Showing papers by "Victor Chang Cardiac Research Institute published in 2009"

Transglutaminases and Disease: Lessons From Genetically Engineered Mouse Models and Inherited Disorders

Abstract: The human transglutaminase (TG) family consists of a structural protein, protein 4.2, that lacks catalytic activity, and eight zymogens/enzymes, designated factor XIII-A (FXIII-A) and TG1-7, that catalyze three types of posttranslational modification reactions: transamidation, esterification, and hydrolysis. These reactions are essential for biological processes such as blood coagulation, skin barrier formation, and extracellular matrix assembly but can also contribute to the pathophysiology of various inflammatory, autoimmune, and degenerative conditions. Some members of the TG family, for example, TG2, can participate in biological processes through actions unrelated to transamidase catalytic activity. We present here a comprehensive review of recent insights into the physiology and pathophysiology of TG family members that have come from studies of genetically engineered mouse models and/or inherited disorders. The review focuses on FXIII-A, TG1, TG2, TG5, and protein 4.2, as mice deficient in TG3, TG4, TG6, or TG7 have not yet been reported, nor have mutations in these proteins been linked to human disease.

Sequential treatment of drug-resistant tumors with targeted minicells containing siRNA or a cytotoxic drug

Abstract: The dose-limiting toxicity of chemotherapeutics, heterogeneity and drug resistance of cancer cells, and difficulties of targeted delivery to tumors all pose daunting challenges to effective cancer therapy. We report that small interfering RNA (siRNA) duplexes readily penetrate intact bacterially derived minicells previously shown to cause tumor stabilization and regression when packaged with chemotherapeutics. When targeted via antibodies to tumor-cell-surface receptors, minicells can specifically and sequentially deliver to tumor xenografts first siRNAs or short hairpin RNA (shRNA)-encoding plasmids to compromise drug resistance by knocking down a multidrug resistance protein. Subsequent administration of targeted minicells containing cytotoxic drugs eliminate formerly drug-resistant tumors. The two waves of treatment, involving minicells loaded with both types of payload, enable complete survival without toxicity in mice with tumor xenografts, while involving several thousandfold less drug, siRNA and antibody than needed for conventional systemic administration of cancer therapies.

Telomerase activity is associated with an increase in DNA methylation at the proximal subtelomere and a reduction in telomeric transcription

Abstract: Tumours and immortalized cells avoid telomere attrition by using either the ribonucleoprotein enzyme telomerase or a recombination-based alternative lengthening of telomeres (ALT) mechanism. Available evidence from mice suggests that the epigenetic state of the telomere may influence the mechanism of telomere maintenance, but this has not been directly tested in human cancer. Here we investigated cytosine methylation directly adjacent to the telomere as a marker of the telomere's epigenetic state in a panel of human cell lines. We find that while ALT cells show highly heterogeneous patterns of subtelomeric methylation, subtelomeric regions in telomerase-positive cells invariably show denser methylation than normal cells, being almost completely methylated. When compared to matched normal and ALT cells, telomerase-positive cells also exhibit reduced levels of the telomeric repeat-containing-RNA (TERRA), whose transcription originates in the subtelomere. Our results are consistent with the notion that TERRA may inhibit telomerase: the heavy cytosine methylation we observe in telomerase-positive cells may reflect selection for TERRA silencing in order to facilitate telomerase activity at the telomere. These data suggest that the epigenetic differences between telomerase-positive and ALT cells may underlie the mechanism of telomere maintenance in human tumorigenesis and highlight the broad reaching consequences of epigenetic dysregulation in cancer.

microRNA-Mediated Messenger RNA Deadenylation Contributes to Translational Repression in Mammalian Cells

