The purpose of this investigation was to examine selected biochemical mechanisms known to influence contractility and energy metabolism in the myocardium, with particular emphasis placed on the regulatory role of protein phosphorylation in the ventricular myocardium. The investigation was conducted in three phases; initially the cardiac contraction cycle was examined to determine whether reported fluctuations in myocardial cAMP levels were associated with other biochemical events known to be cAMP-dependent. The second phase involved the determination of specific kinase activities and endogenous substrates in a highly purified cardiac sarcolemmal preparation. In the final phase, ventricular myocytes were utilized to examine the ability of adenosinergic and muscarinic agonists to influence the isoproterenol-induced increases in protein phosphorylation. Studies in the first phase examined cyclic AMP levels and selected kinase activities in hearts frozen at various stages of the cardiac cycle. An automated clamping device, capable of freezing a perfused rat heart in less than 50 msec, was utilized to separate the cardiac cycle into various phases. Three different timing schemes were employed to divide the cycle into 2 to 4 segments. These different timing schemes revealed no significant differences in cAMP during the cardiac cycle. Myocardial cAMP values ranged from 2.5 to 4.1 pmol/min/mg protein in all phases. However, in one scheme there was a tendency for cAMP to be elevated in early systole, with minimal values occurring diastole. There were also no significant differences seen for either glycogen phosphorylase or cAMP-dependent protein kinase (PKA) activity between various phases of the cardiac cycle. Since no significant fluctuations were observed in the levels of cAMP or the activities of PKA or glycogen phosphorylase during a single cardiac contraction cycle, it would appear that these agents do not exert their effects on cardiac function on a beat to beat basis. The second phase of study examined the nature and function of individual protein kinases in the myocardium. Using a highly purified cardiac sarcolemmal preparation, kinase specific, synthetic substrates were employed to quantify the activities of cAMP-dependent (PKA), calcium/calmodulin-dependent (PKCM), calcium/phospholipid-dependent (PKC) and cGMP-dependent (PKG) protein kinases. Additionally, endogenous protein substrates were examined in this preparation to provide possible insight as to the function of these kinases in the heart. The activities of PKA, PKG, PKCM, and PKC in nmol 32P/min/μg protein were as follows: PKA, 1606; PKG, 35.7; PKCM, 353; and PKC, 13.2. Three endogenous protein substrates of apparent molecular weights of 15kD, 28kD and 92kD were phosphorylated. While no endogenous protein phosphorylation was detectable as a result of cG-PK activity, all of the substrates were phosphorylated, to varying degrees, by both PKA and CACM-PK. PKC phosphorylated only the 15kD substrate. Even though several endogenous kinases are evident in the sarcolemmal preparation, cAMP-dependent protein kinase demonstrates the greatest degree of activity. This kinase also appeared to be the most abundant; however, there is some concern as to the source of these kinases in the membrane preparation since endothelial membranes as well as cardiac membranes appeared to be present. Evidence for endothelial contamination was provided by the finding that the membrane preparation contained appreciable amounts of angiotensin converting enzyme (ACE) activity, an enzyme felt to reside in the vascular endothelium. Since studies with this preparation could not exclude contribution of nonmuscle cell membranes a model consisting solely of dispersed ventricular myocytes was developed. The third phase of these studies examined protein phosphorylation in primary cultures of ventricular myocytes. Specifically, these studies examined protein phosphorylation induced by exposure to isoproterenol (ISO), a catecholamine known to effect changes in the phosphorylation state of proteins in the heart by means of a β-adrenergic-mediated/cAMP-dependent mechanism was examined. Additionally, the effects of phenylisopropy-ladenosine (PIA) and carbamyl choline chloride (CARB) were examined with regard to their anti-adrenergic role(s) in this process. Adherent, collagenase-dispersed, radiolabelled (32p) ventricular myocytes exposed to ISO demonstrated a dose and time dependent increase in 32p incorporation into several endogenous protein substrates. When the myocytes were exposed (60 sec) to either PIA or CARB prior to the exposure to ISO, ISO-induced 32p incorporation into protein substrates of apparent molecular weight of 6kD, 31kD and 155kD was reduced up to 67% when compared to the effects of ISO alone. Additionally, both PIA and CARB attenuated the ISO-induced increase in PKA activity in the myocyte, yet only CARB was seen to produce an inhibitory effect on the ISO-induced increase in cAMP levels in the myocytes. The effects of CARB were dose-dependent and inhibited the effects of ISO on 32p incorporation at all doses tested. PIA elicited biphasic effects: lower PIA concentrations were inhibitory in nature, while higher concentrations of PIA appeared to potentiate the increase in 32p incorporation induced by ISO. Based on electrophoretic mobilities (SDS/PAGE) of the 6kD and the 155kD substrates, these substrates have been tentatively identified as the monomeric form of the sarcoplasmic reticulum-associated protein, phospholamban, and the contractile filament-associated protein, C protein, respectively. The 31kD substrate has been identified, by means of immunoblot, as the contractile filament-associated protein, troponin I. The role of protein phosphorylation in the myocardium involves complex, inter-related mechanisms that encompass extracellular, transmembranal and cytoplasmic elements in the heart. It is well understood that certain mechanisms of the contraction cycle known to vary on a beat to beat basis, such as myosin ATPase, involve changes in protein phosphorylation. However, the nature of the various kinases and substrates examined in this study appear to influence longer-term events of myocardial contractility. Mechanisms coupled with hormone action, modulation of second messenger-dependent components, and factors associated with changes in contractility seen with aging and disease are more likely to exhibit changes similar to those described herein. A better understanding of the underlying biochemistry may provide greater insight into the importance of these metabolic changes.

