Showing papers by "Victor Chang Cardiac Research Institute published in 2002"

Neuregulin 1 and Susceptibility to Schizophrenia

Abstract: The cause of schizophrenia is unknown, but it has a significant genetic component. Pharmacologic studies, studies of gene expression in man, and studies of mouse mutants suggest involvement of glutamate and dopamine neurotransmitter systems. However, so far, strong association has not been found between schizophrenia and variants of the genes encoding components of these systems. Here, we report the results of a genomewide scan of schizophrenia families in Iceland; these results support previous work, done in five populations, showing that schizophrenia maps to chromosome 8p. Extensive fine-mapping of the 8p locus and haplotype-association analysis, supplemented by a transmission/disequilibrium test, identifies neuregulin 1 (NRG1) as a candidate gene for schizophrenia. NRG1 is expressed at central nervous system synapses and has a clear role in the expression and activation of neurotransmitter receptors, including glutamate receptors. Mutant mice heterozygous for either NRG1 or its receptor, ErbB4, show a behavioral phenotype that overlaps with mouse models for schizophrenia. Furthermore, NRG1 hypomorphs have fewer functional NMDA receptors than wild-type mice. We also demonstrate that the behavioral phenotypes of the NRG1 hypomorphs are partially reversible with clozapine, an atypical antipsychotic drug used to treat schizophrenia.

Molecular mechanisms of inherited cardiomyopathies.

Abstract: Cardiomyopathies are diseases of heart muscle that may result from a diverse array of conditions that damage the heart and other organs and impair myocardial function, including infection, ischemia, and toxins. However, they may also occur as primary diseases restricted to striated muscle. Over the past decade, the importance of inherited gene defects in the pathogenesis of primary cardiomyopathies has been recognized, with mutations in some 18 genes having been identified as causing hypertrophic cardiomyopathy (HCM) and/or dilated cardiomyopathy (DCM). Defining the role of these genes in cardiac function and the mechanisms by which mutations in these genes lead to hypertrophy, dilation, and contractile failure are major goals of ongoing research. Pathophysiological mechanisms that have been implicated in HCM and DCM include the following: defective force generation, due to mutations in sarcomeric protein genes; defective force transmission, due to mutations in cytoskeletal protein genes; myocardial energy deficits, due to mutations in ATP regulatory protein genes; and abnormal Ca2+ homeostasis, due to altered availability of Ca2+ and altered myofibrillar Ca2+ sensitivity. Improved understanding that will result from these studies should ultimately lead to new approaches for the diagnosis, prognostic stratification, and treatment of patients with heart failure.

Efficient Cre-mediated deletion in cardiac progenitor cells conferred by a 3'UTR-ires-Cre allele of the homeobox gene Nkx2-5.

Abstract: Conditional gene targeting and transgenic strategies utilizing Cre recombinase have been successfully applied to the analysis of development in mouse embryos. To create a conditional system applicable to heart progenitor cells, a Cre recombinase gene linked at its 5' end to an internal ribosome entry site (IRES) was inserted into the 3' untranslated region of the cardiac homeobox gene Nkx2-5 using gene targeting. Nkx2-5IRESCre mice were fully viable as homozygotes. We evaluated the efficacy of Cre-mediated deletion by crossing Nkx2-5IRESCre mice with the Cre-dependent R26R and Z/AP reporter strains. Efficient deletion was observed in the cardiac crescent and heart tube in both strains. However, the Z/AP locus showed transient resistance to deletion in caudal heart progenitors. Such resistance was not evident at the R26R locus, suggesting that Cre-mediated deletion in myocardium may be locus-dependent. From cardiac crescent stages, deletion was seen not only in myocardium, but also endocardium, dorsal mesocardium and pericardial mesoderm. The Cre domain apparently includes cells dorsal to the heart that have been shown to constitute a secondary heart field, contributing myocardium to the outflow tract. Other sites of Nkx2-5 expression, including pharyngeal endoderm and its derivatives, branchial arch epithelium, stomach, spleen, pancreas and liver, also showed efficient deletion. Our data suggest that the Nkx2-5IRESCre strain will be useful for genetic dissection of the multiple tiers of lineage allocation to the forming heart as well as of molecular interactions within the heart fields and heart tube.