Abstract: Animal microRNAs (miRNAs) typically regulate gene expression by binding to partially complementary target sites in the 3′ untranslated region (UTR) of messenger RNA (mRNA) reducing its translation and stability. They also commonly induce shortening of the mRNA 3′ poly(A) tail, which contributes to their mRNA decay promoting function. The relationship between miRNA-mediated deadenylation and translational repression has been less clear. Using transfection of reporter constructs carrying three imperfectly matching let-7 target sites in the 3′ UTR into mammalian cells we observe rapid target mRNA deadenylation that precedes measureable translational repression by endogenous let-7 miRNA. Depleting cells of the argonaute co-factors RCK or TNRC6A can impair let-7-mediated repression despite ongoing mRNA deadenylation, indicating that deadenylation alone is not sufficient to effect full repression. Nevertheless, the magnitude of translational repression by let-7 is diminished when the target reporter lacks a poly(A) tail. Employing an antisense strategy to block deadenylation of target mRNA with poly(A) tail also partially impairs translational repression. On the one hand, these experiments confirm that tail removal by deadenylation is not strictly required for translational repression. On the other hand they show directly that deadenylation can augment miRNA-mediated translational repression in mammalian cells beyond stimulating mRNA decay. Taken together with published work, these results suggest a dual role of deadenylation in miRNA function: it contributes to translational repression as well as mRNA decay and is thus critically involved in establishing the quantitatively appropriate physiological response to miRNAs.

Does Wave Reflection Dominate Age-Related Change in Aortic Blood Pressure Across the Human Life Span?

Abstract: Aortic systolic and pulse pressure rise with age because of aortic stiffening Two factors are responsible: a larger incident wave because of increased aortic characteristic impedance and premature return of wave reflection from peripheral sites This study aimed to determine the relative contribution of each factor before and after age 60 years Aortic pressure waveforms were generated for 3682 healthy subjects using a generalized transfer function applied to radial pressure waveforms recorded by applanation tonometry Linear regression and product of coefficient mediation analysis were performed in the cross-sectional cohort to determine the yearly contribution of the incident and reflected waves (waves measured as first systolic peak and augmented pressure, respectively) to aortic systolic and pulse pressure elevation with age This was done separately for subjects ≤60 and >60 years of age, with both sexes initially pooled and subsequently separated Analyses were repeated with correction for height, weight, heart rate, and mean arterial pressure Before age 60 years, the reflected wave was a greater ( P P P difference >005) to age-related aortic systolic and pulse pressure elevations This general pattern was observed in both sexes and persisted after correction for confounders Wave reflection is important across the life span, whereas aortic characteristic impedance contributes significantly only beyond age 60 years

Identification and Functional Characterization of Cardiac Troponin I As a Novel Disease Gene in Autosomal Dominant Dilated Cardiomyopathy

Abstract: Rationale: Idiopathic dilated cardiomyopathy (DCM) is inherited in approximately one third of cases, usually as an autosomal dominant trait. More than 30 loci have been identified, several of which encode sarcomeric proteins which can also be mutated to cause hypertrophic cardiomyopathy. One contractile protein gene well known as a hypertrophic cardiomyopathy disease gene, but with no reported mutation in autosomal dominant DCM, is TNNI3 which encodes cardiac troponin I. Objective: To test TNNI3 as a candidate gene, a panel of 96 probands with DCM was analyzed. Methods and Results: Genomic DNA was isolated and TNNI3 exons screened by heteroduplex analysis. Exons with aberrant profiles were sequenced and variants evaluated by segregation analysis and study of normal controls. We report 2 novel TNNI3 missense mutations, Lys36Gln and Asn185Lys, each associated with severe and early onset familial DCM. Of the 5 mutation carriers, cardiac transplantation was required in 3, at ages 6, 15, and 24 years. Analysis...

Progress in the Understanding of the Genetic Etiology of Vertebral Segmentation Disorders in Humans

Abstract: Vertebral malformations contribute substantially to the pathophysiology of kyphosis and scoliosis, common health problems associated with back and neck pain, disability, cosmetic disfigurement, and functional distress. This review explores (1) recent advances in the understanding of the molecular embryology underlying vertebral development and relevance to elucidation of etiologies of several known human vertebral malformation syndromes; (2) outcomes of molecular studies elucidating genetic contributions to congenital and sporadic vertebral malformation; and (3) complex interrelationships between genetic and environmental factors that contribute to the pathogenesis of isolated syndromic and nonsyndromic congenital vertebral malformation. Discussion includes exploration of the importance of establishing improved classification systems for vertebral malformation, future directions in molecular and genetic research approaches to vertebral malformation, and translational value of research efforts to clinical management and genetic counseling of affected individuals and their families.