Introdução: Dos mecanismos fisiopatológicos à estratificação de risco e manejo, ainda persistem hoje muitas lacunas e debates sobre a miocardiopatia não compactada (MNC). Recentemente, a ressonância magnética cardiovascular (RMC) vem sendo amplamente utilizada para aumentar a precisão do diagnóstico de MNC em pacientes com alta probabilidade clínica pré-teste, com valor prognóstico e alta relevância na tomada de decisões clínicas. Objetivo: Este estudo teve como objetivo caracterizar o mapeamento T1 e o volume extracelular (VEC) miocárdico por RMC em pacientes com MNC e investigar como esses marcadores teciduais relacionam-se com a fração de ejeção do ventrículo esquerdo (FEVE) e arritmias ventriculares (AV). Métodos: Foram recrutados prospectivamente 36 pacientes com MNC e 18 controles saudáveis para realizarem uma RMC com mapeamento T1 entre julho de 2013 e setembro de 2016. O VEC foi avaliado apenas para os segmentos do ventrículo esquerdo sem áreas de fibrose macroscópica pela técnica do realce tardio (RT), objetivando-se investigar a presença de fibrose miocárdica intersticial difusa. Para avaliar as diferenças entre os parâmetros de RMC nos pacientes e controles, foram usados o teste t entre as amostras pareadas (Wilcoxon) e um modelo de regressão linear foi construído para investigar a relação entre a FEVE e os achados clínicos e de imagem (inclusive o VEC). Resultados: Os pacientes com MNC apresentaram maiores valores de T1 nativo (1.024 ± 43ms versus 995 ± 22ms, p = 0,01) e VEC (28,0 ± 4,5% vs. 23,5 ± 2,2%, p < 0,001) em relação aos controles. Apenas o VEC foi associado independentemente com a FEVE (beta = -1,3, p = 0,003) na regressão multivariada. Houve uma interessante tendência para a terapia betabloqueadora modificar positivamente a relação entre ECV e LVEF (beta = 4,1, intervalo de confiança de 95%, -0,6 a 8,8), porém com p alto (0,08). Além disso, entre pacientes com MNC e RT ausente (negativo), AV foram associadas com maior VEC (27,7% em pacientes com AV vs 25,8% em pacientes sem AV, p = 0,002). Conclusão: Nos pacientes com MNC, a caracterização tecidual miocárdica por mapeamento T1 sugere uma expansão extracelular por fibrose intersticial difusa no miocardio sem fibrose focal pelo RT, o que foi associada à disfunção ventricular e AV. Tais achados podem dar suporte a um potencial valor do mapeamento T1 no refino da estratificação de risco de pacientes com MNC / Background: From pathophysiological mechanisms to risk stratification and management, much debate and discussion persist regarding non-compaction cardiomyopathy (NCC). Recently, cardiovascular magnetic resonance (CMR) imaging has been widely used to more accurately diagnose NCC in patients with high clinical pre-test probability, with prognostic value and high relevance in the clinical decision making process. Purpose: This study aimed to characterize myocardial T1 mapping and extracellular volume (ECV) fraction by cardiovascular magnetic resonance (CMR), as well as investigate how these tissue markers relate to left ventricular ejection fraction (LVEF) and ventricular arrhythmias (VA) in patients with NCC. Methods: We prospectively recruited 36 patients with NCC and 18 controls to perform a cardiovascular magnetic resonance (CMR) with T1 mapping between July 2013 and September 2016. ECV was quantified in LV segments without late gadolinium enhancement (LGE) areas to investigate diffuse myocardial fibrosis. Differences in CMR parameters between patients and controls were assessed using t-test or Wilcoxon rank-sum test, and a linear regression model was built for LVEF to test the association with ECV and clinical characteristics. Results: Patients with NCC had higher native T1 (1024±43ms vs. 995±22ms, p=0.01) and expanded ECV (28.0±4.5% vs. 23.5±2.2%, p < 0.001) compared to controls. ECV was independently associated with LVEF (beta=-1.3, p=0.003). There was a trend for beta-blocker therapy to modify the relationship between ECV and LVEF (beta=4.1, 95% confidence interval, 0.6 to 8.8, p=0.08). Moreover, among patients without LGE, VA were associated with higher ECV (27.7% with VA vs 25.8% without VA, p=0.002). Conclusion: In NCC patients, tissue characterization by T1 mapping suggests an extracellular expansion by diffuse fibrosis in myocardium without LGE, which was associated with myocardial dysfunction and VA. These findings lend support to the potential role of T1 mapping in refining NCC risk stratification

Made available in DSpace on 2014-12-17T15:16:19Z (GMT). No. of bitstreams: 1 FlavioSS_DISSERT.pdf: 1009224 bytes, checksum: 11ff4662d4d8985817935b34117dbf5f (MD5) Previous issue date: 2014-02-03 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Our aim was to investigate the effects of an aerobic training program on adverse and early left ventricle (LV) remodeling, using an experimental model of short-term type 1 diabetes (T1D). Wistar rats were divided in 4 groups: sedentary control (SC), trained control (TC), sedentary diabetic (SD) and trained diabetic (TD). T1D was induced by streptozotocin (45 mg/kg). The training program consisted of 4 weeks running on a treadmill (13 m/min, 60 min/day, 5 days/week). At the end of the experiments, hearts were collected for analysis of morphology and transcriptional profile of LV, by focusing on its remodeling. Deaths were recorded during the 4-week period. We verified high mortality among animals of DS group, whereas it was significantly