF-1, the severely dysfunctional heart in evolving myocardial infarction is capable of undergoing additional hypertrophy with evidence of improved function, suggesting a beneficial effect. Further investigation of the potential role of growth factor therapy in heart failure appears warranted.

Animals↗
PubMed1995

Divergent pathways mediate the induction of ANF transgenes in neonatal and hypertrophic ventricular myocardium.

To determine whether similar or divergent pathways mediate atrial natriuretic factor (ANF) induction in neonatal and hypertrophied adult ventricular myocardium, and to assess whether studies using an in vitro model system of hypertrophy have fidelity to the in vivo context during pressure overload hypertrophy, we generated transgenic mice which harbor either 638 or 3,003 bp of the rat ANF 5' flanking region ligated upstream from a luciferase reporter. Luciferase activity in the ventricles of day 1 transgenic neonates was 8-24-fold higher than the levels expressed in the ventricles of adult mice. Adult mice expressed the luciferase reporter in an appropriate tissue-specific manner. Transverse aortic constriction of adult mice harboring ANF reporter transgenes demonstrated no significant increase in reporter activity in the ventricle. These findings demonstrate that distinct regions of the ANF 5'-flanking region are required for inducible expression of the ANF gene in the hypertrophic adult ventricle compared with those required for atrial-specific and developmentally appropriate expression in the intact neonatal heart. Furthermore, the cis regulatory elements necessary for induction of ANF expression in endothelin-1 or alpha 1-adrenergically stimulated cultured neonatal ventricular myocytes are not sufficient for induction in the in vivo context of pressure overload hypertrophy.

Animals↗
PubMed1994

Chamber specification of atrial myosin light chain-2 expression precedes septation during murine cardiogenesis.

To study the molecular mechanisms that control patterning of the heart tube during early cardiogenesis, we have used the ventricular myosin regulatory light chain (MLC-2v), which is expressed in the ventricular segment of the primitive heart tube, as a genetic marker for ventricular specification in rodents. To assess whether the atrial isoform, MLC-2a, could also serve as a chamber-specific marker, we cloned an atrial MLC-2 cDNA (554 base pairs) which displayed homology to the human MLC-2a cDNA at both the nucleotide (87%) and amino acid (95%) levels. Northern blot, reverse transcriptase-linked polymerase chain reaction, RNase protection, and Western blot analysis revealed atrial restricted expression in the adult mouse heart, very low levels in aorta, and no detectable expression in ventricle, skeletal muscle, uterus, or liver. In situ hybridization studies during mouse embryogenesis revealed cardiac specific expression throughout days 8-16 postcoitum, with atrial restricted expression from day 12 and qualitatively greater atrial expression than ventricular from day 9. Thus, preferential pattern of expression in the atria occurs prior to septation. The MLC-2a gene was differentially regulated when compared with MLC-2v expression during embryonic stem cell cardiogenesis in vitro with MLC-2a transcript levels detectable from day 6 in suspension cultures as compared with day 9 for MLC-2v. The region-specific expression of the MLC-2a and MLC-2v genes in their respective chambers during early cardiogenesis provides genetic markers for chamber specification (atrial and ventricular) in both the in vitro and in vivo context.

Amino Acid Sequence↗
PubMed1994

Gq- and ras-dependent pathways mediate hypertrophy of neonatal rat ventricular myocytes following alpha 1-adrenergic stimulation.

