The reactive nitrogen species (RNS) nitric oxide (NO), nitroxyl anion (NO") and nitrosonium cation (NO+), modulate a myriad of biological processes. The microsomal haem oxygenases (HO-1, HO-2 and HO-3) oxidatively catabolise haem to bilirubin, carbon monoxide (CO) and ferrous iron (Fe2+). Sensitivity of the inducible isoform (HO-1) to a variety of inducers has identified HO-1 as an effective endogenous cytoprotectant against oxidative stress. Although nitrosative stimuli can enhance HO-1 expression, little is known about the biochemistry and mechanisms of this response. This Thesis examines a number of aspects related to HO-1 and nitrosative stimuli in cardiac cells, including: 1. induction by NO" 2. the biochemistry of NOVNO-mediated induction of HO-1 3. identification of a possible mechanism for the activation of HO-1 by NO congeners 4. the antinitrosative potential of bilirubin and 5. the potential of glyceryl trinitrate (GTN), a clinically used NO donor, to activate the haem oxygenase pathway. These different aspects of HO-1 were addressed using biochemical, molecular biology and cell culture techniques. The results indicate that NO", in analogy with other RNS, is a potent inducer of haem oxygenase activity and HO-1 mRNA and protein expression. A proposed mechanism for this response is modulation of thiol groups within redox-sensitive transcription factors. An antinitrosative and HO-1 inducing capacity was identified for bilirubin and GTN, respectively. Collectively, these findings suggest that the haem oxygenase pathway can act both as a sensor to, and target of, redox based mechanisms involving RNS, and extend our knowledge on the biological function of HO-1 in response to nitrosative stress.

Cardiac Ca2+ cycling consist of a series of tightly controlled events, regulated by a number of key proteins that release and sequester Ca2+ both at the cell surface and via intracellular stores. RyR2 is the main SR Ca2+ release channel. Direct regulation of RyR2 by CaMKIId, an important Ca -dependent enzyme, has previously been demonstrated.

Wnt signalling is required during cardiogenesis for both specification of heart tissue and the morphogenetic movements necessary to form the heart. Canonical signalling is required mid gastrula to restrict the size of the cardiac field whilst Wnt 11 mediated non-canonical signalling is required for cardiac induction. Wnt 11R, which is expressed later, is also thought to signal through the non-canonical pathway to promote heart morphogenesis. Xfz7is expressed during gastrulation in the mesodermal cells fated to become heart. Expression Is maintained in the primary heart field as the dorsolateral mesoderm migrates to the ventral midline. We have used loss of function analysis to determine the role of Xfz7 during heart development. Morpholino mediated knock down of Xfz7 gives a heartless phenotype similar to that of Wnt 11 morpholino.

TGFβ signalling has long been recognised as having a role in heart development. The central TGFβ type II receptor (Tgfbr2) is thought to be essential for epithelialmesenchymal transition (EMT) in the atrioventricular cushions. However, little is known about the roles of Tgfbr2 in heart development in vivo. This is largely because the Tgfbr2 knockout (KO) shows early embryonic lethality during heart valve development. To bypass the early lethality of the TRfbr2 KO mouse and to investigate the role of Tgfbr2 during heart development, a cre/loxP system was used to generate three different conditional knockout mouse models to specifically delete this receptor in (i) the AV myocardium, (ii) the ventricular myocardium and (iii) the endocardium.

No description available.

