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Memoria para optar al título de Químico Farmacéutico / La exposición al frío, activa los nervios simpáticos periféricos y aumenta la tasa de recambio de noradrenalina en órganos con inervación simpática. Este aumento de las concentraciones de noradrenalina en una madre gestante sometida a estrés por frío de 4ºC por 3 horas diarias durante el periodo gestacional, causarían: a.- vasoconstricción a nivel placentario acompañado de una hipoxia fetal, y/o b.- proliferación de la vasculatura placentaría. Tales mecanismos adaptativos debido a la hipoxia fetal, podrían involucrar una entrega mayor de nutrientes y hormonas y gatillar mecanismos adaptativos del feto para sobrevivir a estas nuevas condiciones, especialmente en la función cardiaca. Inicialmente esta adaptación presentará un beneficio en términos de supervivencia, pero posteriormente podría gatillar hipertrofia cardiaca. Así, este estudio evaluó el efecto sobre la morfología cardiaca producida por exposición fetal a estrés por frío crónico (4°C, 3h/día, 21 días), en la descendencia de machos y la respuesta a la estimulación β-adrenérgica (Isoproterenol) en ratas adultas. Para ello, se uso ratas machos Sprague-Dawley en distintas etapas de crecimiento (neonatal, prepúberal y adultas) utilizando ratas de la misma edad- no estresadascomo control. Utilizando parámetros morfométricos (Masa cardiaca total (MCT)/ masa corporal (MC) y masa ventricular (MV)/ masa corporal), medición de área y perímetro en cardiomiocitos y marcadores bioquímicos tales como cadena pesada de β- Miosina (β-MHC) y colágeno tipo I, no se encontraron cambios en todos los grupos de estudio: neonatos, prepúberes y adultos, indicando que no hay hipertrofia ni fibrosis cardiaca. Por último, para evaluar el efecto de la estimulación β-adrenérgica in vivo de los animales, administramos por vía SC, el agonista adrenérgico Isoproterenol (1 mg/Kg peso/día/ 10 días en ratas adultas), para inducir hipertrofia y fibrosis. Las crías prenatalmente expuestas a estrés presentan una mayor respuesta al medicamento expresado como mayor respuesta hipertrófica, pero no así fibrótica en comparación a las ratas del grupo control. Además, presentaron una alta tasa de mortalidad en respuesta al tratamiento sugiriendo fuertemente una mayor sensibilidad a la droga. El conjunto de estos resultados nos sugieren que la sobreactivación simpática durante el embarazo debido al estrés, afecta la función cardíaca de la progenie de una forma que apoya fuertemente una programación de la función cardiaca y con ello, una mayor sensibilidad a una nueva estimulación adrenérgica cuando adultos

The ability of the adult heart to generate new myocytes after injury is not established. Our purpose was to determine if the adult heart has the capacity to generate new myocytes after injury, and to gain insight into their source. Cardiac injury was induced in the adult feline heart by infusing Isoproterenol (ISO) for 10 days with minipumps and then animals were allowed to recover for 7 or 28 days. Cardiac function was measured with echocardiography and proliferative cells were identified by nuclear incorporation of 5-bromodeoxyuridine (BrdU; 7 day minipump infusion). BrdU was infused for 7 days before euthanasia at Day 10 (injury), Day 17 (early recovery), and Day 38 (late recovery) and, with a separate group of animals, was infused during injury and removed at Day 10, with animals euthanized at Day 38 for a pulse-chase experiment. Isoproterenol caused a reduction in cardiac function with evidence of myocyte loss from necrosis. During the injury phase there was a significant increase in the number of proliferative cells in the atria and ventricle, including an increase in cKit+/BrdU+ proliferative cardiac precursor cells, but there was no increase in the number of BrdU+ new myocytes (Day 10). During the first seven days of recovery (Day 17) there was a significant reduction in cellular proliferation (total BrdU+ nuclei, including cKit+/BrdU+ proliferative cardiac precursor cells) but a significant increase in BrdU+ myocytes. There was modest improvement in cardiac structure and function during recovery. At Day 38 (late recovery), overall cell proliferation (BrdU+ cells) was not different than control (BrdU infused from Days 31-38); however, increased numbers of ("bright") BrdU+ myocytes were found at Day 38 in the pulse-chase experiment, when BrdU was infused during injury (and removed at Day 10). Some of the newly formed myocytes (from the pulse-chase group; Day 38), derived from BrdU+ cardiac precursors appear to be transiently proliferative (between Days 10-38) producing a population of "dimly" BrdU+ myocytes in our pulse-chase protocol (BrdU infused during injury, Days 3-10, and removed at Day 10, with heart explant at Day 38). No significant numbers of "dimly" BrdU+ nuclei were found in any of the hearts in which BrdU was infused for 7 days prior to the animal being euthanized (Control, Day 10, Day 17, Day 38). These observations are most consistent with the conclusions stated. Our results also suggest that myocyte regeneration, as defined by BrdU+ myocytes, was more robust in the atria than the ventricle. The reasons for these differences are not clear and deserve additional study. If true, our findings suggest that cardiac precursors isolated and expanded from atrial tissue might be a better source of cells for autologous cardiac cell therapy. In summary, our data shows that the adult heart has the ability to generate new myocytes after injury, suggests that ISO injury activates cardiac precursor cells that can differentiate into new myocytes during cardiac repair, but that the environment of the ISO injured heart blunts the differentiation of cardiac precursors into functional new myocytes. The contribution of new myocytes to improved function of the ventricle would appear to be small, unless we have underestimated the number of these cells. This is quite possible, and further study is warranted to incorporate the number of "dimly" BrdU+ myocytes that may have undergone a proliferative phase as a progenitor cell and/or as an immature cardiac myocyte. Further understanding the factors that limit endogenous new myocyte formation could significantly contribute to new therapeutic applications and improve the quality of life, and potentially the lifespan, of patients in heart failure. / Physiology

The temporal nature of ectopic activity is important to elucidating the mechanisms that can lead to arrhythmogenesis. However, challenges remain in distinguishing between ectopic and non-ectopic beats. A new methodology was developed and validated to distinguish between beat types. Rapid pacing was used to induce both ectopic and non-ectopic beats. Using an electrocardiogram, the post-pacing recovery beat cycle length (RCL) and QRS were normalized to pre-paced R-R and QRS intervals and analyzed using a K-means clustering algorithm. Control hearts only produced beats with RCL ratios that increased with rapid pacing, suggestive of non-ectopic activity. Hypercalcemia and digoxin both produced significantly earlier beats with wider QRS durations, suggestive of ectopic activity. Increasing pacing further shortened RCL during digoxin + hypothermia, a mechanistic identifier of ectopic activity. When tested against a previously validated analysis, our algorithm performed well. Therefore, this electrocardiogram based algorithm distinguishes between ectopic and non-ectopic beats. In a prospective study, tetrodotoxin increased RCL ratio without changing the QRS duration of excited beats, suggesting neuronal sodium channels play an important role in ectopic beat timing. The next goal was to create a consistent model of ectopic activity. Both sympathetic and parasympathetic stimulation independently potentiate arrhythmogenesis, and we investigated the effects of independent and simultaneous stimulation on the temporal nature of arrhythmogenesis. Isoproterenol (ISO), a sympathetic agonist, transiently produced ectopic activity and increased heart rate. Acetylcholine (ACh), a parasympathetic agonist, did not significantly produce ectopic activity but did slow heart rate. ACh added after ISO also transiently produced ectopic activity, while heart rate remained slowed. Importantly, ISO following ACh persistently increased ectopic activity and heart rate. Therefore, ISO following ACh is an ideal model for creating sustained ectopic activity. Mature animals exhibited sustained arrhythmogenesis while young animals did not. When ACh was removed and then followed by ISO, ectopic activity and heart rate transiently increased, similar to ISO alone. This suggests that maintained ACh perfusion can sustain ISO sensitivity, in contrast to ISO perfusion alone. The data in this dissertation provide an insight into the mechanisms that affect the ectopic beat timing and arrhythmia propensity. / Ph. D.