alpha 1-Adrenergic agonists activate a hypertrophic response in cultured neonatal ventricular myocytes, which include an increase in cell size, organization of contractile proteins into sarcomeric units, and the induction of the atrial natriuretic factor (ANF) gene. Previous findings have supported a role for ras in this signaling pathway. Utilizing microinjection techniques to delivery affinity-purified neutralizing antibodies to G alpha q,11 into cultured ventricular myocytes, the current studies demonstrate a functional requirement for the heterotrimeric G protein, Gq, in the alpha 1-adrenergic induction of the ANF gene, changes in cell size, organization of myofilaments, and phosphoinositide hydrolysis. Expression of a constitutively active mutant of G alpha q leads to the expression of ANF protein in these cells. Taken together, these data suggest that G q-dependent pathways are necessary and sufficient to activate defined features of the hypertrophic response. In attempts to further delineate the relative roles of ras and Gq in this pathway, we found that G alpha q is required for alpha 1-adrenergic phosphoinositide hydrolysis, though ras does not appear to be necessary for this response. In addition, we coexpressed an inhibitory ras mutant, along with the constitutively active G alpha q. Expression of ANF protein stimulated by the G alpha q mutant was not inhibited. Thus, both ras- and Gq-dependent pathways are necessary to fully transduce defined features of alpha 1-adrenergic-stimulated hypertrophy of neonatal cardiac ventricular myocytes, but activated Gq may be able to induce ANF expression independent of inhibitory ras.

Animals↗
PubMed1994

RXR alpha mutant mice establish a genetic basis for vitamin A signaling in heart morphogenesis.

We have established a targeted loss-of-function mutation in the RXR alpha gene in the mouse germ line that results in embryonic lethality between E13.5 and E16.5 when bred to homozygosity. The major defect responsible for lethality is hypoplastic development of the ventricular chambers of the heart, which is manifest as a grossly thinned ventricular wall with concurrent defects in ventricular septation. This phenotype is identical to a subset of the effects of embryonic vitamin A deficiency and, therefore, establishes RXR alpha as a genetic component of the vitamin A signaling pathway in cardiac morphogenesis. The cardiac outflow tracts and associated vessels, which are populated by derivatives of the neural crest and which are also sensitive to vitamin A deficiency, are normal in homozygous embryos, indicating the genetic independence of ventricular chamber development. Hepatic differentiation was dramatically but transiently retarded yet is histologically and morphologically normal. These results ascribe an essential function for the RXR alpha gene in embryonic development and provide the first evidence of a requirement for RXR in one of its predicted hormone response pathways.

Animals↗
PubMed1994

Enhanced myocardial function in transgenic mice overexpressing the beta 2-adrenergic receptor.

Transgenic mice were created with cardiac-specific overexpression of the beta 2-adrenergic receptor. This resulted in increased basal myocardial adenylyl cyclase activity, enhanced atrial contractility, and increased left ventricular function in vivo; these parameters at baseline in the transgenic animals were equal to those observed in control animals maximally stimulated with isoproterenol. These results illustrate a useful approach for studying the effect of gene expression on cardiac contractility. Because chronic heart failure in humans is accompanied by a reduction in the number of myocardial beta-adrenergic receptors and in inotropic responsiveness, these results suggest a potential gene therapy approach to this disease state.

Adenylyl Cyclases↗
PubMed1994

Molecular and physiological alterations in murine ventricular dysfunction.

The present study reports the development and characterization of a murine model of right ventricular dysfunction following graded constriction in the pulmonary artery via microsurgical approaches. To analyze in vivo ventricular function, a technique of x-ray contrast microangiography was developed to allow the quantitative analysis of ventricular volumes and of ejection fraction in normal and pressure-overloaded right ventricle. Severe, chronic pulmonary arterial banding for 14 days resulted in right ventricular dilatation and dysfunction, associated with right atrial enlargement, and angiographic evidence of tricuspid regurgitation. These effects were dependent on the extent of hemodynamic overload, since more moderate pulmonary arterial constriction resulted in hypertrophy with maintenance of right ventricular function. With severe pulmonary artery constriction, the murine right ventricle displays a failing heart phenotype including chamber dilation with reduced function that resembles right ventricular dysfunction in man during chronic pulmonary arterial hypertension. Northern and immunoblot analyses demonstrate a marked down-regulation of phospholamban mRNA and its corresponding protein with both levels of constriction, while a less pronounced but significant depression of sarcoplasmic reticulum Ca(2+)-ATPase protein was observed with severe overload, suggesting that this pattern is an early genetic marker of ventricular dysfunction. By coupling mouse genetics with this murine model and the ability to assess cardiac function in vivo, one should be able to test the role of the down-regulation of phospholamban and other defined alterations in the cardiac muscle gene program in the onset of the failing heart phenotype.