reduced in DT group. DS group also showed an increase in cross-sectional area of cardiomyocytes and fibrosis. TD group exhibited reduction in measures of cardiac trophism, but with respect to collagen content, it was similar to CS group. Analysis of gene expression related to cardiac remodeling revealed decreased expression of collagen I and III, as well as low expression of MMP-2 in DS group. TD group showed decreased levels of mRNA for MMP-9, and unchanged gene expression of MMP-2 when compared with the CS group. The expression of MMP-2 and TGF-&#61538;1 were increased in CT group. The ratio between gene expression of collagen I and III was increased in the CT group and decreased in diabetic groups. These results establish early changes of the structure and transcriptional profile of LV myocardium. Moreover, they indicate that aerobic exercise training plays specific protection against mechanisms responsible for cardiac damage observed in T1D / Nosso objetivo foi investigar os efeitos de um programa de treinamento aer?bio sobre o remodelamento adverso e precoce do ventr?culo esquerdo (VE), utilizando modelo experimental de curto prazo de diabetes tipo 1 (DM1). Ratos Wistar foram divididos em 4 grupos: controle sedent?rio (CS), controle treinado (CT), diab?tico sedent?rio (DS) e diab?tico treinado (DT). O DM1 foi induzido por estreptozotocina (45 mg/kg). O programa de treinamento consistiu em 4 semanas de corrida em esteira (13 m/min, 60 min/dia, 5 dias/semana). Ao fim dos experimentos, os cora??es foram coletados para analise da morfologia e do perfil transcricional do VE, com foco em seu remodelamento. Os ?bitos foram registrados durante as 4 semanas. Verificamos alta mortalidade entre os animais do grupo DS, enquanto que esta foi significativamente reduzida no grupo DT. O grupo DS apresentou aumento na ?rea de sec??o transversa dos cardiomi?citos e fibrose. O grupo DT exibiu redu??o das medidas de trofismo card?aco, mas com rela??o ao conte?do col?geno, foi similar ao grupo CS. As an?lises de express?o de genes ligados ao remodelamento card?aco revelaram redu??o na express?o dos col?genos I e III, al?m de baixa express?o da MMP-2, no grupo DS. O grupo DT apresentou diminui??o dos n?veis de mRNA para MMP-9, e express?o g?nica de MMP-2 inalterada, se comparado ao grupo CS. As express?es da MMP-2 e do TGF-&#61538;1 foram aumentadas no grupo CT. A raz?o entre express?o g?nica dos col?genos I e III mostrou-se elevada no grupo CT e reduzida nos grupos diab?ticos. Esses resultados estabelecem altera??es precoces da estrutura e do perfil transcricional do VE. Ainda, indicam que o treinamento aer?bio exerce prote??o espec?fica contra mecanismos respons?veis pelo dano card?aco observado no DM1

Teas and infusions from C. spiralis leaf have largely been used by folk medicine as diuretic, hypotensor, cytotoxic, immunomodulator, antilithiasic, antidiarrheic, antispasmodic, antiurolitic, antimicrobian, antifungic, antioxidant, antileishmania activity, antiinflamatory, and antiedematogenic activity. In spite of these biological effects attributed to the extracts of C. spiralis, nothing so far could be found in the scientific literature dealing with its effects on the mammalian myocardium.The present study aimed to describe the inotropic effects produced by extracts from the C. spiralis leaf on isolated guinea pig atrium, as well as to contribute for a better understanding about its mechanism of action in that tissue. In isolated mouse cardiomyocytes, the effect produced by those extracts on the intracellular calcium transient and on the sarcolemal L-type calcium current were also measured. Experiments performed to evaluate the contractile effects were carried out on isolated atrium from guinea pig (Cavia porcellus). Firstly, our purpose was to determine the most potent fraction obtained from the C. spiralis leaf. This was done by comparing the hydroalchoolic crude extract with the following ones: aqueous, chloroform, and ethyl acetate. A phytochemical analysis was performed on the fraction exhibiting the greater potency. This evaluation followed the procedures proposed by Matos (1997). The content of sodium and potassium in the most potent fraction was determined by flame photometry. In the contractile experiments, the atrial force was measured isometrically. Biological signals were captured, amplified, and then stored in computer to be processed off line. Intracellular calcium transients were studied by confocal microscopy with laser scanning by using the fluorescent dye FLUO 4AM. Calcium inward currents were measured in mouse cardiomyocytes by using patch clamp technique in the whole cell configuration. Yield percentage of the aqueous fraction (AqF) was 69,40%. This fraction showed the most potent depressor effect on the myocardial contractility (EC50 = 305 ± 41,00 mg/L, Hill constant = 1,46 ± 0,19). The following metabolites were found in the AqF: tannins, saponins, and polifenols (flavonol, flavononol, flavone, xanthone, phenol, and flavonoid). The potassium and sodium contents in 1 g/L of AqF were 1,91 and 0,15 mM, respectively. This was not enough to change the myocardial inotropism, even in the highest concentration of AqF used in the experiments. The contraction and the relaxation time, as well as the time related to the excitation-contraction coupling (stimulus-response) were not modified by adding AqF to the organ bath. However, AqF reduced the Efficiency Index for the