Atrial fibrillation (AF), is characterised by rapid, irregular atrial stimulation, which leads to reduced contraction, resulting in a negative force-frequency-relationship (FFR). It is thought that rapid stimulation frequencies, which are synonymous with AF, disturb excitation-contraction-coupling (ECC). Ageing increases both the prevalence and economic impact of AF progressively. Although mechanisms underlying AF have been investigated in human and animal models, the cellular electrophysiology, and molecular changes that render the atria in aged individuals more susceptible to AF, still remain poorly understood. To our knowledge, there are no data investigating the effects of rapid stimulation frequencies on the Ca2+ handling within atrial myocytes, of a larger mammal, which are able to maintain AF, and is physiologically relevant to the human. To investigate this, sheep atrial myocytes were isolated, via enzymatic digestion. Measurements of intracellular Ca2+ ([Ca2+]i) were obtained via analysis of fluorescence (Fluo 5F am). Electrophysiological experiments were performed via the perforated-patch clamp technique, under voltage-clamp stimulation, to assess measurements of [Ca2+]i and trans-sarcolemmal currents. All voltage clamp experiments were performed at 37°C. Analysis of CaMKII inhibition, on Ca2+ wave frequency, was performed in non-stimulated atrial myocytes, at room temperature. Increased stimulation frequency (from 1Hz – 5Hz) had a significant impact on Ca2+ handling and trans-sarcolemmal currents within the atria. A reduction in Ca2+ transient amplitude was observed with increased rate. This was achieved despite increases in SR Ca2+ content, which were due to increased SERCA activity. The reduction in Ca2+ transient amplitude was attributed to reduced L-type Ca2+ current (ICa-L). Ageing augmented the rise in diastolic [Ca2+]i which was observed with rate, but with no further impac on Ca2+ transient amplitude. The rate dependent increase in SR Ca2+ content was augmented with age, and was presumed to be as a result of a reduction in ICa-L.By sensitising the ryanodine receptor (RyR), with low-dose caffeine (500 μmol.l-1), the fractional release of the first Ca2+ transient upon application was exacerbated by an increase in rate. This rapidly decayed to control levels at all stimulation frequencies. This data suggests that increases in RYR sensitivity lead to a greater Ca2+ release from the SR, for a given trigger. By ceasing stimulation there was potentiation of the first Ca2+ transient, post-rest, in comparison to pre-rest at 1Hz. This was augmented by increased rate. As ICa-L was unaltered between pre- and post-rest, within each frequency, it was assumed that the increased SR Ca2+ content with rate, coupled with continued SR Ca2+ uptake during the rest period, enhanced the fractional SR Ca2+ release for a given trigger, thus potentiating the amplitude of the Ca2+ transient. However, this requires further investigation. Other data found that CaMKII inhibition (via KN93) had no effect on Ca2+ wave frequency in control, or heart failure (HF), non-stimulated sheep atrial myocytes, which suggests either CaMKII is not up-regulated in the HF model used, or the concentration of KN93 used was insufficient. Further investigation is required in this area. The alterations in the mechanisms that modulate SR Ca2+ release and uptake are affected by alterations in stimulation frequency, which alter key modulators of contractile force.

In recent years Computational Fluid Dynamics (CFD) has been extensively used in the study of the indoor environment and the thermal comfort conditions for the design of modern buildings, however, there remains the need to thoroughly evaluate the accuracy of the results given by CFD methods. In the present work, numerical investigations of the indoor thermal environment in the atria of two existing buildings and in a simple three-storey atrium building design have been undertaken using CFD techniques. The initial work involved the evaluation of various turbulence models and a radiation model used in CFD simulations for the prediction of the thermal environment in atria of different geometrical configurations in two buildings for which experimental data is available. The airflow patterns and temperature distributions were determined, under both forced and hybrid ventilation conditions and thermal comfort conditions were evaluated. The numerical predictions were compared with the available experimental measurements and, in general, good agreement was obtained between the numerical and experimental results. After the evaluation of the adequacy of available turbulence models and the validation of the accuracy of the CFD model used, a simple full-scale three-storey atrium building was modeled to explore the potential of using buoyancy-driven natural ventilation. The validated CFD model was used to determine the ventilation flow rates, airflow patterns, and temperature distributions in the building. The dynamic effect of the thermal mass of the external walls on the performance of the building was also investigated using transient CFD simulations. Atria with various geometrical configurations were studied in order to investigate the effect of atrium design changes on the air flow and temperature distributions in the simple atrium building considered. A parametric study was carried out to assess the sensitivity of the ventilation performance to the change in various geometric and solar parameters. On the basis of this parametric study, a few changes were carried out in the design of the building to examine their effect on ventilation performance. Finally, the use of night ventilation in the atrium building was explored and it was found that night ventilation can be increased by using hot water circulation in the chimney walls. / Thesis (Ph.D, Mechanical and Materials Engineering) -- Queen's University, 2012-07-22 12:57:00.947