Adenosine Triphosphatases↗
PubMed1994

The basic helix-loop-helix protein upstream stimulating factor regulates the cardiac ventricular myosin light-chain 2 gene via independent cis regulatory elements.

Previous studies have documented that 250 bp of the rat cardiac ventricular myosin light-chain 2 (MLC-2v) promoter is sufficient to confer cardiac muscle-specific expression on a luciferase reporter gene in both transgenic mice and primary cultured neonatal rat myocardial cells. Utilizing ligation-mediated PCR to perform in vivo dimethyl sulfate footprinting, the present study has identified protein-DNA interaction within the position from -176 to -165. This region, identified as MLE1, contains a core sequence, CACGTG, which conforms to the consensus E-box site and is identical to the upstream stimulating factor (USF)-binding site of the adenovirus major late promoter. Transient assays of luciferase reporter genes containing point mutations of the site demonstrate the importance of this cis regulatory element in the transcriptional activation of this cardiac muscle gene in ventricular muscle cells. The protein complex that occupies this site is capable of binding to HF-1a and PRE B sites which are known to be required for cardiac muscle-specific expression of rat MLC-2v and alpha-myosin heavy-chain genes, respectively. This study provides direct evidence that USF, a member of the basic helix-loop-helix leucine zipper family, binds to MLE1, HF-1a, and PRE B sites and suggests that it is a component of protein complexes that may coordinately control the expression of MLC-2v and alpha-myosin heavy-chain genes. The current study also provides evidence that USF can positively and negatively regulate the MLC-2v gene via independent cis regulatory elements.

Animals↗
PubMed1994

Positive regulatory elements (HF-1a and HF-1b) and a novel negative regulatory element (HF-3) mediate ventricular muscle-specific expression of myosin light-chain 2-luciferase fusion genes in transgenic mice.

The cardiac myosin light-chain 2v (MLC-2v) gene has served as a model system to identify the pathways which restrict the expression of cardiac muscle genes to particular chambers of the heart during cardiogenesis. To identify the critical cis regulatory elements which mediate ventricular chamber-specific expression of the MLC-2v gene in the in vivo context, a series of transgenic mice which harbor mutations in putative MLC-2 cis regulatory elements in a 250-bp MLC-2-luciferase fusion gene which is expressed in a ventricular chamber-specific fashion in transgenic mice were generated. These studies demonstrate that both components of HF-1 (HF-1a and HF-1b/MEF-2) are required to maintain ventricular chamber-specific expression and function as positive regulatory elements. Mutations in another conserved element (HF-2) are without statistically significant effect on ventricular chamber expression. Transgenics harboring mutations in the E-box site also displayed significant upregulation of reporter activity in the soleus, gastrocnemius, and uterus, with a borderline effect on expression in liver. Mutations in another conserved element (HF-3) result in a marked (> 75-fold) upregulation of the luciferase reporter activity in the soleus muscle of multiple independent or transgenic founders. Since the HF-3 mutations appeared to have only a marginal effect on luciferase reporter activity in liver tissue, HF-3 appears to function as a novel negative regulatory element to primarily suppress expression in muscle tissues. Thus, a combination of positive (HF-1a/HF-1b) and negative (E-box and HF-3) regulatory elements appear to be required to maintain ventricular chamber-specific expression in the in vivo context.

Animals↗
PubMed1994

Heterokaryons of cardiac myocytes and fibroblasts reveal the lack of dominance of the cardiac muscle phenotype.