Axial skeletal defects caused by mutation in the spondylocostal dysplasia/pudgy gene Dll3 are associated with disruption of the segmentation clock within the presomitic mesoderm.

Abstract: A loss-of-function mutation in the mouse delta-like3 (Dll3) gene has been generated following gene targeting, and results in severe axial skeletal defects These defects, which consist of highly disorganised vertebrae and costal defects, are similar to those associated with the Dll3-dependent pudgy mutant in mouse and with spondylocostal dysplasia (MIM 277300) in humans This study demonstrates that Dll3neo and Dll3pu are functionally equivalent alleles with respect to the skeletal dysplasia, and we suggest that the three human DLL3 mutations associated with spondylocostal dysplasia are also functionally equivalent to the Dll3neo null allele Our phenotypic analysis of Dll3neo/Dll3neo mutants shows that the developmental origins of the skeletal defects lie in delayed and irregular somite formation, which results in the perturbation of anteroposterior somite polarity As the expression of Lfng, Hes1, Hes5 and Hey1 is disrupted in the presomitic mesoderm, we suggest that the somitic aberrations are founded in the disruption of the segmentation clock that intrinsically oscillates within presomitic mesoderm

Transcriptional Interference by Independently Regulated Genes Occurs in Any Relative Arrangement of the Genes and Is Influenced by Chromosomal Integration Position

Abstract: Transcriptional interference is the influence, generally suppressive, of one active transcriptional unit on another unit linked in cis. Its wide occurrence in experimental systems suggests that it may also influence transcription in many loci, but little is known about its precise nature or underlying mechanisms. Here we report a study of the interaction of two nearly identical transcription units juxtaposed in various arrangements. Each reporter gene in the constructs has its own promoter and enhancer and a strong polyadenylation signal. We used recombinase-mediated cassette exchange (RMCE) to insert the constructs into previously tagged genomic sites in cultured cells. This strategy also allows the constructs to be assessed in both orientations with respect to flanking chromatin. In each of the possible arrangements (tandem, divergent, and convergent), the presence of two genes strongly suppresses expression of both genes compared to that of an identical single gene at the same integration site. The suppression is most severe with the convergent arrangement and least severe in total with the divergent arrangement, while the tandem arrangement is most strongly influenced by the integration site and the genes’ orientation within the site. These results suggest that transcriptional interference could underlie some position effects and contribute to the regulation of genes in complex loci.

Pathophysiologic and therapeutic importance of tissue ACE: a consensus report.

Abstract: Angiotensin-converting enzyme (ACE) activation and the de novo production of angiotensin II contribute to cardiovascular disease through direct pathological tissue effects, including vascular remodeling and inflammation, as well as indirect action on nitric oxide bioavailability and its consequences. The endothelium plays a pivotal role in both vascular function and structure; thus, the predominant localization of ACE to the endothelium has implications for the pathobiology of vascular disease, such as coronary artery disease. Numerous experimental studies and clinical trials support the emerging realization that tissue ACE is a vital therapeutic target, and that its inhibition may restore endothelial function or prevent endothelial dysfunction. These effects exceed those attributable to blood pressure reduction alone; hence, ACE inhibitors may exert an important part of their effects through direct tissue action. Pharmacologic studies show that while ACE inhibitors may differ according to their binding affinity for tissue ACE the clinical significance remains to be determined.

Conserved tryptophan in the core domain of transglutaminase is essential for catalytic activity

Abstract: Transglutaminase 2 (TG2) is a distinctive member of the family of Ca2+-dependent enzymes recognized mostly by their abilities to catalyze the posttranslational crosslinking of proteins. TG2 uniquely binds and hydrolyzes GTP; binding GTP inhibits its crosslinking activity but allows it to function in signal transduction (hence the Gh designation). The core domain of TG2 (residues 139–471, rat) comprises the papain-like catalytic triad and the GTP-binding domain (residues 159–173) and contains almost all of the conserved tryptophans of the protein. Examining point mutations at Trp positions 180, 241, 278, 332, and 337 showed that, upon binding 2′-(or 3′)-O-(N-methylanthraniloyl)GTP (mantGTP), the Phe-332 mutant was the weakest (35% less than wild type) in resonance energy transfer from the protein (λexc, max = 290 nm) to the mant fluorophore (λem = 444 nm) and had a reduced affinity for mantGTP. Trp-332, situated near the catalytic center and the nucleotide-binding area of TG2, may be part of the allosteric relay machinery that transmits negative effector signals from nucleotide binding to the active center of TG2. A most important observation was that, whereas no enzyme activity could be detected when Trp-241 was replaced with Ala or Gln, partial preservation of catalytic activity was seen with substitutions by Tyr > Phe > His. The results indicate that Trp-241 is essential for catalysis, possibly by stabilizing the transition states by H-bonding, quadrupole–ion, or van der Waals interactions. This contrasts with the evolutionarily related papain family of cysteine proteases, which uses Gln-19 (papain) for stabilizing the transition state.

