Despite advances in treatment, heart failure remains one of the top killers in Canada. This recognition motivates a new research focus to harness the fundamental repair properties of the human heart, with human cardiac stem cells (CSCs) emerging as a promising cell candidate to regenerate damaged myocardium. The rationale of this approach is simple with ex vivo amplification of CSCs from clinical grade biopsies, followed by delivery to areas of injury, where they engraft and regenerate the heart. Currently, outcomes are limited by modest engraftment and poor long-term survival of the injected CSCs due to on-going cell loss during transplantation. As such, we explored the effect of cell encapsulation to increase CSC engraftment and survival after myocardial injection. Transcript and protein profiling of human atrial appendage sourced CSCs revealed strong expression the pro-survival integrin dimers αVβ3 and α5β1- thus rationalizing the integration of fibronectin and fibrinogen into a supportive intra-capsular matrix. Encapsulation maintained CSC viability and expression of pro-survival transcripts when compared to standard suspended CSCs. Media conditioned by encapsulated CSCs demonstrated superior production of pro-angiogenic/ cardioprotective cytokines, angiogenesis and recruitment of circulating angiogenic cells. Intra-myocardial injection of encapsulated CSCs after experimental myocardial infarction favorably affected long-term retention of CSCs, reduced scar burden and improved overall cardiac function. Taken together, cell encapsulation of CSCs prevents detachment induced cell death while boosting the mechanical retention of CSCs to enhance repair of damaged myocardium.

Congenital heart defects (CHD) are the largest class of birth defects in humans and are a major cause of infant mortality and morbidity. Deciphering the molecular and genetic etiologies central for heart development and the pathogenesis of congenital heart diseases (CHD) is a challenging puzzle. We have previously demonstrated that the zinc-finger kruppel-like transcription factor KLF13, expressed predominantly in the atria, binds evolutionarily conserved regulatory elements known as CACC-boxes and transcriptionally activates several cardiac promoters. KLF13 loss of function in Xenopus embryos was associated with cardiac developmental defects underscoring its critical role in the heart. In the current study, using in vivo and in vitro approaches, we examined KLF13’s mechanisms of action and its interaction with other cardiac regulators. To test the evolutionary conserved role in the mammalian heart, we deleted the Klf13 gene in transgenic mice using homologous recombination. Mice with homozygote deletion of Klf13 were born at reduced frequency owing to severe heart defects. We also report the existence of a novel isoform of KLF13, referred to here as KLF13b. Furthermore, we report that KLF13 interacts biochemically and genetically with the T-box transcription factor TBX5 which is a key regulator of heart development. Our data provide novel insight into the role of KLF13 in cardiac transcription and suggest that KLF13 maybe a genetic modifier of congenital heart disease. Furthering our knowledge of protein-protein interactions and gene transcription will enhance genotype-phenotype correlation and contribute to better understanding of the etiology of CHD.

Heart failure is responsible for one in twenty deaths in the UK, and as the average age of the general population increases that number is predicted to rise over the coming years. Hypertrophic growth is believed to be an adaptive response to a chronic increase in workload under circumstances such as hypertension, yet it is also known to contribute to the pathological progression into heart failure. Abnormal calcium handling is known to play a critical role in determining disease progression, not only through its function as the driving force behind myocardial contraction and relaxation but also through directing the signals which regulate hypertrophic growth. Both isoforms 1 and 4 of the diastolic calcium extrusion pump plasma membrane calcium ATPase (PMCA) are present in the heart, yet unlike in other cell types their contribution to overall calcium clearance is only small; however their role in the disease process is yet to be defined. A novel mouse line was generated in which both PMCA1 and 4 were deleted from the myocardium (PMCA1:4dcko mice). Through comparison with PMCA1 knockout mice (PMCA1cko) this thesis set out to identify the specific function of each pump under normal conditions and during the development of pathological hypertrophy induced by pressure overload through transverse aortic constriction (TAC).Under basal conditions each isoform functioned independently, PMCA1 to extrude calcium during diastole and PMCA4 to regulate calcium levels during systole; however the loss of neither isoform impacted significantly on cardiac function. In response to TAC, PMCA1cko mice progressed rapidly into decompensation and displayed signs of systolic failure after just 2 weeks, whilst cardiac function was preserved in TAC controls. Calcium handling analysis revealed that prior to the onset of failure PMCA1cko mice displayed a distinct lack of adaptive changes to calcium cycling which were present in controls. In stark contrast, the additional loss of PMCA4 led to an attenuated hypertrophic response to TAC in PMCA1:4dcko mice which remarkably preserved cardiac function despite the absence of PMCA1. This adds to accumulating evidence which suggests that the inhibition of PMCA4 may be protective during the development of pathological hypertrophy, whilst highlighting the possibility for a novel role for PMCA1 in coordinating essential adaptations required to enhance calcium cycling in response to the increased demands imposed on the left ventricle during pressure overload.