Objective: Dosing intervals of 12 and 24 hours for atenolol have been recommended, but an evidentiary basis is lacking. To test the hypothesis that repeated, once-daily oral administration of atenolol attenuates the heart rate response to isoproterenol for 24 hours, we performed a double-blind, randomized, placebo-controlled cross-over experiment. Animals: Twenty healthy dogs Procedures: Dogs were randomly assigned to receive either placebo (P) and then atenolol (A), [1 mg/kg PO q24h] or vice versa. Treatment periods were 5-7 days; time between periods was 7 days. Heart rates (bpm) at rest (HRr) and during constant rate [0.2 μg/kg/min] infusion of isoproterenol (HRi) were electrocardiographically obtained 0, 0.25, 3, 6, 12, 18, and 24 hours after final administration of drug or placebo. A mixed model ANOVA was used to evaluate the effects of treatment (Tr), time after drug or placebo administration (t), interaction of treatment and time (Tr*t) as well as period and sequence on HRr and HRi. Results: Sequence or period effects were not detected. There was a significant effect of Tr (p <0.0001) and Tr*t (p <0.0001) on HRi. Atenolol significantly attenuated HRi for 24 hours but did so maximally at 3 hours (least squares means ± SE, A: 146±5 bpm, P: 208±5 bpm); the effect at 24 hours was small (A: 193±5, P: 206±5). Atenolol had a small but significant effect (p <0.0001) on HRr. Conclusions and Clinical Relevance: The results of this study support a dosing interval that is less than 24 hours. / MS / This thesis was designed to test the effects of the drug atenolol on heart rate in dogs. Atenolol is used to reduce the heart rate of dogs with cardiovascular disease. The study used 20 dogs that were given oral capsules in both a placebo (no drug) and atenolol phase of the experiment. The study was designed to control for other causes of slower heart rate and make sure that the investigator did not know which treatment was given to a dog. Placebo dogs had a high heart rate response to the drug isoproterenol whereas atenolol treated dogs had a statistically significant lower heart rate response compared to placebo over a 24 hours period of time. The difference between treatments was small after 24 hours and further work is needed to determine the best time interval between doses of medication.

O aumento da atividade do Sistema Nervoso Simpático e a estimulação crônica dos receptores &#946;-adrenérgicos (&#946;-AR) estão associados a diversas patologias que acometem o sistema cardiovascular. Entretanto, ainda não estão evidenciados os efeitos da hiperatividade &#946;-AR no tecido renal. Assim, o objetivo deste estudo foi investigar os efeitos da ativação crônica &#946;-AR na função e morfologia renal, bem como na expressão do SRA intrarrenal. Os experimentos revelaram que o tratamento com ISO não alterou a pressão arterial, mas induziu hipertrofia cardíaca. ISO reduziu a perfusão glomerular e aumentou a FF, mas não alterou a morfologia renal. Nos estudos de qPCR, ISO aumentou a expressão de RNAm para renina, angiotensinogênio, ECA1, AT1, Nox 4, p22phox, NFB, IL-1&#946;, TGF-&#946; e Bax no córtex. Em conclusão, a ativação crônica &#946;-AR alterou a hemodinâmica renal, sugeriu ativação do SRA intrarrenal e aumento na geração de espécies reativas de oxigênio, o que justifica o aumento da expressão de RNAm para moléculas pró- inflamatórias, pró-fibróticas e pró-apoptóticas. / The increased activity of the Sympathetic Nervous System and the chronic stimulation of &#946;-adrenergic receptors (&#946;-AR) are associated to several pathologies that affect the cardiovascular system. However, the effects of &#946;-AR hyperactivity on renal tissue have not been evidenced yet. Thus, the aim of this study was to investigate the effects of chronic &#946;-AR activation on renal function and morphology, as well as on intrarenal RAS expression. The experiments revealed that ISO treatment did not alter blood pressure, but induced cardiac hypertrophy. ISO reduced glomerular perfusion and increased FF, but did not alter renal morphology. In the qPCR studies, ISO increased the mRNA expression for renin, angiotensinogen, ECA1, AT1, Nox4, p22phox, NFB, IL-1&#946;, TGF-&#946; and Bax in the cortex. In conclusion, chronic &#946;-AR activation altered renal hemodynamics, suggested activation of intrarenal RAS and increased generation of reactive oxygen species, which justifies the increase of mRNA expression for pro-inflammatory, pro-fibrotic and pro- apoptotic molecules.