The stress responses of cardiomyocytes are likely to constitute a significant aspect of the development of cardiac pathologies. Oxidative stress is a common theme in the pathophysiology of ischaemic and non-ischaemic cardiomyopathy. On the other hand, hypertrophic agonists (e.g. endothelin-1, ET-1) are important mediators of the hypertrophic response to hemodynamic overloading, and cardiac pathologies such as heart failure are usually preceded by cardiac hypertrophy. Microarray studies indicate that one of the genes most potently induced by H2O2 (as an oxidative stress) or ET-1 in cardiac myocytes is the transcription factor ATF3. This thesis examines the regulation and role of ATF3 in neonatal rat ventricular cardiomyocytes. The increase in expression of ATF3 mRNA was confirmed in neonatal rat cardiomyocytes exposed to H2O2 or ET-1 using RT-PCR and Q-PCR. Using immunoblotting, it was confirmed that H2O2 or ET-1 increased ATF3 protein expression. ATF3 is an immediate early gene since the upregulation of ATF3 mRNA by ET-1 was not inhibited by cycloheximide (20 μM). Upregulation of ATF3 was inhibited by U0126 (10 μM), suggesting that signalling through ERK1/2 (extracellular signal-regulated kinases 1/2) was required. Other studies suggest that ATF3 downregulates interleukin-6 (IL-6) in human cells. The rat IL-6 promoter possesses an ATF consensus sequence, and chromatin immunoprecipitation (ChIP) analysis indicated that ET-1 or H2O2 increased the association of ATF3 with the IL-6 promoter. IL-6 mRNA and protein expression was transiently upregulated in cardiomyocytes exposed to H2O2 or ET-1, and this was shown to be an immediate early gene response. Following IL-6 mRNA upregulation, its expression was more rapidly downregulated than that of ATF3. The peak of ATF3 protein expression coincided with the return of IL-6 mRNA to basal levels after stimulation with either ET-1 or H2O2. This suggests that ATF3 operates in a negative feedback loop to downregulate IL-6 mRNA expression in cardiomyocytes. This was confirmed by adenoviral-mediated overexpression of full-length ATF3 antisense RNA, which attenuated ATF3 mRNA and protein expression following stimulation with ET-1. Inhibition of ATF3 expression was associated with superinduction of IL-6 mRNA as shown by Q-PCR. Other potential downstream targets of ATF3 were identified from microarray studies using bioinformatics. Those with ATF/CRE consensus sequences in their promoters included epiregulin and leukaemia inhibitory factor (LIF) (also shown to be immediate early genes). Epiregulin and LIF mRNA expression was superinduced by ATF3 antisense RNA. Taken together, these experiments indicate that ATF3 operates in a negative feedback loop by downregulating a cluster of immediate early genes induced by ET-1. Thus, ATF3 may play an important role in the establishment and fine-tuning of an organised and compensatory hypertrophic response in the cardiomyocyte secondary to hypertrophic stimulation.

No description available.

No description available.

Cardiac development is a complex multi-step process involving a diverse network of genes. Defects in cardiac genes can lead to congenital heart defects (CHDs), and understanding the processes involved in normal cardiogenesis is crucial in elucidating the pathogenesis of disease. Mutations in a number of cardiac transcription factors have been associated with CHDs, including TBX5 and GATA-4. These transcription factors are high in the regulatory hierarchy and form a complex that is thought to direct and synergistically regulate common cardiac pathways. However, little is currently known about their joint targets. This study aimed to identify and analyse genes important in cardiogenesis, with focus on the combined targets of TBX5 and GAT A-4. Microarray expression analysis of TBX5/ GATA-4 double overexpression P19 cell lines led to the identification of a large number of genes, of which seven were selected for study; PA2.26, PETA-3, FUCA 1, FN, TPM1, DES, and RBMS1. Expression of these was confirmed in the embryonic chick heart at Hamburger and Hamilton (HH) stages 12 - 26. The cell cycle regulator and transcription factor RBMS1 was selected for investigation of its role in cardiac development. Morpholino knockdown of RBMS1 expression in the developing chick resulted in defects in cardiac looping and atrial septation. Whilst further work is required to strengthen this data, this is a novel finding and opens up possibilities for future research into cardiac transcriptional networks. Microarray expression analysis using an in ovo model of TBX5 and GATA-4 double knockdown led to the identification of a number of interesting putative targets, including TFAP2B, GPC3, and CRABP1, which have known roles in cardiac development. TFAP2B is of particular interest as mutations in this gene are associated with the heart-hand disorder Char syndrome. LOC420770, a novel gene of unknown function, was also identified and displayed expression indicative of a heart-limb profile. Further studies to attempt elucidation of the function of this gene may provide a novel candidate gene for CHD. Investigation of these genes will form the basis of future research, contributing to our current knowledge of the vast network of genes involved in development of the heart.

No description available.