Time domain analysis of the arterial pulse in clinical medicine

Abstract: The arterial pulse at any site is created by an impulse generated by the left ventricle as it ejects blood into the aorta, together with multiple impulses travelling in the opposite direction from reflecting sites in the peripheral circulation. The compound wave at any site depends on the pattern of ventricular ejection, the properties of large arteries, particularly their stiffness (which determines rate of propagation) and the distance to and impedance mismatch at reflecting sites. Physicians are familiar with waveform analysis in the time domain, as in the electrocardiogram (ECG) where the principal features are explicable on the basis of atrial depolarisation followed by ventricular depolarisation, then repolarisation. Effects of cardiac functional and structural disease can be inferred from the ECG. It is more difficult to make similar interpretations from the pulse waveform and clinicians usually use this only to count heart rate, extremes of the pressure pulse to express systolic and diastolic pressure, and (sometimes) time from wave foot to incisural notch to measure time of systole and diastole. More information can be gleaned from the shape of the arterial pressure wave through consideration of the factors which create it--on stiffening of large arteries with age, effects of drugs on smallest arteries, and changes in such arterial properties on left ventricular load and function. Such is a major challenge to future physicians. It is aided by better and more accurate methods for measuring flow and diameter as well as pressure waveforms, and by appropriate use of other analytic techniques such as analysis of the pulse in the frequency domain.

The zebrafish dystrophic mutant softy maintains muscle fibre viability despite basement membrane rupture and muscle detachment.

Abstract: The skeletal muscle basement membrane fulfils several crucial functions during development and in the mature myotome and defects in its composition underlie certain forms of muscular dystrophy. A major component of this extracellular structure is the laminin polymer, which assembles into a resilient meshwork that protects the sarcolemma during contraction. Here we describe a zebrafish mutant, softy, which displays severe embryonic muscle degeneration as a result of initial basement membrane failure. The softy phenotype is caused by a mutation in the lamb2 gene, identifying laminin β2 as an essential component of this basement membrane. Uniquely, softy homozygotes are able to recover and survive to adulthood despite the loss of myofibre adhesion. We identify the formation of ectopic, stable basement membrane attachments as a novel means by which detached fibres are able to maintain viability. This demonstration of a muscular dystrophy model possessing innate fibre viability following muscle detachment suggests basement membrane augmentation as a therapeutic strategy to inhibit myofibre loss.

Common genetic control of haemangioblast and cardiac development in zebrafish

Abstract: Over the past few years it has become clear that over half of the mammalian heart derives from outside the heart field as originally defined. Such a second heart field, however, has not been described in zebrafish, which could explain its smaller, two-chambered heart. Instead, zebrafish have a population of haemangioblasts, which is absent in mammalian embryos, raising the possibility that these cells represent the evolutionary ancestor of the second heart field. Here, we show for the first time that the genetic programmes of these anterior haemangioblasts and the adjacent heart field are co-regulated, by transcription factors previously associated with heart but not blood or endothelial development. We demonstrate that gata4, gata5 and gata6 are essential for anterior haemangioblast specification, and for subsequent myelopoiesis, acting as early as cloche and upstream of scl . The requirement for gata4, gata5 and gata6 in myeloid, endothelial and cardiac specification is in the mesoderm, but these factors also control, from within the endoderm and the yolk syncytial layer, the migration of the cardiac precursors as they differentiate. This genetic link between the blood/endothelial and cardiac programmes supports the notion that this haemangioblast population in zebrafish is an evolutionary antecedent of the second heart field, and has implications for the differentiation of haemangioblasts and cardiomyocytes from pluripotent cells, and for the origins of stem cells in the adult heart.

Not All hERG Pore Domain Mutations Have a Severe Phenotype: G584S Has an Inactivation Gating Defect with Mild Phenotype Compared to G572S, Which Has a Dominant Negative Trafficking Defect and a Severe Phenotype

