M.Sc. (Med. (Paediatric Cardiology)), Faculty of Health Sciences, University of the Witwatersrand, 2008 / Left ventricular (LV) noncompaction is characterized by the presence of an extensive trabecular myocardial layer within the luminal aspect of the compact myocardium of the ventricular wall. The trabeculae are both excessive in number and more prominent than normal. Noncompaction may occur in isolation usually with clinical features of dilated cardiomyopathy, or it may be associated with congenital or acquired heart diseases. Echocardiography is the reference standard for diagnosis, where a ratio of thickness of trabecular-to-compact myocardium (compaction ratio) of >2 is a major diagnostic criterion. Noncompaction is usually considered to result from persistence of the highly trabeculated myocardium found in early cardiogenesis of the human embryo. If persistence of excess trabeculae is the only determinant of the compaction ratio it would be expected that it would remain a consistent measurement in postnatal life. However, temporal changes in the degree of noncompaction in individual case reports have raised the question as to whether the compaction ratio might be sensitive to haemodynamic or other factors. In the present dissertation, I assessed echocardiographically whether the compaction ratio is associated with increases in indices of LV volume preload in 100 children or adolescents with ventricular septal defects (VSD), and 36 with chronic rheumatic heart disease (RHD). Compared to 79 normal controls (compaction ratio=1.4±0.07), patients with VSDs (compaction ratio=2.0±0.2, p<0.0001) and RHD (compaction ratio = 2.0±0.3, p< 0.0001) had a marked increase in the compaction ratio. A compaction ratio>2 was found in 42% of patients with VSDs and 47% with RHD. In VSDs, independent of age and gender, the compaction ratio was positively associated with LV mass index (LVMI) (partial r=0.44, p<0.0001), VSD size (partial r=0.4, p<0.0001), LV end diastolic diameter indexed (LVEDD) (partial r=0.24, p= 0.01), and the presence of additional shunts (partial r=0.21, p=0.02). In RHD, independent of age and gender, the compaction ratio was positively associated with LVEDD (partial r=0.62, p=0.0001), and LVMI (partial r=0.48, p=0.005), and negatively with LV ejection fraction (partial r=0.31, p=0.03). The strong association of indices of LV volume load and the compaction ratio would suggest that haemodynamic influences are contributing to the compaction ratio both in congenital and acquired cardiac disease in childhood. Thus an increased compaction ratio may be the consequence of an increased volume preload, and therefore may not necessarily occur only as a result of persistence of embryonic patterns.

Aim: To describe innovative uses of catheter based treatment in a variety of native and post surgical stenoses in children and young adults with congenital heart disease. Background: Cardiac catheterization in man was first described 1929 and since then there has been a drive to develop endovascular techniques to investigate and treat both congenital and acquired heart disease. Many of the advances are being made in congenital heart disease. Methods: A number of congenital cardiac stenotic lesions were studied including baffle obstruction after atrial switch for transposition of the great arteries, aortic stenosis in infants, coarctation of the aorta, peripheral pulmonary artery stenosis and superior vena caval obstruction. The use of angioplasty balloons, cutting balloons, stents and alternative catheter approaches were investigated for these lesions. Results: Following atrial redirection surgery for transposition of the great arteries balloon angioplasty improved baffle haemodynamics. The technique of anterograde balloon dilation of the aortic valve was developed and had superior outcomes in terms of aortic insufficiency compared to a retrograde approach in neonates with severe aortic valve stenosis. In an animal model of peripheral pulmonary arterial stenosis, the application of cutting balloon angioplasty produced effective relief in a controlled fashion. Balloon mounted stents were used in patients with native and post surgical coarctation of the aorta with significant relief of stenosis and relief of hypertension. Finally, a group of patients with superior vena obstruction syndrome after surgical repair of partial anomalous pulmonary venous drainage had successful treatment using balloon mounted stents. Conclusions: Catheter based treatment of congenital and post surgical vascular stenoses of the heart and great arteries using angioplasty balloons, cutting balloons and balloon mounted stents is safe and appears to be effective in the short and medium term. It may represent a useful alternative to surgery and will reduce the number of surgical procedures required over a lifetime. Future directions will include bio-absorbable stents and hybrid techniques involving surgery.