Atrial fibrillation (AF) is a common and serious arrhythmia. Current treatments are of limited efficacy, and most do not treat the atrial structural remodeling (hypertrophy and fibrosis) that underlies most clinical AF. Our group has created an experimental dog model of atrial mechanical stretch called the simultaneous atrial and ventricular pacing (SAVP) model (which results in atrial fibrosis and susceptibility to AF) in order to study novel treatments for structural remodeling induced AF. Omega-3 polyunsaturated fatty acids (n3 PUFAs), particularly the marine derived forms eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been shown to be effective in treating arrhythmias (including AF) in some animal studies and clinical trials. The mechanism for this effect of n3 PUFAs is not well understood. In this study we sought to characterize the n3 PUFA effect on AF vulnerability, atrial electrophysiology, histology, and gene expression, and determine relevant mechanisms. Dogs were paced for 0, 2, 7 or 14 days and given n3 PUFAs, olive oil or nothing. Prophylactic n3 PUFAs significantly reduced both AF vulnerability and conduction slowing in SAVP dogs (%AF inducibility: 9.2±8.8 vs. 4.7±6.3; global atrial conduction time: 75±11ms vs. 65±6ms [SAVP 14 days vs. SAVP 14 days with n3 PUFAs, P<0.05 for both comparisons]). Prophylactic n3 PUFAs also reduced inflammation (mean CD18 grade: 2.1±0.8 vs. 1.3±0.6 [SAVP 2 days vs. SAVP 2 days with n3 PUFAs, P=0.055]), hypertrophy (myocyte cross-sectional area: 498±64µm2 vs. 322±111µm2 [SAVP 14 days vs. SAVP 14 days with n3 PUFAs, P<0.05]), and fibrosis (%collagen area vs. unpaced dogs: 178±58 vs. 127±37 [SAVP 14 days vs. SAVP 14 days with n3 PUFAs, P<0.05]). N3 PUFAs were also found to reduce the expression of structural remodeling related molecules such as TGF-β, EGF, ERK and Akt. N3 PUFAs given after some pacing had already occurred were found to be less effective at reducing AF vulnerability and structural remodeling. The results of this study suggest that, in the SAVP model, n3 PUFAs reduce vulnerability to AF by attenuation of adverse structural remodeling at the genetic level.

Atrial fibrillation (AF) is a common and serious arrhythmia. Current treatments are of limited efficacy, and most do not treat the atrial structural remodeling (hypertrophy and fibrosis) that underlies most clinical AF. Our group has created an experimental dog model of atrial mechanical stretch called the simultaneous atrial and ventricular pacing (SAVP) model (which results in atrial fibrosis and susceptibility to AF) in order to study novel treatments for structural remodeling induced AF. Omega-3 polyunsaturated fatty acids (n3 PUFAs), particularly the marine derived forms eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been shown to be effective in treating arrhythmias (including AF) in some animal studies and clinical trials. The mechanism for this effect of n3 PUFAs is not well understood. In this study we sought to characterize the n3 PUFA effect on AF vulnerability, atrial electrophysiology, histology, and gene expression, and determine relevant mechanisms. Dogs were paced for 0, 2, 7 or 14 days and given n3 PUFAs, olive oil or nothing. Prophylactic n3 PUFAs significantly reduced both AF vulnerability and conduction slowing in SAVP dogs (%AF inducibility: 9.2±8.8 vs. 4.7±6.3; global atrial conduction time: 75±11ms vs. 65±6ms [SAVP 14 days vs. SAVP 14 days with n3 PUFAs, P<0.05 for both comparisons]). Prophylactic n3 PUFAs also reduced inflammation (mean CD18 grade: 2.1±0.8 vs. 1.3±0.6 [SAVP 2 days vs. SAVP 2 days with n3 PUFAs, P=0.055]), hypertrophy (myocyte cross-sectional area: 498±64µm2 vs. 322±111µm2 [SAVP 14 days vs. SAVP 14 days with n3 PUFAs, P<0.05]), and fibrosis (%collagen area vs. unpaced dogs: 178±58 vs. 127±37 [SAVP 14 days vs. SAVP 14 days with n3 PUFAs, P<0.05]). N3 PUFAs were also found to reduce the expression of structural remodeling related molecules such as TGF-β, EGF, ERK and Akt. N3 PUFAs given after some pacing had already occurred were found to be less effective at reducing AF vulnerability and structural remodeling. The results of this study suggest that, in the SAVP model, n3 PUFAs reduce vulnerability to AF by attenuation of adverse structural remodeling at the genetic level.

