The cardiac conduction system is responsible for maintaining and orchestrating the rhythmic contractions of the heart. Results from lineage tracing studies indicate that precursor cells in the ventricles give rise to both cardiac muscle and conduction cells. Using chick embryonic hearts, we have found that Notch signaling plays an important role in the differentiation of cardiac muscle and conduction cell lineages in the ventricles. Notch1 expression coincides with a conduction marker at early stages of conduction system development. Mis-expression of constitutively active Notch1 (NIC) in early heart tubes exhibited multiple effects on cardiac cell differentiation. Cells expressing NIC had a significant decrease in the expression of cardiac muscle markers, but an increase in the expression of conduction cell markers. Loss-of-function studies further support that Notch1 signaling is important for the differentiation of these cardiac cell types. Functional electrophysiology studies show that the expression of constitutively active Notch1 resulted in abnormalities in ventricular conduction pathway patterns. During cardiogenesis, groups of myocardial cells become separated from each other, and migrate to form the trabeculae. These finger-like projections found within the ventricular chamber coalesce to generate the muscular portions of the interventricular septum, the thickened myocardium, and future sites of the conduction system. We have found that Notch signaling regulates the migration of cardiac cells during cardiogenesis. Over-expression of constitutively active Notch causes cells to localize more centrally within the heart, while loss-of-Notch function results in cells distributed within the periphery of the heart. Staining of heart sections shows that Notch signaling regulates the expression of N-cadherin, the predominant adhesion molecule in cardiomyocytes. We find that the effects of Notch on cell migration are two-fold: delamination and cell motility. Time-lapse studies demonstrate that Notch signaling increases cell motility, but does not affect speed or directionality of migration. Furthermore, we find that the effects of Notch on cell migration is independent of its effects on differentiation.

The tenn Acute Coronary Syndrome (ACS) encompasses a spectrum of patients who present with chest discomfort or other symptoms caused by myocardial ischemia or infarction. Since critical or acute care nurses care for such patients, they should have a thorough knowledge of ACS pathophysiology and current treatments for ACS The purpose of this research study is to explore and describe the knowledge level that the critical care nurses in a state hospital in East Texas feel they have regarding ACS. This study was quantitative, descriptive and contextual in design, in which a sample survey was performed, using a questionnaire based on a literature study. The response of most ofthe critical care nurses tested was that they felt they had insufficient knowledge. An in-service training session has been proposed to help improve the nurses' knowledge and expertise on ACS. / Health Studies / M.A. (Nursing Science)

Made available in DSpace on 2014-06-11T19:28:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-03-05Bitstream added on 2014-06-13T19:16:38Z : No. of bitstreams: 1 soares_pa_me_guara.pdf: 1465480 bytes, checksum: 4168953181ace550160249ec336c443e (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A análise espacial é uma importante ferramenta para estudo das doenças em grupos populacionais. O objetivo deste estudo é analisar a distribuição espacial do número de internações por infarto agudo do miocárdio e doença isquêmica do coração no Vale do Paraíba Paulista, Brasil. Foram utilizadas as estatísticas de internação do Sistema Único de Saúde, no portal DATASUS, segundo sexo e faixa etária acima de 30 anos, em dois períodos distintos: 1999-2000 e 2004-2005. As estatísticas e a distribuição espacial foram realizadas por meio do programa TERRAVIEW. Para minimizar as flutuações aleatórias das taxas, empregou-se o método Bayesano empírico. A unidade de análise do padrão espacial foi constituída pelos 35 municípios que compõem o Vale do Paraíba Paulista. Para análise da autocorrelação espacial foram utilizados: Moran global, Moran local, diagrama de espalhamento de Moran. As distribuições espaciais para infarto agudo do miocárdio e doença isquêmica do coração no Vale do Paraíba Paulista evidenciaram autocorrelação espacial, estatisticamente significativa, para os dois períodos. Os resultados para os dois períodos apontam a existência de um aglomerado central, em que se concentram as maiores taxas, constituídos pelos municípios: Guaratinguetá, Campos do Jordão, São Bento do Sapucaí e Santo Antônio do Pinhal. Os dos fatores individuais e contextuais possivelmente são indicativos da necessidade de incorporação de novos parâmetros, proporcionando novos meios para identificação de áreas problemáticas. / Spatial analysis is an important tool for the study of diseases in population groups. The objective of this study is to analyze the spatial distribution of the number of hospitalizations for acute heart attack of myocardium and ischemic disease of the heart in the Vale do Paraiba Paulista, Brazil. Were used statistics for hospitalization of the Unified Health System, the portal DATASUS, according to sex and age above 30 years, in two distinct periods: 1999-2000 and 2004-2005. The statistics and spatial distribution were performed through the program TERRAVIEW. To minimize the random fluctuations of the rates is the method employed Bayesano empirical. The unit of analysis of the spatial pattern was formed by 35 municipalities that make up the Vale do Paraiba Paulista. For analysis of the spatial autocorrelation were used: Moran global, local Moran, of Moran diagram of scattering. The spatial distributions for acute heart attack of myocardium and ischemic disease of the heart in the Vale do Paraiba Paulista showed spatial autocorrelation, statistically significant for the two periods. The results for both periods indicate the existence of a central cluster, which concentrated the higher rates, made by municipalities: Guaratinguetá, Campos do Jordao, São Bento do Sapucaí, Santo Antonio do Pinhal. The factors of the individual and contextual possibly are indicative of the need to incorporate new parameters, providing new means for identifying problem areas.