Abstract: Distinct Phenotypes in hERG Pore Domain Mutations. Introduction: Mutations in the pore domain of the human ether-a-go-go-related gene (hERG) potassium channel are associated with higher risk of sudden death. However, in many kindreds clinical presentation is variable, making it hard to predict risk. We hypothesized that in vitro phenotyping of the intrinsic severity of individual mutations can assist with risk stratification. Methods and Results: We analyzed 2 hERG pore domain mutations, G572S and G584S. Similar to 90% of hERG missense mutations, G572S-hERG subunits did not traffic to the plasma membrane but could coassemble with WT subunits and resulted in a dominant negative suppression of hERG current density. The G584S-hERG subunits traffic normally but have abnormal inactivation gating. Computer models of human ventricular myocyte action potentials (AP), incorporating Markov models of the hERG mutants, indicate that G572S-hERG channels would cause more severe AP prolongation than that seen with G584ShERG channels. Conclusions: hERG-G572S and -G584S are 2 pore domain mutations that involve the same change in sidechain but have very different in vitro phenotypes; G572S causes a dominant negative trafficking defect, whereas G584S is the first hERG missense mutation where the cause of disease can be exclusively attributed to enhanced inactivation. The G572S mutation is intrinsically more severe than the G584S mutation, consistent with the overall clinical presentation in the 2 small kindreds studied here. Further investigation, involving a larger number of cohorts, to test the hypothesis that in vitro phenotyping of the intrinsic severity of a given mutation will assist with risk stratification is therefore warranted.

Structural and functional characterization of the oxidoreductase alpha-DsbA1 from Wolbachia pipientis.

Abstract: The alpha-proteobacterium Wolbachia pipientis is a highly successful intracellular endosymbiont of invertebrates that manipulates its host's reproductive biology to facilitate its own maternal transmission. The fastidious nature of Wolbachia and the lack of genetic transformation have hampered analysis of the molecular basis of these manipulations. Structure determination of key Wolbachia proteins will enable the development of inhibitors for chemical genetics studies. Wolbachia encodes a homologue (alpha-DsbA1) of the Escherichia coli dithiol oxidase enzyme EcDsbA, essential for the oxidative folding of many exported proteins. We found that the active-site cysteine pair of Wolbachia alpha-DsbA1 has the most reducing redox potential of any characterized DsbA. In addition, Wolbachia alpha-DsbA1 possesses a second disulfide that is highly conserved in alpha-proteobacterial DsbAs but not in other DsbAs. The alpha-DsbA1 structure lacks the characteristic hydrophobic features of EcDsbA, and the protein neither complements EcDsbA deletion mutants in E. coli nor interacts with EcDsbB, the redox partner of EcDsbA. The surface characteristics and redox profile of alpha-DsbA1 indicate that it probably plays a specialized oxidative folding role with a narrow substrate specificity. This first report of a Wolbachia protein structure provides the basis for future chemical genetics studies.

Conformational changes in redox pairs of protein structures.

Abstract: Disulfides are conventionally viewed as structurally stabilizing elements in proteins but emerging evidence suggests two disulfide subproteomes exist. One group mediates the well known role of structural stabilization. A second redox-active group are best known for their catalytic functions but are increasingly being recognized for their roles in regulation of protein function. Redox-active disulfides are, by their very nature, more susceptible to reduction than structural disulfides; and conversely, the Cys pairs that form them are more susceptible to oxidation. In this study, we searched for potentially redox-active Cys Pairs by scanning the Protein Data Bank for structures of proteins in alternate redox states. The PDB contains over 1134 unique redox pairs of proteins, many of which exhibit conformational differences between alternate redox states. Several classes of structural changes were observed, proteins that exhibit: disulfide oxidation following expulsion of metals such as zinc; major reorganisation of the polypeptide backbone in association with disulfide redox-activity; order/disorder transitions; and changes in quaternary structure. Based on evidence gathered supporting disulfide redox activity, we propose disulfides present in alternate redox states are likely to have physiologically relevant redox activity.

Comparison of automated candidate gene prediction systems using genes implicated in type 2 diabetes by genome-wide association studies.