The molecular characterization of a cardiac determination gene has been an elusive goal for the past several years. Prior to cloning of the skeletal muscle determination factor MyoD, the presence of a dominantly acting skeletal muscle determination factor had been inferred from the observation that the skeletal muscle phenotype was dominant in skeletal muscle-fibroblast heterokaryons (H. M. Blau, G. K. Pavlath, E. C. Hardeman, C.-P. Chiu, L. Siberstein, S. G. Webster, S. C. Miller, and D. Webster, Science 230:758-766, 1985). In these experiments, we have examined cardiac-fibroblast heterokaryons to investigate the existence of a dominantly acting cardiac determination factor. We have employed a novel experimental approach using primary embryonic fibroblasts from transgenic mice as a means of assaying for the activation of a cardiac promoter-luciferase reporter transgene within fibroblast nuclei. This approach provides a potential means of genetic selection for a dominantly acting positive factor and can be generalized to other systems. We have examined the expression of three markers of the cardiac lineage: a myofibrillar protein promoter (MLC2), a secreted protein (ANF), and a transcription factor (MEF2). MEF2 is specific to both cardiac and skeletal muscle cells. Our results indicate that in a majority of heterokaryons with an equal ratio of cardiac to fibroblast nuclei, none of these cardiac markers are expressed, indicating that the cardiac phenotype is not dominant over the embryonic fibroblast phenotype. The distinction from previous results with skeletal muscle is emphasized by our results with MEF2, which is dominantly expressed in skeletal muscle-fibroblast but not cardiac-fibroblast heterokaryons, supporting its divergent regulation in the two cell types.

Animals↗
PubMed1994

Genetic engineering of cardiac muscle cells: in vitro and in vivo.

Over the last few years many of the difficulties encountered in applying molecular biology techniques to cardiac muscle have been addressed. Although continuous cardiac cell lines are still, as yet, unavailable, progress in embryonic stem cell systems and in our fundamental understanding of the critical steps that lead to cessation of cardiac myocyte cell division suggests that one may soon emerge. The cis-acting promoter elements and transacting factors responsible for cardiac specificity and inducible expression of cardiac genes are being characterized at a rapid pace. Transgenic models of cardiac disease are being generated and new insights are being made towards the goal of gene therapy in the heart. As the repertoire of techniques for transferring DNA into heart cells continues to expand, so the prospect for efficient and long-term expression of exogenous DNA in heart cells improves. The recent rapid rate of progress promises that ideas once fanciful may ultimately be within our reach.

Animals↗
PubMed1993

In vitro chamber specification during embryonic stem cell cardiogenesis. Expression of the ventricular myosin light chain-2 gene is independent of heart tube formation.

The molecular cues that control patterning of the heart tube during early cardiogenesis are largely unknown. The present study has explored the embryonic stem (ES) cell differentiation system to determine if this in vitro model could be useful in studying the process of regional specification of cardiac muscle cells at the earliest possible stages. As assessed by polymerase chain reaction, ribonuclease protection, in situ hybridization, and immunohistochemical analyses, ES cell differentiation into embryoid bodies is characterized by the transcriptional and translational activation of the ventricular regulatory (phosphorylatable) myosin light chain gene, demonstrating that ventricular specification occurs during ES cell cardiogenesis. The finding of a ventricular-specific marker in an in vitro system in the absence of an intact heart tube provides evidence for cardiac regional specification independent of positional cues or physiologic stimuli. The temporal expression of the myogenic regulatory factors, myogenin and MyoD, suggests activation of the skeletal muscle program following cardiac myogenesis in vitro, indicating temporal fidelity to the progression of in vivo myogenesis. These data establish the mouse embryonic stem cell system as a model for cardiac chamber specification and suggest a promising approach in the study of regional specification in genetically engineered cardiac muscle cells.

Animals↗
PubMed1993

The alpha 1A-adrenergic receptor subtype mediates biochemical, molecular, and morphologic features of cultured myocardial cell hypertrophy.