Orientador: Heitor Moreno Junior / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T16:25:53Z (GMT). No. of bitstreams: 1 GazzotoFilho_Ademir_M.pdf: 876670 bytes, checksum: e7b954abb9a6ffd97c187bf6ceadaa8a (MD5) Previous issue date: 2009 / Resumo: O óxido nítrico (NO) é um mediador biológico que atua como molécula chave em muitos processos fisiopatológicos, como a regulação do tônus vascular, neurotransmissão, aprendizado, memória, dentre outros. Por causa de sua importância e envolvimento nos mecanismos fisiológicos e patológicos, a sua regulação e síntese têm sido extensivamente estudadas. A inibição crônica do NO por administração via oral do inibidor inespecífico da NO-sintase, a nitro-L-arginina metil éster (L-NAME) resulta em cardiopatia hipertensiva em ratos. A hipertensão renovascular [dois rins e um clipe (2R1C)] e o diabetes melito (DM) associadas, induzem a anormalidades morfológicas semelhantes às descritas no modelo de cardiomiopatia induzida por L-NAME em ratos, mas seus efeitos hemodinâmicos ainda são controversos. O presente estudo avaliou a função cardíaca e vascular nestes dois modelos de cardiomiopatia após oito semanas de tratamento. Foram utilizados ratos Wistar, divididos em 5 Grupos: Grupo Controle; Grupo L-NAME: 60mg/kg/dia de L-NAME; Grupo 2R1C: com estenose cirúrgica de artéria renal para indução de hipertensão arterial renovascular; Grupo DM: animais que receberam estreptozotocina (60 mg/kg intra-venoso), Grupo 2R1C+DM: animais submetidos à cirurgia para indução de hipertensão arterial renovascular e que receberam estreptozotocina. Foram avaliados os seguintes parâmetros: pressão arterial média (PAM), débito cardíaco (DC), resistência vascular periférica total (RVPT), dP/dt positivo e dP/dt negativo em coração isolado. Em todos os grupos foram encontrados aumento na PAM, RVPT e redução do DC depois de oito semanas em relação ao Grupo Controle. Os grupos L-NAME e 2R1C+DM apresentaram aumento na PAM (175,4±29,7 e 158,7±16,7 mmHg, respectivamente) e redução do DC após a oitava semana. A RVPT aumentou em ambos os grupos. Uma diminuição da dP/dt positiva foi observada no grupo 2R1C+DM (1895±98 mmHg/s, p<0,05) vs. Grupo Controle (2534±120 mmHg/s, p<0,05). A dP/dt negativa diminuiu nos grupos L-NAME e 2R1C+DM vs. Grupo Controle (1490±104 e 1460±94 mmHg/s, respectivamente vs. 2080±92 mmHg/s, p<0,05). O presente estudo demonstrou que, apesar das semelhanças morfológicas entre os grupos 2R1C+DM e L-NAME, este último não mimetiza as alterações hemodinâmicas dos modelos diabético-hipertenso renovascular associados em ratos. / Abstract: Nitric oxide (NO) is a biological mediator that acts as a key molecule in many pathophysiological processes such as regulation of vascular tone, neurotransmission, learning, memory, and others. Because of its importance and involvement in physiological and pathological mechanisms, its regulation and synthesis have been extensively studied. Inhibition of NO by chronic oral administration of the nonspecific inhibitor of NO-synthase, the nitro-L-arginine methyl ester (L-NAME) results in hypertensive cardiomyopathy in rats. The renovascular hypertension [two kidneys and clip (2K1C)] and diabetes mellitus (DM) associated induce morphological abnormalities similar to those described in the model of cardiomyopathy induced by L-NAME in rats, but its hemodynamic effects are still controversial. This study evaluated the cardiac and vascular function in these two models of cardiomyopathy after eight weeks of treatment. We evaluate the following parameters: mean arterial pressure (MAP), cardiac output (CO), total total peripheral vascular resistance (TPVR), positive and negative dP/dt in isolated heart. In all groups were observed increased MAP, TPVR and reduced DC after eight weeks in the control group. Wistar rats were divided into the following groups: Control; L-NAME: 60mg/kg/day; 2K1C+DM: streptozotocin (60 mg/kg) and one renal artery clipped. The following parameters were measured: mean arterial pressure (MAP), heart rate, cardiac output (CO) and total peripheral vascular resistance (TPVR). Positive and negative dP/dt was also evaluated in an isolated heart. L-NAME and 2K1C+DM groups had increased MAP (175.4±29.7 and 158.7±16.7 mmHg, respectively) and reduced CO after the 8th week. TPVR was increased in both groups. A decrease in positive dP/dt was found in the 2K1C+DM (1895±98 mmHg/s, p<0.05) vs. Control group (2534±120 mmHg/s, p< 0.05). Negative dP/dt was decreased in the L-NAME and 2K1C+DM groups vs. Control group (1490±104 and 1460±94 mmHg/s, respectively vs. 2080±92 mmHg/s, p<0.05). Decrease in positive dP/dt 2R1C+DM group compared to Control. The negative dP/dt decreased in L-NAME and 2R1C+DM groups. This study demonstrated that despite the morphological similarities between 2K1C+DM and LNAME groups; the L-NAME group did not mimics the hemodynamic changes in hypertension n-diabetic- renovascular model associated in rats. / Mestrado / Mestre em Farmacologia