δ Tryptase Is Expressed in Multiple Human Tissues, and a Recombinant Form Has Proteolytic Activity

Abstract: Tryptases are neutral serine proteases selectively expressed in mast cells and have been implicated in the development of a number of inflammatory diseases including asthma. It has recently been established that the number of genes encoding human mast cell tryptases is much larger than originally believed, but it is not clear how many of these genes are expressed. A recent report suggested that the transcript for at least one of these genes, originally named mMCP-7-like tryptase, is not expressed. To further address this question, we screened tissue-specific RNA samples by RT-PCR, using primers designed to match the putative exonic sequence of this gene. We successfully generated and cloned the correctly sized RT-PCR product from mRNA isolated from the human mast cell-I cell line. Two distinct clones were identified whose nucleotide sequence matched the published sequence of the mMCP-7-like I and mMCP-7-like II genes. Transcripts were detected in a wide variety of human tissues including lung, heart, stomach, spleen, skin, and colon. A polyclonal antipeptide Ab that specifically recognizes the translated product of this transcript was used to demonstrate its expression in mast cells that reside in the colon, lung, and inflamed synovium. A recombinant form of this protein expressed in bacterial cells was able to cleave a synthetic trypsin-sensitive substrate, D-Ile-Phe-Lys pNA. These results suggest that the range of functional tryptases is larger than previously recognized. For simplicity, we suggest that the gene, transcripts, and corresponding protein product be named delta tryptase.

Developmental paradigms in heart disease: insights from tinman.

Abstract: Congenital heart disease is a significant cause of morbidity and mortality in humans, and gene mutations that underlie some of these anomalies are now being described. The NKX2.5 gene, which encodes a homeobox transcription factor, was initially discovered in mice through its similarity to the tinman gene of the fruitfly Drosophila. Tinman is required for formation of the dorsal pulsatile vessel or 'heart' of the fly. Tinman and NKX2.5 share structural and functional features, and in mice the gene is required for normal cardiac looping and differentiation of chamber myocardium. Humans with heterozygous mutations in the NKX2.5 gene generally have a disorder involving progressive atrio-ventricular conduction block and atrial septal defect, although sometimes other abnormalities including tetralogy of Fallot. The TBX5 gene, which encodes another cardiac transcription factor that collaborates with NKX2.5, is also an important cardiac disease gene, with heterozygous mutations responsible for Holt-Oram (hand/heart) syndrome. These contributions to human pathology underscore the relevance of studying biological phenomena in lower organisms, and examination of other genes acting in this and associated pathways will expand our knowledge of congenital abnormalities and disease predisposition, and improve genetic counseling.

NK-2 class homeobox genes and pharyngeal/oral patterning: Nkx2-3 is required for salivary gland and tooth morphogenesis

Abstract: Head development in vertebrates requires reciprocal patterning interactions between cranial neural crest and the ectodermal, mesodermal and endodermal components of the branchial arches. Patterning elements within the pharyngeal endoderm and oral ectoderm appear to play defining roles in this process. Several homeobox genes of the NK-2 class (Nkx2-1, Nkx2-3, Nkx2-5 and Nkx2-6) are expressed regionally in the developing pharynx, and Nkx2-1 mutants and Nkx2-5/Nkx2-6 double mutants show loss of thyroid and distal lung progenitors, and pharyngeal cell viability, respectively. Here we examined the expression and genetic role of Nkx2-3 in pharyngeal development. Nkx2-3 was expressed in the pharyngeal floor and pouches, as well as in oral and branchial arch ectoderm. Expression persisted in the developing thyroid until birth, in mucous-forming cells of the lingual and sublingual salivary glands, and in odontogenic epithelium of the mandible. Examination of Nkx2-3 null mice revealed defects in maturation and cellular organisation of the sublingual glands. Furthermore, cusps were absent from mandibular molars and the third molar was occasionally missing. These data suggest roles for Nkx2-3 during pharyngeal organogenesis, although the considerable potential for genetic redundancy within and outside of this gene family may mask earlier functions in organ specification.