contraction and relaxation phases. The Neyler & Merrillees protocol was employed to evaluate the AqF effect on the calcium inward current in myocardial cells. Our results showed that AqF is able to completely abolish the Bowditch phenomenon, suggesting that it could be acting by reducing the sarcolemal calcium current. Supported by those experimental evidences, experiments were proposed to better understand the relationship between AqF and calcium mechanisms in cardiac cells. The following results were obtained with 1,5 g/L AqF: 1) AqF completely abolished the positive inotropic effect induced by isoproterenol (10-1 to 103 pM); 2) AqF shifted rightwardly the concentration-effect curve for CaCl2 (0.5 to 7.0 mM) and increased the EC50 from 1.12 ± 0.07 (Hill = 1.5) to 7.23 ± 0.47 mM (Hill = 7.4) (n = 3; p < 0.05); 3) AqF completely abolished the positive inotropic effect of (-) BAY K8644 (5 to 2000 nM); 4) AqF reduced the intracellular fluorescence from 4.66 ±1.17 to 3.74 ± 1.0 a.u. (n = 30 cells, 4 mice, p < 0.05); 5) AqF did not modify the decay rate of the fluorescent signal (892 ± 37 to 930 ± 30 ms, n = 30 cells, 4 mice, p > 0.05), indicating that it does not interfiere with the calcium removal from the sarcoplasm; 6) AqF reduced the calcium inward current through L-type calcium channels from 6,29 ± 0,34 to 4,9 ± 0,2 A/F (23% , n = 5 animals, p < 0,05). This study brought us unto the following conclusions: 1) AqF is the most potent fraction obtained from C. spirallis leaves; 2) AqF contains the following secondary metabolites: tannins, saponins, and poliphenols; 3) AqF reduces the contraction force of the guinea pig left atrium; 4) AqF acts on the myocardium contractility by reducing the calcium entry in myocardial cells during contraction. / Preparados de Costus spiralis têm sido usados pela medicina popular (diurético, hipotensor, citotóxico, imunomodulador, antilitiásico, antidiarréico, antiespasmódico, antiurolítico, antimicrobiano, antifúngico, antioxidante, antileishmânia, anti-inflamatório e antiedematogênico). Apesar da gama de ações a eles atribuídas, nada pôde ser encontrado na literatura científica com respeito ao possível efeito dos Este trabalho visou determinar os efeitos inotrópicos obtidos das folhas de C. spiralis, que apresentava maior potência, bem como contribuir para o mecanismo de ação desse preparado no miocárdio de mamíferos. Os experimentos sobre contração foram realizados em átrio esquerdo de cobaia (Cavia porcellus), enquanto que as medidas de transiente de cálcio intracelular e de corrente de membrana foram feitas em cardiomiócitos de camundongo. A investigação fitoquímica do preparado mais ativo foi conduzida segundo Matos (1997). Os teores de sódio e de potássio presentes na fração mais potente, foram determinados por fotometria de chama. A força de contração atrial foi captada isometricamente e, depois amplificada, foi armazenada em computador. O transiente de cálcio intracelular foi avaliado com microscopia confocal de varredura a laser. As correntes de cálcio sarcolemais foram medidas em cardiomiócitos submetidos à técnica do patch clamp ( whole cell ). A fração aquosa (FAq) foi a que apresentou maior rendimento (69,40%) e a que exerceu maior efeito inotrópico negativo (CE50 = 305 ± 41,00 mg/L, Hill = 1,46 ± 0,19). Na sua constituição foram detectadas as seguintes classes de metabólitos secundários: taninos e saponinas, com reação fortemente positiva, e os polifenóis, com reação positiva (flavonóis, flavononóis, flavonas, xantonas, fenóis e flavonóides). Em 1 g/L de FAq foram encontrados 1,91 mM de potássio e 0,15 mM de sódio. A adição de FAq ao Tyrode não modificou significativamente a concentração desses íons. Os tempos de contração e de relaxamento, bem como o tempo de acoplamento eletromecânico não foram alterados pela FAq. Contudo, ela reduziu os Índices de Eficiência da contração e do relaxamento. A FAq aboliu completamente o fenômeno de Bowditch induzido por alta frequência de estimulação, indicando que ela reduz a entrada desse íon nas células. Com base nessa evidência, foram realizados protocolos para aprofundar o conhecimento sobre a participação das correntes de cálcio no mecanismo cardiodepressor da FAq. Esta fração produziu os seguintes resultados: 1) aboliu completamente o efeito inotrópico positivo do isoproterenol (10-1 a 103 pM); 2) deslocou para a direita a curva concentração-efeito para o CaCl2 (0,5 a 7,0 mM), aumentando a CE50 de 1,12 ± 0,07 (Hill = 1,5) para 7,23 ± 0,47 mM (Hill = 7,4) (n = 3; p < 0,05); 3) aboliu completamente o efeito inotrópico positivo do (-) BAY K8644 (5 a 2000 nM); 4) reduziu em cerca de 20% o pico da fluorescência intracelular correspondente ao transiente de cálcio citoplasmático (controle: n = 30 células; teste: n = 27 células; 4 animais); 5) não modificou a velocidade de decaimento do sinal de fluorescência, o que significa que ela não interfere com o funcionamento da bomba de cálcio do retículo sarcoplasmático; 6) reduziu em 23% a densidade de corrente de cálcio tipo-L que variou de -6,29 ± 0,34 para -4,9 ± 0,2 A/F (n = 5 animais, p < 0,05). 1) a FAq foi a fração com maior potência inotrópica; 2) os principais metabólitos secundários presentes na FAq foram taninos, saponinas e polifenóis; 3) a FAq reduz a força de contração do átrio; 4) o mecanismo da ação cardiodepressora da FAq sobre a contratilidade miocárdica se deve à diminuição da disponibilização do cálcio durante a contração.