Abstract: Automated candidate gene prediction systems allow geneticists to hone in on disease genes more rapidly by identifying the most probable candidate genes linked to the disease phenotypes under investigation. Here we assessed the ability of eight different candidate gene prediction systems to predict disease genes in intervals previously associated with type 2 diabetes by benchmarking their performance against genes implicated by recent genome-wide association studies. Using a search space of 9556 genes, all but one of the systems pruned the genome in favour of genes associated with moderate to highly significant SNPs. Of the 11 genes associated with highly significant SNPs identified by the genome-wide association studies, eight were flagged as likely candidates by at least one of the prediction systems. A list of candidates produced by a previous consensus approach did not match any of the genes implicated by 706 moderate to highly significant SNPs flagged by the genome-wide association studies. We prioritized genes associated with medium significance SNPs. The study appraises the relative success of several candidate gene prediction systems against independent genetic data. Even when confronted with challengingly large intervals, the candidate gene prediction systems can successfully select likely disease genes. Furthermore, they can be used to filter statistically less-well-supported genetic data to select more likely candidates. We suggest consensus approaches fail because they penalize novel predictions made from independent underlying databases. To realize their full potential further work needs to be done on prioritization and annotation of genes.

c-Kit function is necessary for in vitro myogenic differentiation of bone marrow hematopoietic cells.

Abstract: In recent years, the differentiation of bone marrow cells (BMCs) into myocytes has been extensively investigated, but the findings remain inconclusive. The purpose of this study was to determine the conditions necessary to induce myogenic differentiation in short-term cultures of adult BMCs, and to identify the BMC subpopulation responsible for this phenomenon. We report that high-density cultures of murine hematopoietic BMCs gave rise to spontaneous beating cell clusters in the presence of vascular endothelial and fibroblast growth factors. These clusters originated from c-kitpos cells. The formation of the clusters could be completely blocked by adding a c-kit/tyrosine kinase inhibitor, Gleevec (imatinib mesylate; Novartis International, Basel, Switzerland, http://www.novartis.com), to the culture. Cluster formation was also blunted in BMCs from c-kit-deficient (KitW/KitW-v) mice. Clustered cells expressed cardiomyocyte-specific transcription factor genes Gata-4 and Nkx2.5, sarcomeric proteins β-MHC and MLC-2v, and ANF and connexin-43. Immunostaining revealed α-sarcomeric actinin expression in more than 90% of clustered cells. Under electron microscopy, the clustered cells exhibited a sarcomeric myofiber arrangement and z-bands. This study defines the microenvironment required to achieve a reproducible in vitro model of beating, myogenic cell clusters. This model could be used to examine the mechanisms responsible for the postnatal myogenic differentiation of BMCs. Our results identify c-kitpos bone marrow hematopoietic cells as the source of the myogenic clusters. STEM CELLS 2009;27:1911–1920

Placental Insufficiency Associated with Loss of Cited1 Causes Renal Medullary Dysplasia

Abstract: A number of studies have shown that placental insufficiency affects embryonic patterning of the kidney and leads to a decreased number of functioning nephrons in adulthood; however, there is circumstantial evidence that placental insufficiency may also affect renal medullary growth, which could account for cases of unexplained renal medullary dysplasia and for abnormalities in renal function among infants who had experienced intrauterine growth retardation. We observed that mice with late gestational placental insufficiency associated with genetic loss of Cited1 expression in the placenta had renal medullary dysplasia. This was not caused by lower urinary tract obstruction or by defects in branching of the ureteric bud during early nephrogenesis but was associated with decreased tissue oxygenation and increased apoptosis in the expanding renal medulla. Loss of placental Cited1 was required for Cited1 mutants to develop renal dysplasia, and this was not dependent on alterations in embryonic Cited1 expression. Taken together, these findings suggest that renal medullary dysplasia in Cited1 mutant mice is a direct consequence of decreased tissue oxygenation resulting from placental insufficiency.

Concentration dependent effect of GsMTx4 on mechanosensitive channels of small conductance in E. coli spheroplasts

Abstract: The spider peptide GsMTx4, at saturating concentration of 5 muM, is an effective and specific inhibitor for stretch-activated mechanosensitive (MS) channels found in a variety of eukaryotic cells. Although the structure of the peptide has been solved, the mode of action remains to be determined. Because of its amphipathic structure, the peptide is proposed to interact with lipids at the boundaries of the MS channel proteins. In addition, GsMTx4 has antimicrobial effects, inhibiting growth of several species of bacteria in the range of 5-64 microM. Previous studies on prokaryotic MS channels, which serve as model systems to explore the principle of MS channel gating, have shown that various amphipathic compounds acting at the protein-lipid interface affect MS channel gating. We have therefore analyzed the effect of different concentrations of extracellular GsMTx4 on MS channels of small conductance, MscS and MscK, in the cytoplasmic membrane of wild-type E. coli spheroplasts using the patch-clamp technique. Our study shows that the peptide GsMTx4 exhibits a biphasic response in which peptide concentration determines inhibition or potentiation of activity in prokaryotic MS channels. At low peptide concentrations of 2 and 4 microM the gating of the prokaryotic MS channels was hampered, manifested by a decrease in pressure sensitivity. In contrast, application of peptide at concentrations of 12 and 20 microM facilitated prokaryotic MS channel opening by increasing the pressure sensitivity.