Diverse requirements for Notch signalling in mammals.

Abstract: The Notch signalling pathway has a central role in a wide variety of developmental processes and it is not therefore surprising that mutations in components of this pathway can cause dramatic human genetic disorders. One developmental process in which the Notch pathway is involved at multiple levels is somitogenesis, the mechanism by which the embryo is divided into segments that ultimately form structures such as the axial skeleton and skeletal muscle of the trunk. We are investigating the human genetic disorder spondylocostal dysplasia (SCD), which is a group of malsegmentation syndromes that occur when this process is disrupted. Mutations in the Notch ligand DELTA-LIKE 3 (DLL3) are responsible for cases of autosomal recessive SCD type I (SCDO1), and we are using information derived from these mutations to study the structure of the DLL3 protein. To aid in elucidation of the underlying developmental defect in SCDO1, we have generated a mouse model by targeted deletion of the Dll3 gene (Dunwoodie et al., 2002). These mice show segmentation defects similar to those seen in SCDO1. In addition, these mice have a distinct set of neural defects that may be useful in future neurological assessment of affected individuals. Finally, since not all cases of SCD are due to mutation of DLL3, we are investigating various genes to find other candidates involved in this genetic disease.

Mutation of a Single TMVI Residue, Phe282, in the β2-Adrenergic Receptor Results in Structurally Distinct Activated Receptor Conformations†

Abstract: We showed previously that Phe303 in transmembrane segment (TM) VI of the α1B-adrenergic receptor (α1B-AR), a residue conserved in many G protein-coupled receptors (GPCRs), is critically involved in coupling agonist binding with TM helical movement and G protein activation. Here the equivalent residue, Phe282, in the β2-AR was evaluated by mutation to glycine, asparagine, alanine, or leucine. Except for F282N, which exhibits attenuated basal and maximal isoproterenol stimulation, the Phe282 mutants display varying degrees of constitutive activity (F282L > F282A > F282G), and as shown by the results of substituted cysteine accessibility method (SCAM) studies, induce movement of endogenous cysteine(s) into the water-accessible ligand-binding pocket. For F282A, movement is confined to Cys285 in TMVI, whereas F282L induces movement of both Cys285 in TMVI and Cys327 in TMVII. Further, engineered cysteine-sensor studies indicate that F282L causes movement of TMVI, both above and below an apparent kink-inducing T...

Phe(303) in TMVI of the alpha(1B)-adrenergic receptor is a key residue coupling TM helical movements to G-protein activation.

Abstract: We showed previously that Phe(303) in transmembrane segment (TM) VI of the alpha(1B)-adrenergic receptor, a highly conserved residue in G-protein-coupled receptors (GPCRs), is critically involved in receptor-activation and G-protein-coupling [Chen, S. H., Lin, F., Xu, M., Hwa, J., and Graham, R. M. (2000) EMBO J. 19, 4265-4271]. Here, we show that saturation mutagenesis of Phe(303) results in a series of mutants with different levels of constitutive activity for inositol phosphate (IP) signaling. Mutants F303G and F303N showed neither basal nor agonist-stimulated IP turnover, whereas F303A displayed increased basal activity but an attenuated maximal response to (-)-epinephrine-stimulation. F303L, on the other hand, showed all features of a typical constitutively active GPCR with markedly increased basal activity and increased potency and efficacy of agonist-stimulated IP signaling. All mutants displayed higher agonist-binding affinities than the wild-type receptor, and by thermal stability studies, those able to signal showed increased susceptibility to inactivation with an order of sensitivity (F303L > F303A > WT) directly related to their degree of constitutive activity. Using the substituted cysteine accessibility method (SCAM) and equilibrium binding studies, we further show that the F303A and F303L mutants result in TM helical movements that differ in accordance with their degree of constitutive activity. These findings, therefore, confirm and extend our previous data implicating Phe(303) as a key residue coupling TM helical movements to G-protein-activation.