Orientador: Kleber Gomes Franchini / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T13:49:24Z (GMT). No. of bitstreams: 1 Clemente_CarolinaFernandaManfrediZambon_D.pdf: 14683053 bytes, checksum: 740572fd1e2c19884bc045a5fafe828f (MD5) Previous issue date: 2008 / Resumo: Doenças do coração cursam frequentemente com hipertrofia do miocárdio. Estímulos mecânicos e neuro-humorais são sinalizadores críticos para o crescimento hipertrófico dos cardiomiócitos nos vários processos patológicos. Neste contexto, estudos indicam que a quinase de adesão focal (FAK), uma proteína tirosino-quinase que participa dos mecanismos de sinalização por integrinas, é uma mediadora importante do crescimento hipertrófico do ventrículo esquerdo (VE). Este estudo teve como objetivo avaliar a influência da FAK na indução da hipertrofia e na deterioração do VE induzidas por sobrecarga pressórica crônica em camundongos através de estratégia de interferência por RNA. A coarctação da aorta em camundongos induziu a hipertrofia do VE acompanhada pelo aumento da expressão e atividade da FAK no miocárdio. A infusão de siRNA específico para FAK (siRNAFAK), via veia jugular, levou ao silenciamento gênico prolongado da FAK (~70%) no VE normal e também no hipertrófico. O knockdown da FAK foi confirmado nos miócitos e fibroblastos cardíacos provenientes do VE de camundongos. O silenciamento da FAK foi acompanhado tanto da prevenção como regressão da hipertrofia do VE. A função do VE foi preservada e a taxa de sobrevivência foi maior nos camundongos tratados com siRNAFAK, apesar da persistência da sobrecarga pressórica. Estes achados foram paralelos a atenuação do crescimento hipertrófico dos cardiomiócitos e da expressão do marcador de hipertrofia ß-MHC no VE sob sobrecarga pressórica. O silenciamento da FAK também atenuou o aumento da fibrose intersticial, conteúdo de colágeno e atividade da metaloproteinase-2 (MMP-2) no VE submetido ao estímulo mecânico. Em fibroblastos extraídos de corações hipertróficos, o silenciamento da FAK foi concomitante à diminuição da expressão de MMP-2. Assim, estes dados indicam que a sinalização mediada pela FAK é necessária não apenas para o desenvolvimento, mas também para sustentar a hipertrofia em resposta a sobrecarga pressórica crônica / Abstract: Hypertrophy is a critical event in the onset of failure in chronically overloaded hearts. Mechanical stress and neurohumoral factors signaling factors have been considered the main triggering stimuli for the installation of hypertrophy in cardiac myocytes in a variety of pathological process. In this context, focal adhesion kinase (FAK), a key protein of the integrin signaling pathway, has attracted particular attention as a mediator of hypertrophy induced by increased load. This study was performed to address the influence of FAK in the pathophysiology of cardiac hypertrophy and failure induced by chronic pressure overload in mice using RNA interference methodology. Aortic constriction in mice induced left ventricle (LV) hypertrophy and increased expression and phosphorylation of FAK. Intrajugular delivery of specific small interfering RNA induced prolonged FAK silencing (~70%) in both normal and hypertrophic LVs. Studies in cardiac myocytes and fibroblasts harvested from LVs confirmed the ability of the systemically administered specific small interfering RNA to silence FAK in both cell types. Myocardial FAK silencing was accompanied by prevention, as well as reversal, of load-induced left ventricular hypertrophy. The function of LVs was preserved and the survival rate was higher in banded mice treated with small interfering RNA targeted to FAK, despite the persistent pressure overload. Further analysis indicated attenuation of cardiac myocyte hypertrophic growth and of the rise in the expression of ß-myosin heavy chain in overloaded LVs. Moreover, FAK silencing was demonstrated to attenuate the rise in the fibrosis, collagen content, and activity of matrix metalloproteinase-2 in overloaded LVs, as well as the rise of matrix metalloproteinase-2 protein expression in fibroblasts harvested from overloaded LVs. This study indicate that FAK is necessary not only to the development but also to sustain LV hypertrophy in response to chronic pressure overload / Doutorado / Medicina Experimental / Doutor em Fisiopatologia Medica