Orientador: Kleber Gomes Franchini / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-06T17:54:14Z (GMT). No. of bitstreams: 1 Lopes_MauricioMarson_D.pdf: 2463239 bytes, checksum: 06bb8a40d038270d697d2a632626c188 (MD5) Previous issue date: 2006 / Resumo: A insuficiência cardíaca é um dos maiores problemas de saúde pública da atualidade. Esforços têm sido feitos para a obtenção do entendimento dos mecanismos que resultam no comprometimento funcional e estrutural do miocárdio, responsáveis, em última instância, pela síndrome clínica. Doenças das válvulas constituem importante contingente dentre as causas de disfunção miocárdica que resulta insuficiência cardíaca. Tipicamente, as doenças das válvulas resultam em sobrecargas mecânicas das câmaras ventriculares que têm papel central no estabelecimento e evolução das alterações estruturais que acompanham a deterioração funcional do miocárdio e, por conseguinte, a da função global das câmaras cardíacas. É fato bem estabelecido na prática clínica a estreita relação entre a presença e a intensidade de comprometimento da função das câmaras ventriculares e o prognóstico clínico ou cirúrgico dos pacientes. Portanto, o esclarecimento dos mecanismos moleculares e celulares que resultam em alterações estruturais irreversíveis, bem como o estabelecimento de indicadores clínico-laboratoriais de alterações precoces é área de grande interesse prático. No presente estudo foram avaliados pacientes portadores de insuficiência mitral com indicação clínica de tratamento cirúrgico, com o objetivo de avaliar alterações estruturais do miocárdio e sua relação com a função ventricular e a expressão e atividade da quinase de adesão focal, uma tirosino-quinase envolvida em mecanismos de sinalização de estímulos mecânicos. Pacientes com insuficiência mitral (21) foram avaliados clinica e ecocardiograficamente no período pré-operatório e em seguimento de um ano após a cirurgia. Foram realizadas biópsias intra-operatórias do ventrículo esquerdo para análise histo-morfométrica e bioquímica da expressão e atividade da quinase de adesão focal e quantificação da área ocupada pelo interstício miocárdico. A maioria dos pacientes apresentava sintomas importantes de insuficiência cardíaca (classe funcional III-IV) no pré-operatório de cirurgia para correção da insuficiência mitral. A média da fração de ejeção e a do diâmetro sistólico final do ventrículo esquerdo encontravam-se dentro da faixa de normalidade. Havia sinais de remodelamento excêntrico do ventrículo esquerdo e aumento dos níveis médios de pressão sistólica da artéria pulmonar. Esses pacientes foram acompanhados por 12 meses após a cirurgia valvar mitral, com avaliações clínicas e ecocardiográficas a cada três meses. Foram verificadas reduções significativas no número e intensidade dos sintomas no período de seguimento pós-operatório. Houve também alteração das características ecocardiográficas indicativas de redução da intensidade do remodelamento no período de seguimento pós-operatório. Foi detectada correlação negativa entre a pressão da artéria pulmonar e o diâmetro diastólico final do ventrículo esquerdo no período pré-operatório. Constatou-se redução significativa da pressão da artéria pulmonar no seguimento pós-operatório. A análise histo-morfométrica das amostras de biópsias subendocárdicas dos pacientes com insuficiência mitral revelou intensa degeneração do miocárdio, com sinais de degeneração e fibrose intersticial. Houve correlação negativa entre a intensidade da fibrose e a fração de ejeção do ventrículo esquerdo no pré-operatório. Detectou-se aumento na expressão da quinase de adesão focal (FAK) em amostras de biópsias de ventrículo esquerdo de pacientes com insuficiência mitral, em comparação com a expressão no miocárdio de pacientes com estenose mitral (grupo de seis pacientes submetidos a biópsia intra-operatória). Além de aumento na expressão, também verificamos aumento na fosforilação em tirosina da FAK com anticorpo fosfo-específico para a tirosina 397, indicativo de aumento da atividade da enzima. Na análise quantitativa de amostras preparadas através de imuno-histoquímica com os anticorpos anti-FAK e anti-vimentina (marcador de fibroblasto) verificamos aumento da marcação com anticorpo anti-FAK nas células intersticiais e também nos cardiomiócitos de amostras de pacientes com insuficiência mitral. Os novos achados do presente estudo são: 1) correlação negativa entre o nível da pressão da artéria pulmonar e do diâmetro do ventrículo esquerdo; 2) correlação entre a intensidade da fibrose e a disfunção ventricular esquerda e 3) aumento da atividade da quinase de adesão focal, correlacionado à intensidade da fibrose intersticial em pacientes com insuficiência mitral com indicação cirúrgica. Estes achados permitem-nos concluir que a elevação precoce da pressão na artéria pulmonar pode constituir-se em marcador clínico de valor para indicação precoce de cirurgia em portadores de insuficiência mitral na ausência de intenso remodelamento excêntrico do ventrículo esquerdo. Por outro lado, a correlação estreita entre a área do interstício subendocárdico e a disfunção ventricular sugere que a avaliação da intensidade da fibrose no ato operatório pode ser um bom marcador de prognóstico. Finalmente, o aumento da atividade da FAK, principalmente no interstício, sugere que esta enzima e suas vias de sinalização intracelular podem contribuir para a gênese da fibrose intersticial. Este resultado aponta para a possibilidade de que modalidades terapêuticas específicas para FAK possam ter papel no tratamento pós-operatório de pacientes com insuficiência mitral e cardíaca / Abstract: Heart failure is one of the biggest problems faced by public health. Efforts have been made in order to achieve full understanding of the mechanisms that result in the myocardium structural and functional deterioration, which is responsible for advanced symptoms. Valvular diseases are important causes of myocardium dysfunction, which result in heart failure. Typically, valvular diseases result in mechanic overload of the ventricular chambers, which play a central role in the setting up and evolution of the structural alterations that follow myocardium functional deterioration and, consequently, the global dysfunction of the cardiac chambers. The close relation between the ventricular dysfunction and the clinical or surgical prognosis of the patient is well documented in clinical practice. Therefore, the understanding of the cellular and molecular mechanisms that result in the irreversible structural