The preload recruitable stroke work relationship as a measure of left ventricular contractile dysfunction in porcine cardiac allografts

Abstract: Objective: Paradoxically, it has been reported that after 1.5–4 h of hypothermic ischaemic preservation there is complete recovery of contractile function in canine cardiac allografts, as assessed by the preload recruitable stroke work (PRSW) relationship. This raises questions about the suitability of the canine heart as a model for preservation research and the PRSW relationship as an end-point. The aim of the present study was to evaluate the PRSW relationship as an index of left ventricular contractility in porcine cardiac allografts. Methods: Eighteen orthotopic heart transplants were performed in inbred Westran pigs. Brain death was induced in the donor pigs 1 h prior to explantation. The donor hearts were arrested with extracellular cardioplegia, which was stored in ice prior to administration. On explantation, the donor hearts were immersed in cardioplegia and stored in ice. The donor hearts were subjected to either 4 (IT4, n ¼ 6), 6 (IT6, n ¼ 9) or 14 (IT14, n ¼ 3) h of ischaemia. Post-transplant, all hearts were supported with dobutamine (10 mcg/kg per min). The PRSW relationship was derived from pressure-volume loops obtained by epicardial sonomicrometry and transmyocardial micromanometry. Multiple linear regression was used to describe and compare the PRSW relationship before brain death in the donor and after weaning from bypass in the recipient. Results: Eleven hearts were weaned successfully from cardiopulmonary bypass: IT4 100% (6/6), IT6 56% (5/9) and IT14 0% (0/3) (IT4 versus IT14: P ¼ 0:012). Analysis of the PRSW relationship revealed a reduction in contractility in both the IT4 and IT6 groups (both P , 0:0001), but a greater reduction in the IT6 group (P , 0:0001). Notably, the volume-axis intercept of the PRSW relationship was found to be a better discriminator of post-preservation contractile dysfunction than the slope of the PRSW relationship. Conclusions: The porcine heart’s susceptibility to ischaemic injury makes it ideal for evaluating the effect of different preservation strategies on contractile recovery. The PRSW relationship can be used to evaluate the differences in contractile recovery, though the nature of the effect of ischaemic preservation necessitates analysis by multiple linear regression. q 2002 Elsevier Science B.V. All rights reserved.

16 – Molecular Determinants of Cardiac Development and Congenital Disease

Abstract: This chapter discusses the molecular determinants of cardiac development and congential disease. The development of the mammalian heart is an exquisite example of how form is established during organ ontogeny and how relationships between form and function are realized. Although heart development has been studied at the morphological level since antiquity, its patterning principles are still far from clear. The details of the genes that guide heart myogenesis and morphogenesis are emerging at a rapid rate, contributing clues to patterning events and substantially informing the understanding of congenital, acquired, and maladaptive cardiac diseases. This chapter outlines recent progress on dissecting the molecular programs that guide heart formation and morphogenesis with emphasis on cardiac induction and the emergence of pattern through cellular and molecular interactions within the heart field and early heart tube. The information provides an essential framework that will allow genetic programs to be meaningfully linked to the emergence of heart form and function, and to the interpretation and amelioration of congenital heart disease.

Essential role for the lymphostromal plasma membrane Ly-6 superfamily molecule thymic shared antigen 1 in development of the embryonic adrenal gland.

Abstract: Thymic shared antigen 1 (TSA-1) is a plasma membrane protein of the Ly-6 superfamily expressed on thymocytes, thymic stromal cells, and other cells of the hematopoietic system. TSA-1 is also expressed in other nonhematopoietic tissues, in particular, embryonic and adult adrenal glands. To address the function of TSA-1, we generated mutant mice in which TSA-1 expression was inactivated by gene targeting. Here we show that deletion of both TSA-1 alleles results in abnormal adrenal gland development and midgestational lethality due to cardiac abnormalities. We also report that TSA-1-deficient adrenal glands have significantly reduced levels of the catecholamines noradrenaline and adrenaline. We conclude that TSA-1 is required for normal embryonic development but that deletion of its expression does not obviously impair lymphoid development.