Cardiovascular disease remains the leading cause of death in the developed world. In this thesis computational modelling techniques were used to study the mechanisms and genesis of atrial arrhythmias. It is separated into 2 parts: (1) The mechanistic links between mutations of the fast Na+ channel (INa) and the ability of the sinoatrial node to pace the surrounding atrial muscle were investigated. The mutations were separated into two groups, one for the mutations affecting the steady-state activation, and the other for those affecting steady-state inactivation. On the single cell level it was found that all mutations slowed the pacing rate of the sinoatrial node in a similar way, but at the 2D level the two mutation groups modulated the excitation of the tissue differently. One caused a conduction block between the sinoatrial node and atrium, where the other abolished pacemaking all together. (2) A new set of mathematical models were then developed for the sheep atria. This was incorporated into an anatomically detailed 3D geometry of the whole sheep atria to form a platform suitable for the study of clinical atrial fibrillation, and other atrial arrhythmias. Due to the lack of single cell electrophysiology data available, a method of cross-species modelling was utilised. A biophysically detailed model of the 3D sheep atria was created, and used in a preliminary study into the susceptibility of tissue to atrial fibrillation from the rapid pacing of the pulmonary vein area. It was found that both electrical heterogeneity and the complex fibre structure of the atria need to be considered for sustained atrial fibrillation to be seen.

This thesis showed that dogs with natural infection with B. canis had both renal and cardiac dysfunction, both of which can be classified as complications of babesiosis and would thus necessitate supportive therapy. This thesis demonstrated that RTE celluria, proteinuria, and variable enzymuria and azotaemia occur in dogs with babesiosis. However, these were all minimal changes and all could be consistent with hypoxia, reduced GFR, or reduced cardiac output This thesis showed that dogs with naturally occurring babesiosis had significant urine met-haemoglobin with no evidence of blood met-haemoglobin. The possibility would be that the urinary methaemoglobin was either produced in the kidney or possibly by oxidation of haemoglobin to met-haemoglobin in the bladder. It has been shown experimentally that met-haemoglobin can be toxic. The combination of reduced GFR, anaemic hypoxia, and met-haemoglobin can all act synergistically to cause renal damage. Renal haemodynamics are also much more likely to be abnormal when cardiac dysfunction is present Reduced renal blood flow and glomerular filtration rate are evidence of redistribution of blood flow that commonly occurs in early heart failure. An important finding in this thesis was that dogs with babesiosis had lower serum sodium than control dogs but there was no difference between mild, severe, or complicated cases of babesiosis. In addition, dogs with babesiosis had a lower fractional clearance of sodium than Clinically healthy control dogs, which can be interpreted as sodium retention by the kidneys. This sodium retention would also result in water retention , which will result in an expansion of the plasma volume. In the past heart lesions in canine babesiosis were regarded as rare complications, with the majority of lesions being reported as incidental findings at post-mortem examination of complicated babesiosis cases. This thesis has demonstrated that cardiac lesions to be common in canine babesiosis. This thesis showed that that ECG changes in babesiosis were similar to the pattern described for myocarditis and myocardial ischaemia, and together with the histopathological findings indicated that the heart suffers from the same pathological processes described in other organs in canine babesiosis, namely inflammation and hypoxia. As the clinical application of the ECG changes found in this thesis was limited, cardiovascular assessment should be based on functional monitoring rather than ECG. Using cardiac troponin as a marker of myocardial injury, this thesis showed that myocardial cell injury occurs with canine babesiosis. Cardiac troponins, especially troponin I, are sensitive markers of myocardial injury in canine babesiosis, and the magnitude of elevation of plasma troponin I concentrations appears to be proportional to the severity of the disease. ECG changes and serum cardiac troponin were correlated with histopathology. On cardiac histopathology from dogs that succumbed to babesiosis, haemorrhage, necrosis, inflammation and fibrin microthrombi in the myocardium were documented, all of which would have resulted in ECG changes and elevations in cardiac troponin. Myocardial infarction causes left ventricular failure, which will result in hypotension and an expansion of the plasma volume due to homeostatic mechanisms. This thesis showed that dogs with babesiosis had hypoalbuminaemia, which may be because of intravascular volume dilution due to fluid retention. In disease hypoalbuminaemia can occur as a negative acute-phase protein. In the light of the cardiac changes, hyponatraemia, and hypotension, a probable cause would be fluid retention due to myocardial disease. This thesis showed that dogs with babesiosis had left ventricular lesions, which can result in systolic heart failure. / Thesis (PhD)--University of Pretoria, 2005. / Veterinary Tropical Diseases / unrestricted