Este estudo teve por objetivo avaliar a eficácia de determinado impresso na fixação dec conhecimentos e adoção de práticas de saúde. Para se atingir esse objetivo foi elaborado o Manual \"Gestante Cardíaca\" para educação de pacientes do Programa \"Cardiopatia e Gravidez\" do Instituto \"Dante Pazzanese\"de Cardiologia. As gestantes cardíacas inscritas no Programa foram divididas em dois grupos. Após consulta médica onde receberam orientação, um dos grupos recebeu o Manual. Conhecimentos e práticas das gestantes foram testados quando ingressaram no Programa e retestados na última consulta. As gestantes do grupo que recebeu o impresso tiveram maior ganho em conhecimentos (82,4 por cento ) e adotaram mais priticas (94,4 por cento ) que as do outro grupo (2,0 por cento e 40,5 por cento ). Os resultados obtidos parecem sugerir a eficácia do Manual para fixar conhecimentos e estimular a gestante cárdiaca na adoção das práticas recomendadas. O processo de elaboraçio do Manual é descrito no trabalho. / The purpose of this study was to evaluate the efficacy of a printed material to reinforce knowledge and in the adoption of health practices. A manual designed to cardiac pregnant women of the Program \"Cardiopathy and Pregnancy\" of the Institute of Cardiology \"Dante Pazzanese\" of São Paulo was prepared. The women were divided into two groups, one of which received the Manual after medical consultation. Both groups were subjected to orientation. Womens\' knowledge and practices were tested at the first consultation and retested at the last one. The group of women who had rcceived the material gained more in knowledge (82,4 per cent ) and adopted more practices (94,4 per cent ) than the group who did not receive the Manual (2,0 per cent and 40,5 per cent ). The results obtained suggest the efficacy of the printed material to reinforce knowledge and stimulate recommended practices. The Manual elaboration process is described in this paper.

Cardiovascular disease is the leading cause of death in patients with underlying chronic kidney disease (CKD). Up to one third of patients presenting with an acute coronary syndrome have CKD stage 3-5. Outcomes following acute myocardial infarction in patients with underlying CKD remain poor. CKD patients are routinely excluded from clinical trials in novel cardioprotective strategies resulting in a paucity of prospective data on which to base guidelines for clinical practice. The aims of this work were to: • Establish and characterise two models of chronic uraemia in rodents: the subtotal nephrectomy model and the adenine diet model. • Determine the effects of underlying chronic uraemia on myocardial ischaemia tolerance. • Examine pharmacological cardioprotective strategies in the context of underlying uraemia using a PARP inhibitor • Investigate the cardioprotective effects of ischaemic conditioning in the context of uraemia. Ischaemic preconditioning and postconditioning protocols were used in both uraemic and non-uraemic animals in a model of acute myocardial infarction. • Preliminary work, using standard molecular biological techniques, was carried out in order to confirm the putative survival pathways responsible for the effect of preconditioning. • Investigate the effect of combining early and late remote ischaemic preconditioning to identify whether summation of these strategies could provide additional tissue protection in a model of acute kidney injury. The results demonstrate that both models develop a uraemic phenotype. Subtotal nephrectomy animals exhibit reduced ischaemia tolerance. PARP inhibition as a pharmacological post conditioning agent was shown to be ineffective at conferring tissue protection, whereas both ischaemic preconditioning and postconditioning were effective cytoprotective strategies in both non-uraemic and uraemic animals. Furthermore, additional benefit was seen when early and late remote preconditioning were summated in a rodent model of acute kidney injury. This work provides a basis for future clinical trials in cardioprotection in the context of underlying CKD.

Cardiovascular disease is the leading cause of unnatural death worldwide. Damage to the endothelial glycocalyx impairs endothelial functions and thereafter leads to the development of cardiovascular diseases. Despite this, many issues remain to be explored in our understanding of the metabolism and vasculoprotective potential of the glycocalyx. This study focuses on the recovery and structural stability of the glycocalyx, and its role in electric field-directed cell migration in vitro. The integrity of the glycocalyx is compromised following trypsin treatment during cell passages. Results from our study show that cell seeding density affects the recovery speed of the glycocalyx in the first 48h. Higher cell density results in more rapid recovery of the glycocalyx. Regardless of the initial cell seeding density, a well-developed glycocalyx layer is observed when cell confluence is reached. Micropatterning is used to study effects of the cell shape on the recovery of the glycocalyx. Elliptical patterns have been used to conform endothelial cells to torpedo shapes, mimicking their morphology under a shear flow. More rapid development of the glycocalyx on elliptical cells is observed than that on circular shaped cells during the early stage of recovery. Effects of the actin cytoskeleton on the stability of the glycocalyx are investigated, following our interest in shedding of the glycocalyx in abnormal vascular microenvironment. Rapid depolymerisation of the actin cytoskeleton leads to cell retraction within 10mins, with the glycocalyx preserved on the cell surface. This is also seen during 24h persistent actin disruption under static conditions. However, when endothelial cells are subjected to 24h steady laminar shear stress, the glycocalyx is seen to shift to the downstream of the cell surface in the control group, and with actin depolymerisation, significant shedding of the glycocalyx from the luminal surface of the cell is observed. This happens together with the loss of focal adhesions on the basal membrane. Using a custom designed electric field (EF) chamber, I demonstrate that the cell migration speed increases by 30~40% following 5h of EF exposure. Cells also show preferred movement towards the anode. However, both are abolished after the enzymatic removal of the glycocalyx, indicating that the speedup and the directional cell migration in applied EF require the presence of the glycocalyx. Even distribution of the glycocalyx on the cell surface at the end of the EF stimulation suggests that EF-directed cell migration is not related to the polarization of the glycocalyx on the cell membrane. All these findings provide a better understanding of the glycocalyx, which will help to develop new strategies for protection of the glycocalyx, restoration of endothelial functions and finally prevention of cardiovascular diseases.