Mutagenesis screening in mice is an effective means of identifying essential genes in cardiovascular development. The l11Jus8 (L8) mutant mouse line was originally isolated from a region-specific N-ethyl-N-nitrosourea (ENU) chemical mutagenesis screen and exhibited an autosomal recessive mid-gestational embryonic lethal phenotype characterised by haemorrhage in the thoracic cavity, blood pooling in the heart, right ventricular dysmorphology and yolk sac vascular degeneration. Prior work mapped the L8 mutation to a ~2.77Mb region on mouse chromosome 11. The aim of this study was to further characterise the L8 mutant phenotype and identify the L8 causative mutation. Phenotypic characterisation conducted here confirmed mid-gestational lethality, haemorrhage and yolk sac vascular degeneration in L8 mutants. Histological analysis of L8 mutants demonstrated presence of fragmented cell nuclei and loss of myocardial integrity in embryonic atrial myocardium. Areas of fragmented cell nuclei did not exhibit positive staining for apoptosis. Furthermore, L8 mutants did not appear to experience typical cardiac defects in aspects including myocardial or smooth muscle differentiation, cell proliferation, ECM production, myocardial hypoplasia/hyperplasia, basement membrane components or observable aberrations in cardiac conduction. L8 mutants exhibited atypical cardiac defects including sudden cessation of heartbeat with morphological indicators of necrosis such as swelling of mitochondria and release of microparticles both from atrial myocardial cells. The L8 mutant appears to represent a novel combination of cardiac defects or novel defects with secondary cardiac phenotypes. Sequencing of the coding exons and splice junctions of 22 candidate genes within the ~2.77Mb L8 locus did not identify the causative mutation. The L8 locus was therefore further refined to a ~1.16Mb region including 20 genes. Sanger sequencing of 10 of these genes plus targeted sequence capture and SOLiD sequencing of the region did not identify a potential L8 mutation. Given the refinement of the candidate locus and advances in sequencing technology and analysis, further sequencing will likely identify the L8 mutation and confirm the cause of the embryonic lethal phenotype.

The murine model has been used to investigate the role of cardiac sensory axons in various disease states. However, the distribution and morphological structures of cardiac nociceptive axons in normal murine tissues have not yet been well characterized. In this study, whole-mount atria from FVB mice were processed with calcitonin gene-related peptide (CGRP) and substance P (SP) primary antibodies followed by secondary antibodies, and then examined using confocal microscopy. We found: 1) Large CGRP-IR axon bundles entered the atria with the major veins, and these large bundles bifurcated into small bundles and single axons that formed terminal end-nets and free endings in the epicardium. Varicose CGRP-IR axons had close contacts with muscle fibers, and some CGRP-IR axons formed varicosities around principle neurons (PNs) within intrinsic cardiac ganglia (ICGs). 2) SP-IR axons also were found in the same regions of the atria, attached to veins, and within cardiac ganglia. Similar to CGRP-IR axons, these SP-IR axons formed terminal end-nets and free endings in the atrial epicardium and myocardium. Within ICGs, SP-IR axons formed varicose endings around PNs. However, SP-IR nerve fibers were less abundant than CGRP-IR fibers in the atria. 3) None of the PNs were CGRP-IR or SP-IR. 4) CGRP-IR and SP-IR often colocalized in terminal varicosities around PNs. Collectively, our data document the distribution pattern and morphology of CGRP-IR and SP-IR axons and terminals in different regions of the atria. This knowledge provides useful information for CGRP-IR and SP-IR axons that can be referred to in future studies of pathological remodeling.