No description available.

Atrioventricular nodal reentrant tachycardia (AVNRT) is an abnormal heart rhythm caused by aberrant electrical conduction within the AV node. AVNRT is the most common type of paroxysmal supraventricular tachycardia (PSVT), with approximately 50,000 new cases per year in the United States. Catheter ablation of AV node tissue has become the first-line definitive treatment for AVNRT, owing to its high efficacy, tolerability, and safety. Two modalities of ablation, radiofrequency (RF) and cryoablation are commonly utilized in clinical practice with high levels of success in treating AVNRT. To date, studies on the two modalities have compared metrics such as acute success rate, procedure time, fluoroscopy time, and recurrence of AVNRT. Recurrence of AVNRT has been observed as far as 10 years after RF ablation. In patients with a history of RF ablation for AVNRT, rates of atrial fibrillation are higher than that of the general population. However, long-term studies directly comparing RF and cryoablation outcomes do not exist. This retrospective cohort study is designed to examine the rates of AVNRT recurrence and new arrythmias in patients 10 to 15 years after receiving either RF or cryoablation. To accomplish this, eligible participants will have their medical records reviewed for documentation of AVNRT recurrence, atrial fibrillation, atrial flutter, and complete AV block requiring pacemaker implantation. In doing so, we hope to give providers more insight into the risk profiles for each modality.

Mitochondrial dysfunction is a common feature in a wide range of disorders and diseases from obesity, diabetes, cancer to cardiovascular diseases. The overall goal of my doctoral research has been to investigate mitochondrial metabolic dysfunction in skeletal and cardiac muscles in the context of chronic disease development. Perinatal nutrition is well known to affect risk for insulin resistance, obesity, and cardiovascular disease during adulthood. The underlying mechanisms however, are poorly understood. Previous research from our lab showed that the in utero maternal undernutrition mouse model is one in which skeletal and cardiac muscle physiology and metabolism is impaired. Here we used this model to study the impact of in utero undernutrition on offspring skeletal primary muscle cells and to determine if there is a cell autonomous phenotype. Metabolic analyses using extracellular flux technologies revealed a shift from oxidative to glycolytic metabolism in primary myotubes. Gene expression profiling identified significant changes in mRNA expression, including an upregulation of cell stress and OXPHOS genes and a downregulation of cell division genes. However, there were no changes in levels of marker proteins for mitochondrial oxidative phosphorylation (OXPHOS). Findings are consistent with the conclusion that susceptibility to metabolic disease in adulthood can be caused at least in part by muscle defects that are programmed in utero and mediated by impaired mitochondrial function. In my second project, the effects of the absence of glutaredoxin-2 (Grx2) on redox homeostasis and on mitochondrial dynamics and energetics in cardiac muscle from mice were investigated. Previous work in our lab established that Grx2-deficient mice exhibit fibrotic cardiac hypertrophy, and hypertension, and that complex I of OXPHOS is defective in isolated mitochondria. Here we studied the role of Grx2 in the control of mitochondrial structure and function in intact cells and tissue, as well as the role of GRX2 in human heart disease. We demonstrated that the absence of Grx2 impacts mitochondrial fusion, ultrastructure and energetics in mouse primary cardiomyocytes and cardiac tissue and that provision of the glutathione precursor, N-acetylcysteine (NAC) did not restore glutathione redox or prevent impairments. Furthermore we used data from the human Genotype-Tissue Expression consortium to show that low GRX2 expression is associated with increased fibrosis, hypertrophy, and infarct in the left ventricle. Altogether, our results indicate that GRX2 plays a major role in cardiac mitochondrial structure and function, and protects against left ventricle pathologies in humans. In my third project, we collaborated with cardiac surgeon, Dr. Calum Redpath, of the Ottawa Heart Institute to study atrial mitochondrial metabolism in atrial fibrillation patients with and without type 2 diabetes (T2DM). T2DM is a major risk factor for atrial fibrillation, but the causes are poorly understood. Atrial appendages from coronary artery bypass graft surgery were collected and analyzed. We showed an impaired complex I respiration in diabetic patients with atrial fibrillation compared to diabetic patients without atrial fibrillation. In addition, and for the first time in atrial fibrillation patients, mitochondrial supercomplexes were studied; results showed no differences in the assembly of the “traditional” complexes but a decrease in the formation of “high oligomeric” complexes. A strong trend for increased protein oxidation was also observed. There were no changes in markers for OXPHOS protein levels. Overall findings reveal novel aspects of mitochondrial dysfunction in atrial fibrillation and diabetes in humans. Overall, our results reveal that in utero undernutrition affects the programming of skeletal muscle primary cells, thereby increasing susceptibility to metabolic diseases. In addition, we show that GRX2 impacts cardiac mitochondrial dynamics and energetics in both mice and humans. Finally, we show impaired mitochondrial function and supercomplex assembly in humans with atrial fibrillation and T2DM. Ultimately, understanding the mechanisms causing mitochondrial dysfunction in muscle tissues during chronic disease development will increase our capacity to identify effective prevention and treatment strategies.