Orientadores: José Wilson Magalhães Bassani, Rosana Almada Bassani / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Elétrica e de Computação / Made available in DSpace on 2018-08-20T23:57:01Z (GMT). No. of bitstreams: 1 Goulart_JairTrape_M.pdf: 1965789 bytes, checksum: 94fe3a318201b7addef8365fbeea0f40 (MD5) Previous issue date: 2012 / Resumo: A aplicação de campos elétricos de alta intensidade (CEAI) no coração é realizada frequentemente durante tentativas de reversão da fibrilação, e para gerar sincronismo cardíaco com o uso de marca-passos. Entretanto, este procedimento rotineiro pode causar danos ao miocárdio. O objetivo deste trabalho foi investigar se a estimulação ?-adrenérgica afeta a vulnerabilidade de miócitos ventriculares ao CEAI, uma vez que, durante a fibrilação ventricular, há aumento reflexo do tônus simpático, em virtude da consequente hipotensão arterial, e a administração de catecolaminas é um procedimento comum neste tipo de emergência. Miócitos foram isolados do ventrículo esquerdo de ratos Wistar adultos por digestão enzimática. As células foram perfundidas com solução de Tyrode a 23 °C e estimuladas a 0,5 Hz. Aplicou-se CEAI em intensidades crescentes até que fosse induzida uma lesão letal na célula. A probabilidade de morte celular foi determinada em função da amplitude do CEIA e da máxima variação estimada do potencial de membrana (?Vmax) por análise de sobrevivência (curva de letalidade). Destas curvas, obteve-se o parâmetro EL50 (valor de CEAI ou ?Vmax com probabilidade de letalidade de 0,5). A estimulação ?-adrenérgica, pela perfusão com 10 nM de isoproterenol (ISO), foi realizada na ausência e presença de 150 nM de metoprolol (MET, bloqueador de adrenoceptores ?1), 10 ?M de H-89 (inibidor de PKA) e 500 nM de BIS I (inibidor de PKC), bem como após protocolo para minimizar o aumento de mobilização celular de Ca2+. As curvas foram comparadas por teste de Mantel-Cox. O tratamento com ISO promoveu o deslocamento da curva para a direita (p < 0,01) e EL50 aumentou de 85 para 100 V/cm. MET e H-89 aboliram o efeito protetor do ISO, e, quando perfundidos na ausência de ISO, não tiveram quaisquer efeitos sobre a indução de letalidade por CEAI. A redução da mobilização de Ca2+ e o tratamento com BIS I não alteraram as curvas de letalidade, na presença ou ausência de ISO, mas provocaram significante aumento no tempo de recuperação das células após um choque não letal. As curvas em função de ?Vmax foram similares àquelas em função da intensidade do campo. Os resultados mostram que a estimulação ?-adrenérgica é capaz de proteger os miócitos dos efeitos deletérios do CEAI, permitindo que a célula suporte maiores ?Vmax. A proteção parece ser mediada por adrenoceptores ?1 e PKA, e possivelmente envolve ativação de mecanismos de reparo. Estas vias de proteção podem ser futuramente exploradas para atenuar os danos miocárdicos causados pela desfibrilação/cardioversão / Abstract: The myocardium is exposed to high-intensity electric fields (HIEF) during cardiac electric defibrillation, which may reverse life-threatening arrhythmias, but also cause cell damage. Impairment of cardiac pumping during ventricular fibrillation is usually associated with high sympathetic tone, and catecholamine infusion is a common procedure for the emergencial treatment of this arrhyhmia. The present study was carried out to investigate whether adrenergic stimulation affects the vulnerability of ventricular myocytes to the lethal effect of HIEF. Left ventricular myocytes were isolated from adult Wistar rat hearts by collagenase digestion. Under perfusion with Tyrode's solution at 23 ºC, cyclic contractile activity was evoked by stimulation at 0.5 Hz. A HIEF pulse was then applied, after which cells were allowed to rest and recover from the shock. Afterward, the protocol was repeated increasing HIEF amplitude until cell death ensued. The probability of cell death as a function of the field intensity or the estimated maximum change in membrane potential, ?Vmax (lethality curve) was