alpha 1-Adrenergic agonists induce a hypertrophic phenotype in cultured neonatal rat ventricular myocytes. Quantifiable markers of this phenotype include stimulation of phosphoinositide hydrolysis, transcriptional induction of atrial natriuretic factor (ANF) gene expression, and an increase in myocardial cell size. The aim of the present work was to determine which alpha 1-adrenergic receptor subtype mediates the acquisition of these parameters of myocardial cell hypertrophy. Phosphoinositide hydrolysis is inhibited by low concentrations of 5-methylurapidil (log Ki = -8.7) and (+)-niguldipine (log Ki = -10.6). The alpha-adrenergic receptor-induced increase in transcriptional activation of an ANF luciferase reporter gene is inhibited over the same range of concentrations of 5-methylurapidil (log Ki = -8.2) and (+)-niguldipine (log Ki = -11.2) that inhibit phosphoinositide hydrolysis. In addition, the increase in cell size that accompanies alpha-adrenergic receptor stimulation of cultured ventricular myocytes is blocked by similar concentrations of 5-methylurapidil (log Ki = -8.0) and (+)-niguldipine (log Ki = -10.6). In contrast, treatment with the alpha 1B selective antagonist chlorethylclonidine at a concentration of 10 microM had no effect on the adrenergically mediated induction of ANF luciferase reporter gene expression or the adrenergically induced increase in myocardial cell size. These findings demonstrate that pharmacologically identifiable alpha 1A-adrenergic receptors mediate not only the early effects of alpha 1-adrenergic stimulation such as phosphoinositide hydrolysis, but that they activate the signaling pathways that control transcriptional induction of the ANF luciferase reporter gene and an increase in myocardial cell size. Studies using alpha 1-adrenergic receptor cDNAs to delineate and alter the direct interaction of this receptor subtype with proximal signaling molecules, e.g. GTP binding proteins, should provide a powerful means of assessing their role in the induction of the molecular and morphologic parameters of myocardial cell hypertrophy.

Animals↗
PubMed1993

Positional specification of ventricular myosin light chain 2 expression in the primitive murine heart tube.

To study the process of ventricular specification during cardiogenesis, we examined the in situ expression of cardiac ventricular myosin light chain 2 (MLC-2v) mRNA during murine embryogenesis. As assessed by hybridization with a specific MLC-2v riboprobe, mRNA expression can be found in the ventricular region at day 8.0 postcoitum (pc). MLC-2v expression is high in the ventricular portion of the heart tube, with no detectable expression in the atrial or sinus venosus regions. The proximal outflow tract of the heart tube also expresses MLC-2v mRNA at minimally detectable levels at this time but then displays a temporally and spatially distinct pattern with expression well established in the proximal out-flow tract region adjacent to the ventricular segment by days 9-10 pc, eventually reaching levels comparable to the trabeculated ventricular myocardium. By day 11 pc, prior to the completion of septation, expression then becomes restricted to the ventricular region at and below the level of the atrioventricular cushion. Transgenic mice harboring a 250-base-pair MLC-2v promoter fragment fused to a luciferase reporter gene demonstrate reporter gene activity from at least day 9 pc. Ventricular region-restricted expression of the luciferase reporter in the embryonic heart, as assessed by immunofluorescence and direct assay of reporter activity in microdissected atrial and ventricular muscle specimens, was confirmed from at least day 15 pc on. Taken together, this provides evidence for early positional specification of MLC-2v gene expression in the primitive heart tube and indicates regional specification of part of the ventricular muscle gene program can precede ventricular septation during mammalian cardiogenesis. Since the 250-base-pair promoter fragment is active developmentally in transgenic mice, this establishes it as a molecular target for the process of ventricular specification in the developing heart tube.

Animals↗
PubMed1993

HRas-dependent pathways can activate morphological and genetic markers of cardiac muscle cell hypertrophy.

We have investigated the role of the proto-oncogene HRas in cardiac cell growth and hypertrophy. By direct needle microinjection of activated Ras protein into primary neonatal rat ventricular cardiac myocytes, we find that, unlike many other cell types, Ras does not induce DNA synthesis in these cells. However, injection of activated Ras does induce expression of both the c-Fos and atrial natriuretic factor (ANF) genes. Expression of both these genes is associated with the hypertrophic response in ventricular myocytes suggesting that Ras is involved in the hypertrophic signalling pathway. Ras injection also causes morphological changes in the cells so that they increase in profile and show changes in the organization of the contractile apparatus. Further support for a role for Ras in the hypertrophic response was obtained from studies showing that activated Ras stimulates ANF promoter activity in transient transfection assays. We also show that a dominant interfering Ras mutant inhibits the hypertrophic stimulation of the ANF promoter by phenylephrine, indicating a role for Ras in the hypertrophic effect of an alpha-adrenergic agonist.

Animals↗