O coração é formado por tecido muscular especializado e por um esqueleto de tecido conjuntivo que sustenta e dá inserção a musculatura. Este tecido conjuntivo é formado predominantemente por fibras colágenas tipo I, tipo III e fibras elásticas, as quais influenciam diretamente as propriedades estruturais e funcionais do miocárdio. Algumas doenças podem provocar alterações qualitativas e quantitativas no colágeno comprometendo a funcionalidade do órgão. Diante da inexistência de informações sobre os efeitos diretos do diabetes na estrutura atrial e a concentração de pesquisas em torno dos ventrículos, especialmente o esquerdo, que está ligado à circulação sistema, a proposta deste trabalho foi analisar as alterações morfológicas quantitativas e qualitativas do colágeno nas câmaras atriais comparando as diferenças entre os grupos de cães sadios e diabéticos. Além disto, esta pesquisa verificou a distribuição de outros constituintes do átrio como fibras elásticas e colágenas do tipo III. Foram utilizados corações de cães sem raça definida, machos e fêmeas, sadios e diabéticos. As estruturas foram examinadas por microscopia óptica, imunofluorescência e microscopia eletrônica de varredura. A taxa de colágeno nos átrios de cães diabéticos foi maior do que nos átrios de cães sadios. Isso indica que há uma mudança estrutural nos átrios de animais com doença metabólica, a partir da verificação de uma proteína estrutural importante como o colágeno. / The heart is composed of specialized muscle tissue and a skeleton of connective tissue that supports and gives muscle insertion. This tissue connective is formed predominantly of collagen type I, type III and elastic fibers, which directly influence the structural and functional properties of the myocardium. Some diseases can cause qualitative and quantitative changes in collagen compromising the functionality of the organ. Due to the lack of information on the direct effects of diabetes on atrial structure and the concentration of researches about the ventricles, especially the left, which is connected to the macro circulation system, the purpose of this study was to analyse the morphological quantitative and qualitative collagen in the chambers atrial comparing the differences between the groups of healthy and diabetic dogs. Moreover, this study found the distribution of other constituents like elastic and collagen type III fibers. We used hearts of mongreal dogs, males and females, healthy and diabetics. The structures were examined by light microscopy, immunofluorescence and scanning electron microscopy. The rate of collagen in the atria of diabetic dogs was greater than in the healthy dogs. This indicates that there is a structural change in the atria of animals with metabolic disease, from the finding of a major structural protein like collagen.

Gap junctions provide direct electrical and biochemical communication between cardiomyocytes in the heart. Connexin40 (Cx40) is the major connexin in the atria of the heart and little is known regarding its regulation. Thus, the goal was to investigate the regulation of Cx40 in both physiological and pathophysiological conditions. The first objective of this thesis was to determine whether Cx40 gap junctions were regulated by â-adrenergic receptor activation. Cx40 has previously been shown to be acutely activated by cAMP, this cAMP-induced increase in Cx40-mediated cell-to-cell dye transfer has been shown to be effected through the â-adrenergic receptor-adenylyl cyclase- Protein Kinase A (PKA) pathway in Cx40-transfected HeLa cells. The second objective of this thesis was to determine whether Cx40 gap junctions were regulated by intracellular Ca2+ concentration ([Ca2+]i ). [Ca2+]i was increased by addition of the ionophore ionomycin and elevating extracellular calcium [Ca2+]o from 1.8 mM to 21.8 mM. This resulted in an elevation of [Ca2+]i and effected an inhibition of Cx40-mediated cell-to-cell dye transfer (IC50 of 500 ± 0.72 nM) which was Calmodulin-dependent. The third objective of this thesis was to determine whether Cx40 gap junctions were regulated by ischemia. Inducing ischemia chemically by inhibiting the electron transport chain with sodium cyanide and glycolysis with iodoacetate and 2-deoxyglucose effected an inhibition of Cx40-mediated cell-to-cell dye transfer that was shown to be Calmodulin dependent. The main conclusions of this thesis were: (1) â-adrenergic receptor activation increases Cx40-mediated cell-to-cell dye transfer which requires the activation of PKA; (2) A sustained elevation in [Ca2+]i causes a partial inhibition of Cx40 gap junction-mediated cell-to-cell dye transfer which was Ca2+-and Calmodulin dependent; (3) Chemical ischemia causes a partial inhibition of Cx40 gap junction-mediated cell-to-cell dye transfer which was shown to be Calmodulin-dependent.