alterations, as well as the setting up of clinical-laboratorial indicators of early alterations is an area of great practical interest. In the present study patients presenting mitral valve disease with clinic indication for surgical treatment, aiming at evaluating structural alterations of the myocardium and their relations to the ventricular function, and the expression and activity of focal adhesion kinase, a tyrosine-kinase involved in mechanisms of mechanic stimulus signalization. Patients suffering from mitral regurgitation (21) were evaluated by clinical exams and echocardiography in the pre-operative period and underwent clinical and echocardiograph exams for a period of one year after surgery. Intra-operative biopsies of the left ventricle were performed in order to carry out histomorphometric and biochemical analyses of the expression and activity of focal adhesion kinase and quantification of the area occupied by the myocardium interstice. The majority of the patients presented advanced symptoms of heart failure (CF III-IV) in the pre-operative for the for mitral failure correction. The average ejection fraction and the left ventricle end systolic volume were found within normality range. There were signs of eccentric remodeling of the left ventricle and increase in the mean levels of systolic pressure of the pulmonary artery. There was a follow up of these patients during three months after mitral valve surgery with clinical and echocardiograph assessments every three months. Significant reductions of symptoms in number and intensity in the post-operative outcome were verified. There was negative correlation between the pressure of the pulmonary artery and the left ventricle final diastolic diameter the pre-operative period. There was significant decrease in the pressure of the pulmonary artery in the post-operative period. The histomorphometric analysis of subendocardial biopsy samples originated from patients with mitral disease showed intense myocardium degeneration, with signs of degeneration and interstitial scar tissue. There was negative correlation between the intensity of the scar tissue and the ejection fraction of the left ventricle in pre-operative. There was increase in the expression of focal adhesion kinase (FAK) in samples of left ventricle biopsies coming from patients with mitral regurgitation in comparison to myocardium expression of patients presenting mitral stenosis (group of six patients submitted to intra-operative biopsy). Besides the increase in expression, there was also increase in tyrosine phosphorylation from FAK with phosphor-specific antibody for tyrosine 397, which indicates increase in the enzymatic activity. The quantitative analysis of samples through imunohistochemistry with anti-FAK antibodies and anti-vimentine (fibroblast marker) showed increase in the marking of interstitial cells with anti-FAK antibody and also in the samples of cardiomyocytes from patients with mitral regurgitation. The new findings of this study are: 1) negative correlation between the level of the pulmonary artery and the left ventricle diameter; 2) correlation between the scar tissue intensity and the left ventricle dysfunction and 3) increase of the focal adhesion kinase (FAK) activity correlated to the interstitial scar tissue intensity in patients with mitral regurgitation having surgical indication. These findings lead to the conclusion that the early increase of pressure in the pulmonary artery may be a valuable clinical marker to indicate precocious surgery in carriers of mitral disease when there is lack of intense eccentric remodeling of the left ventricle. On the other hand, the close correlation between the subendocardial interstitial area and the ventricular dysfunction suggest that the assessment of the scar tissue intensity during the open heart surgery act may be a good prognostic marker. Finally, the increase in the FAK activity, mainly interstitial, suggests that this enzyme and its intracellular signalizing pathway may contribute to interstitial scar tissue genesis. This result points at the possibility that specific therapeutics conditions for FAK can play a role in the post-operative treatment of patients presenting mitral disease and heart failure / Doutorado / Medicina Experimental / Doutor em Fisiopatologia Medica

Made available in DSpace on 2014-07-29T15:07:44Z (GMT). No. of bitstreams: 1 Leandro_Franco.pdf: 1233771 bytes, checksum: d1a3e21430ce8cb834c7bb95cd287196 (MD5) Previous issue date: 2008-03-05 / Changes in physiological parameters of electrocardiography, echocardiography and biochemistry markers were evaluated in dogs anesthetized with different associations of atropine, xylazine and S-ketamine. Twenty three healthy female dogs randomly distributed in four groups named as GI-6, GII-6, GIII-6 and GIV-5 were treated respectively with atropine and S-ketamine (0,04mg/kg; 10 mg/kg); S-ketamine (10 mg/kg); atropine, xylazine and S-ketamine (0,04mg/kg; 1,1 mg/kg; 10 mg/kg) and xylazine and S-ketamine (1,1 mg/kg; 10 mg/kg). Ten minutes after induction, 5mg/kg of S-ketamine was administered to animals from all groups. Measurements of electrocardiogram, echocardiogram and serum activity of aspartate aminotransferase (AST), creatine kinase (CK) and creatine kinase MB isoenzyme (CK-MB) were evaluated for 36 hours. Concerned to electrocardiography, the proposed anesthetic protocols showed significant changes especially in atrioventricular conduction period (PR interval) and systole period (QT interval), mostly in GIV. In relation to echocardiography, the main alterations happened in following variables, final stroke volume, ejection fraction and cardiac output, predominantly in GIV. In serum biochemistry analysis, it was observed alterations in CK and CK-MB values in all groups, which maintained changed for a longer time in GIII and GIV. Thus, under conditions of this study, it can be conclude that atropine, xylazine and S-ketamine association (GIII) determined lower effects on heart, while marking alterations occurred in animals from GIV / Avaliaram-se as alterações eletrocardiográficas, ecocardiográficas e de marcadores bioquímicos em cadelas anestesiadas com atropina-xilazina-cetamina-S (+) em diferentes associações. Utilizaram-se 23 cadelas, clinicamente saudáveis, distribuídas aleatoriamente em quatro grupos experimentais (GI-n=6, GII-n=6, GIII-n=6 e GIV-n=5). Os animais do GI, GII, GIII e GIV foram tratados respectivamente com atropina-cetamina-S (+) (0,04 mg/kg 10 mg/kg), cetamina-S (+) (10 