determined by survival analysis. The protocol was carried out during exposure to 10 nM isoproternol (ISO), in the absence and presence of the ?1-adrenoceptor blocker metoprolol (MET, 150 nM), the PKA inhibitor H-89 (10 ?M), the PKC inhibitor BIS I (500 nM), or after a protocol designed to attenuate the increase in cell Ca2+ mobilization by ISO. Lethality curves were compared with the Mantel-Cox test. Exposure to ISO produced a marked rightward shift of the lethality curves (p< 0.01), and the field intensity associated with the cell death probability of 0.5 (EL50) was increased from 85 to 100 V/cm. Treatment with MET or H-89 alone did not significantly affect the curves, but completely abolished ISO protective effect. Decreasing Ca2+ mobilization and BIS I treatment did not produce significant effects on the lethality curves, either in the absence or presence of ISO, but they increased the time spent to cell recovery after a non-lethal shock. Similar results were obtained for the lethality curve as a function of ?Vmax. Therefore, ?-adrenergic stimulation confers protection to ventricular myocytes from the lethal effects of HIEF, even though cells apparently experience higher variations of ?Vmax, which should lead to extensive electroporation. This effect, which seems to be mediated by ?1-adrenoceptors and dependent on PKA activation, possibly involving recruitment of membrane repair mechanisms. This protective effect may be further explored as a means to attenuate HIEF deleterious effects on cardiac function that frequently result from electric defibrillation/cardioversion / Mestrado / Engenharia Biomedica / Mestre em Engenharia Elétrica

A growing body of evidence has now accumulated supporting the involvement of oxygen-derived free radicals in the development of myocardial ischemia/reperfusion (I/R) injury. We have, therefore, undertaken the present study to examine (1) I/R-related alterations in myocardial antioxidant capacity in pentobarbital anesthetized open-chest rabbits subjected to left circumflex coronary artery ligation followed by reperfusion; (2) the protective effects of pretreatrnent with allopurinol or the 21-aminosteroid U74006F; (3) alternative mechanisms to xanthine oxidase inhibition for allopurinol protection against I/R injury; and (4) the effect of allopurinol treatment on the antioxidant capacity of erythrocytes in pigs used in a heart-lung transplantation study. In the rabbit myocardium, a marked impairment in myocardial antioxidant capacity developed in association with the onset of irreversible injury, as reflected in the enhancement in glutathione (GSH) depletion and formation of thiobarbituric acid-reactive substances (TBARS) following in vitro incubation of tissue homogenate with tert-butylhydroperoxide (TBHP). During the course of post-ischemic reperfusion, the protracted time-course of alterations in antioxidant capacity dissociated them from the early burst of radical formation known to occur at the onset of post-ischemic reperfusion of the myocardium. When the time-dependent changes in functional indices of antioxidant status (TBHP-induced GSH depletion and formation of TBARS) were analysed in relation to activities of antioxidant enzymes, evidence suggestive of functionally relevant impairments in Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and glutathione reductase (GRD) activities was found. These results and our demonstration of significant decreases in the activity of GSH-dependent antioxidant enzymes under acidotic conditions suggest that a transient impairment in the functioning of antioxidant enzymes may be involved in triggering irreversible myocardial I/R injury. Repetitive brief episodes of I/R produced a progressive decrease in myocardial ATP levels, which was not associated with any detectable changes in myocardial antioxidant capacity. Ischemic preconditioning produced by brief episodes of I/R did not affect the severity of subsequently induced I/R injury. These results suggest that brief episodes of myocardial ischemia do not produce oxidative tissue damage and the ischemia-induced depletion in myocardial ATP level is at least partially dissociable from the I/R-related impairment in tissue antioxidant capacity. Isolated Langendorff-perfused rabbit hearts subjected to I/R did not show any changes in antioxidant capacity. However, when intact hearts were subjected to ischemia in vivo and a subsequent reperfusion in vitro, an