Este estudo teve como objetivo avaliar as alterações do complexo de glicoproteínas associadas à distrofina que conferem estabilidade estrutural aos cardiomiócitos na isquemia miocárdica induzida pelo isoproterenol. Materiais e Métodos: Ratos Wistar machos foram divididos em dois grupos: grupo controle (SAL), injeção subcutânea de salina, e grupo isoproterenol (ISO), injeção subcutânea de isoproterenol (85mg/kg) diluído em água destilada, em dois dias consecutivos separados por intervalo de 24hs. Os ratos foram mortos 24 horas após a segunda injeção de salina ou isoproterenol. Os corações foram rapidamente excisados, lavados em salina gelada, pesados e colocados em formol PBS por 24hs a 4ºC e incluídos em parafina ou Historesina. Para análise morfométrica, os corações foram cortados transversalmente na porção medioventricular, equidistante entre o ápice e a base, e incluídos em parafina. Áreas dos ventrículos direito e esquerdo, espessura de parede livre dos ventrículos e septo interventricular foram medidas. Os corações cortados frontalmente nas metades anterior e posterior e incluídos em Historesina foram utilizados para avaliação das áreas de miocitólise. Porções hemiventriculares foram congeladas para as reações de imunofluorescência com os seguintes marcadores: distrofina, ?-1 integrina, ?-actina sarcomérica, ?-sarcoglicana, ?-distroglicana, merosina laminina, albumina, CD68, CD45, CD4 e eNOS. A apoptose foi avaliada através do método de TUNEL. A função cardíaca, as dimensões das cavidades ventriculares e a mobilidade de parede foram analisadas através da ecocardiografia. A análise estatística foi realizada através do teste t de Student, com nível de significância de 5%. Resultados e Conclusão: Houve diferença significativa no peso do coração, na taxa de crescimento corporal, na área do ventrículo esquerdo e na espessura de parede do ventrículo direito entre os grupos. Não houve diferença estatística significativa na espessura da parede do ventrículo esquerdo e septo, mas observou-se tendência à diminuição. As áreas de miocitólise representaram 26,89%, 36,12%, 28,15% no ventrículo direito, septo e ventrículo esquerdo, respectivamente. A imunofluorescência mostrou que a distrofina foi a estrutura mais sensível ao dano provocado pelo isoproterenol, seguida pela perda completa da actina. A redução na expressão de ?-sarcoglicana, ?-distroglicana, ?-1 integrina e laminina, foram considerados como epifenômenos. A expressão de eNOS estava praticamente ausente nas áreas de miocitólise. A expressão aumentada de eNOS nos pequenos vasos ao redor das áreas de miocitólise sugere uma resposta compensatória à isquemia provocada pelo isoproterenol na tentativa de melhora do fluxo sangüíneo para as áreas de lesão. Foi observada alteração na permeabilidade sarcolemal nos cardiomiócitos dos animais tratados com isoproterenol com acúmulo de albumina no espaço intracelular. Observou-se que os cardiomiócitos e os macrófagos estavam constante e claramente marcados para apoptose nas áreas de miocitólise. Na ecocardiografia, os diâmetros sistólico e diastólico do ventrículo esquerdo foram significativemente maiores no grupo ISO em comparação com os controles. A fração de ejeção não foi diferente entre os grupos. O escore de mobilidade de parede mostrou hipocinesia ou acinesia nos segmentos apicais nos corações do grupo ISO. Essas mudanças, relacionadas à isquemia, podem explicar as graves alterações na integridade estrutural do sarcolema dos cardiomiócitos e a lesão induzida pelo isoproterenol. Mecanismos compensatórios no curto período de nosso experimento poderiam manter a função cardíaca normal apesar das graves alterações morfológicas