Orientador: Silméia Garcia Zanati Bazan / Resumo: Fundamento: A fibrilação atrial (FA) é uma arritmia supraventricular caracterizada por uma completa desorganização na atividade elétrica atrial, fazendo com que os átrios percam sua capacidade de contração, não gerando sístole atrial. Sua prevalência aumenta com a idade e geralmente está associada a doenças estruturais cardíacas, trazendo prejuízos hemodinâmicos e complicações tromboembólicas. A anticoagulação oral (ACO) é capaz de prevenir os eventos tromboembólicos e sua monitorização é realizada através do INR (International Normalized Ratio). Objetivos: 1-avaliar a estabilidade do INR dos pacientes anticoagulados portadores de fibrilação atrial (FA) não valvar; 2- avaliar as complicações tromboembólicas ou hemorrágicas nestes pacientes; 3- identificar o subgrupo de maior risco para eventos tromboembólicos ou hemorrágicos. Casuística e Métodos: Foram revisados os prontuários médicos de 203 pacientes atendidos no ambulatório de anticoagulação da Disciplina de Cardiologia do Hospital das Clínicas da Faculdade de Medicina de Botucatu – UNESP (HC-FMB-UNESP) no período de janeiro de 2009 a janeiro de 2015, e calculado o tempo de permanência na faixa terapêutica (Time in Therapeutic Range – TTR), utilizando-se o método consagrado na literatura descrito por Rosendaal, uma interpolação linear para atribuir um valor de INR a cada dia do intervalo entre as aferições registradas. Resultados: O valor de TTR mediano foi de 53(10-88) e médio de 52,21%. Foram analisados os fatores que in... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Background: Atrial fibrillation (AF) is a supraventricular arrhythmia characterized by complete disorganization of atrial electrical activity, causing the atriums to lose their contractile capacity, thereby not generating atrial systole. Its prevalence increases with age and may be associated with cardiac structural diseases, provoking hemodynamic damage and thromboembolic complications. Oral anticoagulation (OAC) is capable of preventing thromboembolic events and is monitored through the INR (International Normalized Ratio). Objectives: 1- to evaluate INR stability in anticoagulated patients with non-valvular Atrial Fibrillation (AF); 2- to evaluate thromboembolic and hemorrhagic complications in these patients; 3- to identify the subgroup of higher risk for thromboembolic or hemorrhagic events. Methods: The medical charts of 203 patients attended at the anticoagulation ambulatory unit in the Cardiology Department at HC-FMB-UNESP in the period from January 2009 to January 2015 were reviewed; the Time in Therapeutic Range (TTR) was calculated by the method esteemed in the literature described by Rosendaal, a linear interpolation to attribute an INR value to each day of the interval between the recorded measurements. Results: The median TTR value was 53 (10-88) and the mean 52.21%. The factors that influenced the TTR value were analyzed in this population, showing that the patients presenting INR instability in the adaptation phase had a lower mean TTR (46.83%) than those with... (Complete abstract click electronic access below) / Mestre

O coração é formado por tecido muscular especializado e por um esqueleto de tecido conjuntivo que sustenta e dá inserção a musculatura. Este tecido conjuntivo é formado predominantemente por fibras colágenas tipo I, tipo III e fibras elásticas, as quais influenciam diretamente as propriedades estruturais e funcionais do miocárdio. Algumas doenças podem provocar alterações qualitativas e quantitativas no colágeno comprometendo a funcionalidade do órgão. Diante da inexistência de informações sobre os efeitos diretos do diabetes na estrutura atrial e a concentração de pesquisas em torno dos ventrículos, especialmente o esquerdo, que está ligado à circulação sistema, a proposta deste trabalho foi analisar as alterações morfológicas quantitativas e qualitativas do colágeno nas câmaras atriais comparando as diferenças entre os grupos de cães sadios e diabéticos. Além disto, esta pesquisa verificou a distribuição de outros constituintes do átrio como fibras elásticas e colágenas do tipo III. Foram utilizados corações de cães sem raça definida, machos e fêmeas, sadios e diabéticos. As estruturas foram examinadas por microscopia óptica, imunofluorescência e microscopia eletrônica de varredura. A taxa de colágeno nos átrios de cães diabéticos foi maior do que nos átrios de cães sadios. Isso indica que há uma mudança estrutural nos átrios de animais com doença metabólica, a partir da verificação de uma proteína estrutural importante como o colágeno. / The heart is composed of specialized muscle tissue and a skeleton of connective tissue that supports and gives muscle insertion. This tissue connective is formed predominantly of collagen type I, type III and elastic fibers, which directly influence the structural and functional properties of the myocardium. Some diseases can cause qualitative and quantitative changes in collagen compromising the functionality of the organ. Due to the lack of information on the direct effects of diabetes on atrial structure and the concentration of researches about the ventricles, especially the left, which is connected to the macro circulation system, the purpose of this study was to analyse the morphological quantitative and qualitative collagen in the chambers atrial comparing the differences between the groups of healthy and diabetic dogs. Moreover, this study found the distribution of other constituents like elastic and collagen type III fibers. We used hearts of mongreal dogs, males and females, healthy and diabetics. The structures were examined by light microscopy, immunofluorescence and scanning electron microscopy. The rate of collagen in the atria of diabetic dogs was greater than in the healthy dogs. This indicates that there is a structural change in the atria of animals with metabolic disease, from the finding of a major structural protein like collagen.