impairment in myocardial antioxidant capacity became apparent. These results suggest that blood elements, possibly activated neutrophils, may be a crucial factor involved in the development of I/R-induced oxidant injury. Chronic allopurinol pretreatment (1 mg/ml in drinking water or approximately 75 mg/kg/day) for 7 days prior to ischemia provided significant protection against I/R-induced alterations in myocardial antioxidant capacity, but not the decrease in tissue ATP levels. This chronic allopurinol regimen was found to enhance myocardial GRD activity in nonischemic tissue. In addition, both allopurinol and oxypurinol inhibited the transition metal ion-catalysed ascorbate oxidation and lipid peroxidation in vitro, likely as a consequence of their metal chelating properties. Similarly, myoglobin-TBHP-catalysed oxidation of uric acid and lipid peroxidation were also suppressed by allopurinol. All these suggest that allopurinol may favorably alter myocardial antioxidant capacity directly by virtue of its transition metal chelating properties and its antioxidant actions in myoglobin-mediated oxidative processes. The acute administration of 21-aminosteroid U74006F (3 mg/kg, i.v) under conditions comparable to those known to protect against trauma-induced damage in the central nervous system failed to reduce manifestations of oxidative injury in rabbit hearts subjected to ischemia and reperfusion. Although reactive oxy-radicals have been implicated in both types of tissue damage, the observed difference in susceptibility to protection by this steroidal antioxidant suggests that the molecular mechanisms involved are not identical. In the heart-lung transplantation study, erythrocytes from allopurinol-treated pigs (given repeatedly at an oral dose of 50 mg/kg) showed a time/dose-dependent increase in antioxidant capacity as reflected in the decrease in malondialdehyde production following in vitro oxidative challenge. The extent of red cell protection in both donor and recipient animals correlated significantly with the functional viability of the transplanted lung tissue, as assessed by tissue water content. These results suggest that the measurement of erythrocyte antioxidant capacity may provide an useful assessment of generalized alterations in tissue antioxidant status produced by pharmacological interventions. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

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 isolated perfused working heart was used to study hypertensive- diabetes induced alterations in cardiac function at 6 and 12 weeks after the induction of diabetes. There was no difference in cardiac function between normotensive Wistar and spontaneously hypertensive (SHR) diabetic rats at 6 weeks after diabetes induction. Wistar-Kyoto (WKY) rats were also included as normotensive controls in our 12-week study. Successful induction of diabetes was confirmed by the presence of hyperglycemia, hypoinsulinemia, glycosuria and increased haemoglobin glycosylation in all three diabetic groups. However, quantitation of various parameters of heart function revealed highly significant differences between SHR diabetic animals and all other groups, associated with an increased mortality. Serum lipids were elevated in SHR and Wistar and unaffected in WKY diabetic rats. Furthermore, thyroid hormone levels were not depressed in WKY diabetic rats and could explain the lack of cardiac dysfunction in these animals. The data provide further evidence that the combination of hypertension and diabetes mellitus produces greater myocardial dysfunction than is seen with either disease alone and is associated with a significant mortality. The effects of hydralazine on blood lipids, systolic pressure and cardiac performance were assessed in male Wistar rats, 6 weeks after they were made diabetic with streptozotocin (STZ). When hydralazine was administered for a 6-week period to the diabetic rats, their blood lipids were not significantly different from that of non-diabetic rats despite a low serum insulin. In contrast, blood lipids were elevated in the diabetic rats that were not treated with hydralazine; these animals also had low insulin levels. Cardiac performance was depressed in the untreated diabetic animals, but the cardiac performance of the hydralazine-treated diabetic animals showed a definite improvement which could be partly explained by their normal thyroid status in contrast to the