mg/kg), atropina-xilazina-cetamina-S (+) (0,04mg/kg 1,1 mg/kg -10 mg/kg) e xilazina-cetamina-S (+) (1,1 mg/kg -10 mg/kg). Após dez minutos da aplicação da indução, foram reaplicados 5,0 mg/kg de cetamina-S (+) nos animais de ambos os grupos. Avaliaram-se os animais por um período de 36 horas, por meio de eletrocardiograma, ecocardiograma e avaliações da atividade sérica de aspartatoaminotransferase (AST), creatinoquinase (CK) e creatinoquinase fração-MB (CK-MB). Relativamente à eletrocardiografia, os protocolos anestésicos estudados desencadearam mudanças significativas especialmente no tempo de condução atrioventricular, evidenciado pelo aumento da duração do intervalo PR, e período de sístole, evidenciado pelo aumento da duração do intervalo QT, sugerindo um quadro de sobrecarga ventricular, predominantemente evidenciados nos animais do grupo tratado com a associação xilazina-cetamina-S (+). Quanto à avaliação ecocardiográfica, as principais alterações foram evidenciadas entre as variáveis, volume sistólico final, fração de ejeção e débito cardíaco, predominantemente nos animais xilazina-cetamina-S (+). Com relação à avaliação bioquímica, notou-se que independente do tratamento adotado foram observadas alterações nos valores de CK e CK-MB, permanecendo alteradas por um período maior nos animais dos grupos tratados com atropina-xilazina-cetamina-S(+) ou xilazina-cetamina-S (+). Desse modo, diante das condições em que o estudo foi realizado permite-se concluir que dentre os grupos estudados, a associação atropina-xilazina-cetamina-S (+) desencadeou menores efeitos sobre o coração, enquanto que as alterações mais significativas ocorreram nos animais tratados com xilazina-cetamina-S(+)

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-03-02T12:05:16Z No. of bitstreams: 1 joaorafaelalves.pdf: 18835102 bytes, checksum: c04fec309271ef247ba877e3cd5e2ba8 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-03-06T19:33:05Z (GMT) No. of bitstreams: 1 joaorafaelalves.pdf: 18835102 bytes, checksum: c04fec309271ef247ba877e3cd5e2ba8 (MD5) / Made available in DSpace on 2017-03-06T19:33:05Z (GMT). No. of bitstreams: 1 joaorafaelalves.pdf: 18835102 bytes, checksum: c04fec309271ef247ba877e3cd5e2ba8 (MD5) Previous issue date: 2014-09-30 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Apresenta-se neste trabalho um modelo computacional simplificado que caracteriza a dinâmica espaço-temporal da perfusão sanguínea no miocárdio cardíaco. Especificamente, este modelo visa reproduzir imagens qualitativas obtidas através de exames de contraste, os quais são amplamente utilizados na medicina clínica para avaliar a perfusão cardíaca. A aplicação do contraste permite a detecção de regiões isquêmicas, fibroses e tumores. Aqui foca-se também no caso de um infarto subendocárdico. Para efeitos da modelagem, considera-se o tecido do miocárdio cardíaco como um meio poroso, isto é, uma região sólida com espaços vazios. Para este fim, a modelagem foi baseada em equações diferenciais e na Lei de Darcy, a qual correlacionada a permeabilidade do tecido, a diferença de pressão e o fluxo de sangue no tecido cardíaco. / We present a simplifed computational model that characterizes the spatio-temporal dynamics of blood perfusion in cardiac myocardium. Specifically, we are interested in reproducing qualitative images obtained by contrast-enhanced exams, which are widely used in clinical medicine to evaluate the blood perfusion in the heart. The aplication of contrast allows the detection of injuries, ischemic regions, fibrosis or tumors. Here we focus on the pathological case associated to subendocardial infarct. In our modelling, we will consider the tissue of cardiac myocardium as a porous media, i.e., a solid region with empty spaces. To this end, the modelling was based on differential equations and Darcy's Law, which correlates tissue permeability, pressure difference and the blood ow in the cardiac tissue.

Background: Different types of cardiomyopathies are associated with variable hypertrophic response. <p>A number of growth factors are thought to play a role in pathologic cardiac remodeling. <p>Aims: We compared the modulation of the TGF-ƒÒ superfamily and IGF-1 signaling pathways and their target genes, the cell cycle regulatory proteins in tachycardia-induced dilated cardiomyopathy, a model with no detectable hypertrophy and in ischemic cardiomyopathy, a model with a marked hypertrophic reaction. <p>Methods: In the first study, endomyocardial biopsies were obtained weekly in 15 dogs, during the development of tachycardiomyopaty. Genes involved in the myostatin-TGF-ƒÒ-Activin-A/Smad signaling pathway, p21 and cyclin D were quantified and correlated to echocardiographic measures of hypertrophy. In the second study, myocardial tissue samples were obtained in 8 dogs with a healed myocardial infarction, in 8 dogs with heart failure induced by overpacing and in 7 healthy dogs. We measured gene expression of IGF-1, its receptor (IGF-1R) and cyclins A, B, D1, D2, D3 and E and correlated them to the level of hypertrophy. <p>Results: Tachycardiomyopathy was characterized by chambers dilation with no identifiable hypertrophy. Ischemic cardiomyopathy was characterized by eccentric hypertrophy. In tachycardiomyopathy, Activin-A mRNA was 4-fold higher than at baseline. Smad7 was overexpressed in severe heart failure; p21, a direct target gene of the Smad pathway was upregulated 8-fold and cyclin D1 was down-regulated. In that model, IGF-1 was overexpressed but neither IGF-1R nor any of the cyclins studied.<p> In ischemic cardiomyopathy, IGF-1, IGF-R, and cyclins B, D1, D3 and E gene expression were upregulated.