The name of William Harvey is imortal, and it is fitting that a quotation form his epoch-making 'De Motu Cordis et Sanguinis' should preface this thesis. the discoverer of the circulation did not fall to point out the difference between foetal and postnatal conditions of the heart and great vessels. Harvey, however, was not particularly concerned with the problems of the foetal circulation, and devoted only a passing glance to the subject, using foetal conditions to illustrate his general argument about the circulation of the blood. The subsequent progress of though on the subject of foetal circulation has been admirably set out in the first chapter of Barclay, Frankin, and Prichard's book 'The Foetal Circulation', published in 1944, but there is no doubt that the major advance since Harvey's time is represented by the cine-radiographic observations made by the authors of this book on the foetal lamb. They provided, for this species, a convincing and complete picture of the pattern of the foetal circulation, together with the change brought on by allowing the foetus to breathe and by severing the umbilical cord, thus simulating the event of birth.

Cardiac stem cells (CSCs)-based therapy provides a promising avenue for the management of ischemic heart diseases. However, engrafted CSCs are subjected to acute cell apoptosis in the ischemic microenvironment. Here, stem cell antigen 1 positive (Sca-1+) CSCs proved to own therapy potential were cultured and treated with H2O2 to mimic the ischemia situation. As autophagy inhibitor, 3-methyladenine (3MA), inhibited H2O2-induced CSCs apoptosis, thus we demonstrated that H2O2 induced autophagy-dependent apoptosis in CSCs, and continued to find key proteins responsible for the crosstalk between autophagy and apoptosis. Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2), increased upon cardiomyocyte injury with unknown functions in CSCs, was increased by H2O2. NR4A2 siRNA attenuated H2O2 induced autophagy and apoptosis in CSCs, which suggested an important role of NR4A2 in CSCs survival in ischemia conditions. Reactive oxygen species (ROS) and NF- κB (P65) subunit were both increased by H2O2. Either the ROS scavenger, N-acetyl-lcysteine (NAC) or NF-κB signaling inhibitor, bay11-7082 could attenuate H2O2-induced autophagy and apoptosis in CSCs, which suggested they were involved in this process. Furthermore, NAC inhibited NF-κB activities, while bay11-7082 inhibited NR4A2 expression, which revealed a ROS/NF-κB/NR4A2 pathway responsible for H2O2- induced autophagy and apoptosis in CSCs. Our study supports a new clue enhancing the survival rate of CSCs in the infarcted myocardium for cell therapy in ischemic cardiomyopathy.