untreated diabetic animals which were slightly hypothyroid. Blood pressure was elevated only in the untreated diabetic animals. Thus hydralazine controlled the high serum lipids and blood pressure and improved cardiac performance in STZ diabetic rats. To examine the influence of sex differences in the STZ model of diabetes, we studied left ventricular function in hearts from 6 week male and female rats. Significantly lower values for +dP/dt occurred in male diabetic rats compared with their own controls or female diabetics at most left atrial filling pressures. Decreases in this value for female diabetic rats compared to their own controls occurred only at high left atrial pressures. It appears that diabetes mellitus produces greater myocardial dysfunction in male diabetic rats. / Pharmaceutical Sciences, Faculty of / Graduate

Antibodies to tropomyosin, cardiac troponin I, myosin, and the beta-adrenergic receptors have been implicated in myocarditis, dilated cardiomyopathy, and heart failure. However, in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), there are only a few studies on how autoantibodies play a role in autoimmune mediated heart disease, despite the prevalence of these conditions. Ro52 antibodies have been implicated in fetal heart block, but their role in adult myocarditis remains elusive. In this study, we look beyond Ro52 and characterized the relevant autoantibodies in adult patients with SLE and RA myocarditis. An optimized immunoprecipitation followed by liquid chromatography mass spectrometry methodology was performed to determine putative auto-antigens in the human heart. The quantity and specificity of auto-antibodies was correlated with clinical measures of myocardial cellular infiltration, as determined by fluorodeoxyglucose (FDG)-positron emission tomography (PET) in patients with SLE and RA. We created autoantibody profiles that are complimentary to SLE and RA patients' clinical profile. Autoantibodies that correlated with cellular infiltration included TPI1, TPM1, MYL2, XRCC6 and APOA4. We then explored methodologies for testing patient autoantibodies using engineered cardiac tissues derived from human induced pluripotent stem cells (iPSCs). These tissues are increasingly used for drug discovery, pharmacology and in models of development and disease. While there are numerous platforms with engineered cardiac tissues, they often require expensive and non-conventional equipment and utilize complex video processing algorithms. As a result, only specialized academic labs have been able to harness this technology. In addition, methodologies and tissue features have been challenging to reproduce between different groups and models. Here, we describe a facile technology (milliPillar) that covers the entire pipeline required for studies of engineered cardiac tissues: (i) platform fabrication, (ii) cardiac tissue generation, (iii) electrical stimulation, (iv) automated real-time data acquisition, and (v) advanced video analyses. We validate these methodologies and demonstrate the versatility of the platform by showcasing the fabrication of tissues in different hydrogel materials and by using cardiomyocytes derived from different iPSC lines in combination with different types of stromal cells. We also validate the long-term culture (100 days) of tissues within the platform and provide protocols for automated analysis of force generation and calcium flux using both brightfield and fluorescent imaging. Lastly, we demonstrate the compatibility of the milliPillar platform with electromechanical stimulation to enhance cardiac tissue function. milliPillar tissues were cultured in the presence of patient autoantibodies to recapitulate the phenotype of myocardial disease, and the calcium transients and force generation were measured. Our results indicated that milliPillar tissues exhibited a decrease in force generation after 6 days in culture with SLE autoantibodies. Separately, our results indicated a prolonged calcium transient after 7 days in culture with SLE and RA autoantibodies. Changes to the downstroke of the calcium transient correlated most with patients’ autoantibody profiles and cellular infiltration. We confirmed autoantibody binding to live tissues/cells in 25% of the patients with SLE and myocarditis. Finally, we used changes in cardiac tissue function in the presence of autoantibodies to classify patients with SLE myocarditis with an accuracy of 87.5%.