<p> In tachycardiomyopathy, Activin-A and p21 were inversely correlated to the thickness of the interventricular septum. In normal dogs and in the both models of cardiomyopathy, IGF-1R was correlated to the thickness of the interventricular septum and to cyclins. <p>Conclusions: Taken together, these results agree with the notion that Activin-A, IGF and cyclins are involved in the modulation of hypertrophic response observed in cardiomyopathies. <p> / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Le succès de la chirurgie cardiaque dépend essentiellement de la préservation des tissus durant l'opération. Afin d'optimiser cette dernière, une protection myocardique est appliquée à travers la perfusion d'une cardioplégie hypothermique lui permettant de tolérer l'ischémie provoquée par l'absence de vascularisation. Malgré ces progrès techniques et les connaissances accumulées, la chirurgie cardiaque reste une chirurgie à risque dont les complications sont parfois de nature inconnue. Il n'existe pourtant pas de suivi opérationnel de la protection myocardique et les seules indications disponibles pour le chirurgien sont l'arrêt contractile du cœur en début de cardioplégie ou l'apparition de fibrillations cardiaques dénonçant une inefficacité à long terme de la cardioplégie. Parmi les paramètres de mesure pouvant contribuer à la surveillance du myocarde sous cardioplégie, le suivi du changement de température induit par celle-ci pourrait devenir un indicateur pertinent. L'objectif de ce travail de thèse est donc de mettre en place un dispositif de suivi thermique du cœur permettant d'évaluer concrètement la diffusion et l'efficacité de perfusion cardioplégique durant la chirurgie cardiovasculaire.Parmi les méthodes de suivi thermique non invasives implantables en salle d'opération, la thermométrie par ultrasons (TUS) et la thermographie par infrarouges (TIR) ont retenu notre attention. Dans un premier temps, une méthode directe de thermométrie par ultrasons basée sur le principe "d'écho tracking" est expérimentée via un capteur ultrasonore $2,25 MHz mono-élément sur une gamme de température de 10 à 30°C. Cependant la caractérisation du muscle cardiaque par ultrasons et les expérimentations menées en laboratoire ont mis en avant la complexité du milieu nécessitant une calibration préalable, difficile à mettre en place en conditions opératoires. La technique présente tout de même une précision de mesure satisfaisante pour une variation de température inférieure à 10°C. Un modèle d'ajustement est alors proposé afin d'identifier la tendance de réchauffement interne du cœur à partir de la température de surface mesurable via une caméra thermique à infrarouge et par l'estimation volumique du milieu par ultrasons. Le modèle est validé dans un premier temps par simulation par éléments finis et dans un second temps durant des expérimentations sur fantômes et cœur in-vitro.Au final, le dispositif multi-physique TIR et TUS est expérimenté sur modèle in-vivo lors d'opérations, durant lesquelles des sessions de refroidissement par cardioplégie hypothermique sont effectuées. L'étude se conclut sur des premiers résultats de suivi thermique satisfaisants où les températures surfaciques acquises par TIR et l'estimation de l'épaisseur des parois ventriculaires par ultrasons permettent d'obtenir efficacement la tendance de réchauffement du myocarde. Le dispositif multi-physique a également démontré la possibilité d'appliquer une calibration directe de la thermométrie ultrasonore. Cette étude de faisabilité a démontré la possibilité d'un monitoring thermique du cœur de manière non invasive et applicable en temps réel. Ce dispositif pourrait être, après adaptation spécifique, implémenté durant les interventions cardio-vasculaire et fournir un indicateur précieux aux chirurgiens quant à l'efficacité de la protection myocardique. / The success of cardiac surgery essentially depends on tissue preservation. To optimize this latter, a myocardial protection is applied thanks to a hypothermic cardioplegia that confers a marked protective effect to the heart under ischemia. Despite these technical developments, cardiac surgery still presents risks and complications are sometimes of an unknown nature. However, no operational real-time monitoring of myocardial tissue exists. Among the metrics that could be analyzed, the temperature change measurement may be a relevant indicator. The objective of the present thesis is therefore to establish a thermal monitoring system of the heart in a way to evaluate the quality of the cardioplegia perfusion during cardiovascular surgery.Among the non-invasive thermal monitoring methods implantable in the operating room, the ultrasonic thermometry and infrared thermography caught our attention. In the first stages of the study, a direct method of ultrasonic thermometry based on the echo tracking is performed on in-vitro samples with 2.25 MHz ultrasonic sensor in a temperature range between 10 to 30°C. The ultrasonic characterization of the heart muscle and the laboratory experiments brought to the forth an environmental complexity requiring prior calibration that may be difficult to implement in operational conditions. However, the technique has a satisfactory measurement accuracy for temperature variations of less than 10°C.An adjustment model is then proposed to identify the in-depth warming trend of the heart thanks to the surface temperature measurement performed by thermal infrared camera and the medium thickness estimated by ultrasound. The model is validated by finite element simulations and experiments realized on ghosts and in-vitro heart samples.Finally, the device is experimented on in-vivo animal models while hypothermic cardioplegia were conducted. Conclusive results were obtained on the heart thermal monitoring. In particular, the surface temperatures acquired by infrared thermography and the thickness estimation of the ventricular walls by ultrasound allowed an estimation of the myocardial-warming trend.This feasibility study has demonstrated the possibility of heart thermal monitoring, noninvasively and appropriate in real time. After specific adaptation, this device could be implemented during cardiovascular interventions and provide a valuable indicator for the surgeons about the efficacy of myocardial protection.

In mammals, myocardial cell death due to infarction results in scar formation and little regenerative response. In contrast, zebrafish have a high capacity to regenerate the heart after surgical resection of myocardial tissue. However, whether zebrafish can also regenerate lesions caused by cell death has not been tested. Here, we present a simple method for induction of necrotic lesions in the adult zebrafish heart based on cryoinjury. Despite widespread tissue death and loss of cardiomyocytes caused by these lesions, zebrafish display a robust regenerative response, which results in substantial clearing of the necrotic tissue and little scar formation. The cellular mechanisms underlying regeneration appear to be similar to those activated in response to ventricular resection. In particular, the epicardium activates a developmental gene program, proliferates and covers the lesion. Concomitantly, mature uninjured cardiomyocytes become proliferative and invade the lesion. Our injury model will be a useful tool to study the molecular mechanisms of natural heart regeneration in response to necrotic cell death.