Insights into the characteristics of mammalian cardiomyocyte terminal differentiation shown through the study of mice with a dysfunctional c-kit.

Abstract: Mammalian cardiomyocytes withdraw from the cell cycle soon after birth. This process is called terminal differentiation. The c-kit, a receptor tyrosine kinase, is expressed on cardiomyocytes immediately after birth but for only a few days. In mice with genetic c-kit dysfunction, adult cardiomyocytes are phenotypically indistinguishable from those of wild type mice, except that they are capable of proliferation in vivo after acute pressure overload. This review explores the idea that postnatal cardiomyocyte differentiation and cell cycle withdrawal are distinct processes and that terminal differentiation may not simply be due to altered expression of genes that regulate the cell cycle but could involve c-kit induced epigenetic change.

The Ccr4-Pop2-NOT mRNA Deadenylase Contributes to Septin Organization in Saccharomyces cerevisiae

Abstract: In yeast, assembly of the septins at the cell cortex is required for a series of key cell cycle events: bud-site selection, the morphogenesis and mitotic exit checkpoints, and cytokinesis. Here we establish that the Ccr4-Pop2-NOT mRNA deadenylase contributes to septin organization. mRNAs encoding regulators of septin assembly (Ccd42, Cdc24, Rga1, Rga2, Bem3, Gin4, Cla4, and Elm1) presented with short poly(A) tails at steady state in wild-type (wt) cells, suggesting their translation could be restricted by deadenylation. Deadenylation of septin regulators was dependent on the major cellular mRNA deadenylase Ccr4-Pop2-NOT, whereas the alternative deadenylase Pan2 played a minor role. Consistent with deadenylation of septin regulators being important for function, deletion of deadenylase subunits CCR4 or POP2, but not PAN2, resulted in septin morphology defects (e.g., ectopic bud-localized septin rings), particularly upon activation of the Cdc28-inhibitory kinase Swe1. Aberrant septin staining was also observed in the deadenylase-dead ccr4-1 mutant, demonstrating the deadenylase activity of Ccr4-Pop2 is required. Moreover, ccr4Δ, pop2Δ, and ccr4-1 mutants showed aberrant cell morphology previously observed in septin assembly mutants and exhibited genetic interactions with mutations that compromise septin assembly (shs1Δ, cla4Δ, elm1Δ, and gin4Δ). Mutations in the Not subunits of Ccr4-Pop2-NOT, which are thought to predominantly function in transcriptional control, also resulted in septin organization defects. Therefore, both mRNA deadenylase and transcriptional functions of Ccr4-Pop2-NOT contribute to septin organization in yeast.

Molecular analysis of PRKAG2, LAMP2, and NKX2-5 genes in a cohort of 125 patients with accessory atrioventricular connection.

Abstract: Molecular Analysis of PRKAG2, LAMP2, and NKX2-5 Genes in a Cohort of 125 Patients With Accessory Atrioventricular Connection Giorgia Esposito, Giorgia Grutter, Fabrizio Drago, Mauro W. Costa, Antonella De Santis, Giovanna Bosco, Bruno Marino, Emanuele Bellacchio, Francesca Lepri, Richard P. Harvey, Anna Sarkozy,* and Bruno Dallapiccola IRCCS-Casa Sollievo della Sofferenza, San Giovanni Rotondo, Rome, Italy Casa Sollievo della Sofferenza-Mendel Institute, Rome, Italy Department of Experimental Medicine and Pathology, University ‘‘La Sapienza’’, Rome, Italy Department of Pediatric Cardiology, Bambino Gesu? Hospital, IRCCS, Rome, Italy Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, Australia Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Section of Pediatric Cardiology, Department of Pediatrics, University ‘‘La Sapienza’’, Rome, Italy