Desmosomes are macromolecular, dynamic and adaptable complexes that connect intermediate filaments of neighboring cells in a variety of tissues, generating a large mechanically resilient structure. The importance of maintaining desmosome homeostasis for tissue integrity and optimal organ function has been revealed through the identification of desmosome-associated disorders and mechanistic studies into desmosome regulation. This thesis focuses on inherited skin and heart conditions linked to mutations in desmosomal genes or in genes believed to be implicated in desmosome regulation. Part of this thesis is focused on the molecular analysis and identification of novel desmosomal mutations in patients clinically diagnosed with Arrhythmogenic Right Ventricular Cardiomyopathy, and the genetic diagnosis of patients with hypotrichosis, hypotrichosis and PPK or acral peeling skin syndrome. Patients were analysed using a number of different genetic techniques including custom capture array, HaloPlex targeted resequencing, exome capture and Sanger sequencing. Both novel and previously reported mutations were identified in DSP, DSC2, DSG2, PKP2, DSG4 or CSTA in patients diagnosed with these disorders. The molecular mechanisms behind mutations in the protease inhibitors cystatin A and calpastatin, leading to the skin disorders exfoliative ichthyosis and PLACK syndrome, were also investigated. In vitro analysis, using siRNA-mediated knockdown in the immortalised keratinocyte cell line HaCaT, demonstrated that these mutations, affecting the structure and function of the protease inhibitors, lead to deficient intercellular adhesion, possibly through the indirect regulation of desmosomal complexes through their target proteases.

The development and function of the heart is governed by a conserved set of transcription factors (TFs) that regulate gene expression in a cell-type, time point and stimulus driven manner. Of these core cardiac TFs, the most ubiquitously expressed is the zinc finger protein GATA4. In cardiomyocytes, GATA4 is central to proliferation, differentiation, hypertrophy and induction of pro-survival pathways. In cardiac endothelial cells, it is required for valve and septal development, although the exact mechanisms remain unclear. To regulate such a wide array of functions in a spatially and temporally controlled manner, GATA4 interacts with specific protein partners, the majority of whom have been identified in cardiomyocytes. However, a complete understanding of the protein interactome of GATA4, particularly in cardiac endothelial cells, has not yet been achieved. Using a mass spectrometry-based approach, we have identified a series of novel GATA4 interacting partners in cardiac endothelial cells. 3xFlag GATA4 was stably overexpressed via retroviral transduction in the TC13 cardiac endothelial precursor cell line, immunoprecipitated from nuclear protein extracts and sent for HPLC-ESI-MS/MS. Several novel GATA4 interacting partners were identified including the chaperone protein Heat Shock Protein 70 (HSP70), the inducible orphan nuclear receptor Nerve Growth Factor 1β (NGFIβ, NUR77) and the Drosophila-Binding/Human Splicing protein family members Non-POU Domain Containing Octamer Binding Protein (NONO) and Paraspeckle 1 (PSPC1). Chapter 1 discusses the interaction between GATA4 and HSP70 and its role in cardiomyocyte survival upon exposure to chemotherapeutic agent Doxorubicin (DOX). HSP70 binds directly to GATA4, preventing DOX-mediated cleavage and degradation by Caspase-1, cardiomyocyte cell death and heart failure. Chapter 2 focuses on the cooperative interaction between GATA4 and NUR77 in cardiac microvascular endothelial cells and its central role in myocardial angiogenesis in response to pressure overload. The GATA4-NUR77 complex transactivates the promoter of Angiopoietin-Like 7 (ANGPTL7), a secreted pro-angiogenic chemotactic factor, triggering endothelial cell proliferation and tube formation in cultured cardiac endothelial cells and increasing myocardial capillary density in vivo. Chapter 3 discusses the interaction between GATA4 and the DBHS proteins NONO and PSPC1 in the regulation of cardiac development. These proteins play opposing roles when bound to GATA4 as PSPC1 enhances GATA4 activation of critical cardiac promoter targets and NONO acts as a rheostat to repress GATA4 activity. In vivo, loss of NONO results in left ventricular non-compaction consistent with humans with loss-of-function mutations. However, simultaneous Gata4 haploinsufficiency partially rescues this phenotype. Together, this data identifies multiple novel cell type and time point specific GATA4 protein partners and sheds light on GATA4 regulatory mechanisms in cardiac development and disease.

Introduction There has been much discussion on personalised medicine; however use of genotype in risk prediction for coronary heart disease (CHD) has not resulted in appreciable improvements over non-genetic risk factors. The primary aim was to determine whether candidate single nucleotide polymorphisms (SNPs) identified from genome-wide association studies improved prediction of CHD over conventional risk factors (CRF). The secondary aim was to determine whether the use of apolipoproteins or lipoprotein(a) improved risk prediction of CHD. Methods Analyses used the Edinburgh Heart Disease Prevention Study (EHDPS), with 1592 men aged 30-59 and follow-up after 20 years; and the Edinburgh Artery Study (EAS), with 1592 men and women aged 54-75 and 15 years of follow-up. Candidate SNPs were identified by systematic literature reviews. CHD status was evaluated as severe (myocardial infarction or coronary revascularisation), and any (severe CHD, angina or non-specified ischaemic heart disease). Cox proportional hazards models were used to evaluate addition of candidate SNPs or lipids to models containing CRF. Results A group of genome-wide significant SNPs resulted in a non-significant improvement in C-index for severe CHD (0.038, p=0.082), and a significant improvement in C-index for any CHD (0.042, p=0.016); the associated net reclassification improvements (NRI) were 20.5% and 18.7%, respectively. Regression trees identified SNPs that were predictive of the remaining variance after adjusting for CRF; this resulted in a significant improvement in C-index for any CHD (0.031, p=0.008). The NRI were 11.0% and 9.6% for severe and any CHD, respectively. When compared with HDL cholesterol/total cholesterol, apolipoprotein AI/total cholesterol yielded a NRI of 3.3% for severe CHD. Lipoprotein(a) improved prediction of severe CHD, with a non-significant improvement in C-index (0.020, p=0.087), and NRI of 11.8%. Conclusion The results of this study indicate that a well selected group of candidate SNPs can improve risk prediction for CHD over-and-above CRF. The inclusion of lipoprotein(a), along with CRF, appeared to improve prediction of severe CHD, but not any CHD.

Heart disease is still the number one killer in the United States. Recent research has suggested that adhering to a plant-based diet can prevent, treat, and reverse heart disease. In order to further clarify these findings, an analysis was made of the components of a plant-based diet and such dietary effects in relation to being a possible treatment for heart disease. Based on a comprehensive investigation of this area of study, an extensive body of evidence supports the finding that a whole-food, plant-based diet can significantly lower the risk of heart disease, mainly by reducing blood levels of lipids and cholesterol associated with atherosclerosis. Comparison was made between the efficacy of the plant-based diet versus more conventional approaches such as medication and surgery. Further clinical trials are needed to validate the findings of adopting this diet in the prevention and treatment of heart disease.

Giant Cell arteritis (GCA) is a systemic inflammatory disease that affects arteries of medium- and large size. Symptoms of GCA such as headache and fever usually promptly improve with treatment of glucocorticoids. Apart from advanced age, female sex and Northern-European descent, risk factors for GCA are unknown. Most studies have found that life expectancy for patients with GCA is not reduced compared with the general population and studies on cardiovascular disease in GCA have provided conflicting results. Data for the studies of this thesis are drawn from the Reykjavik Study (RS) that is a general population-based cohort study with continuous surveillance for coronary heart disease and vital status. Subjects born in 1907–1934 and living in Reykjavik, Iceland or adjacent communities in 1966 were invited for study visit from 1967-1994. Information on cardiovascular risk factors were collected at study visit. Diagnosis of GCA for this study was based on re-examination of all temporal arteries biopsies (TAB) from members of the RS cohort; however, information was also obtained from the original pathology report. Of 19,360 subjects included in the RS, 194 developed GCA during the follow-up period. Body mass index was inversely associated with the occurrence of GCA. Among men, but not women, hypertension was associated and smoking inversely associated with the occurrence of GCA. Among women, but not men, GCA was associated with coronary heart disease. Subjects with GCA had approximately 50% increase in mortality risk compared with the general population. Increase mortality was mainly observed among GCA patients based on the diagnosis of re-examination of TAB; however, no such an association was found if diagnosis of GCA was made based on the original pathology report. Those subjects were likely not clinically diagnosed with GCA, signaling that treatment for GCA might be beneficial with respect to mortality risk.