encontradas. / This study tested the hypothesis that the dystrophin-glycoprotein complex that confers structural stability in cardiomyocytes was affected in the isoproterenol-induced myocardial ischemia. Materials and Methods: Male Wistar rats were divided in control group (SAL), injected subcutaneously with physiological saline, and isoproterenol-treated group (ISO), injected with isoproterenol (85mg/Kg) diluted in distilled water, in two consecutive days, separated by a 24-hour interval. These rats were killed 24 hours after the second injection of isoproterenol or physiological saline. The hearts were rapidly removed, rinsed in ice-cold 0.9% saline solution, weighed, and fixed as a whole in phosphate-buffered for 24 hours at 4oC. For morphometric analysis, the hearts cut into two fragments by a midventricular coronal section and embedded in paraffin. The absolute thicknesses of the septum and left and right ventricular walls and the areas of each ventricular chamber were measured. The hearts for Historesin embedding were frontally cut into anterior and posterior halves for analysis of myocytolytic areas. Hearts frontally cut were frozen for immunofluorescence study using primary antibodies against dystrophin, ?-1 integrin, ?- sarcomeric actin, ?-sarcoglycan, ?-dystroglycan, merosin laminin, albumin, CD68, CD45, CD4 e eNOS. The occurrence of apoptotic cells was evaluated by TUNEL method. The cardiac function, LV dimensions and wall motion segmented score were analyzed by echocardiography. For analysis of differences between the two groups the Student\'s t-test was performed and the level of significance of 5% was chosen to denote difference between means. Results and Conclusion: There was significant difference in the heart weight, in the heart ratio, in the LV area and right ventricular (RV) thicknesses between the two groups. No statistical difference was observed in the thicknesses of the free wall of the LV and septum, although tended to be lower in isoproterenol-treated myocardium. The percentage of myocytolysis in the LV, septum, and RV with myocytolysis in isoproterenol treated rats was: 26.89%, 36.12%, 28.15%, respectively. Immunofluorescence demonstrated that loss of dystrophin was the primary event in the myocytolytic process. Decreased expression of ?-dystroglycan, ?-sarcoglycan, ?-1 integrin and laminin occurred, appearing as epiphenomena. The eNOS expression was almost completely absent in the myocytolytic foci. eNOS expression was enhanced in blood vessels of cardiomyocytes through the entire myocardium of rats given isoproterenol. This is likely a compensatory response to the ischemic insult elicited by isoproterenol administration. In the myocytolytic foci a positive reaction for apoptosis was constantly and clearly noted in cardiomyocytes and macrophages. The echocardiography showed that diastolic and systolic LV dimensions in ISO-group were significantly higher in comparison with control group. The ejection fraction was not different between groups. The wall motion segmented score showed hypokinesis or akinesis in the apical segments in the hearts of ISO-group as compared with controls. These changes, related to ischemic injury, can explain the severe alterations in the structural integrity of the sarcolemma of cardiomyocytes and hence severe and irreversible injury induced by isoproterenol. Compensatory mechanisms in the short time of our experiment could maintain the normal cardiac function in spite of severe myocardial morphological changes.

Neste estudo investigou-se: 1) os subtipos de receptores b-adrenérgicos (b-AR) envolvidos nas alterações de reatividade vascular induzida pela hiperativação dos receptores b-AR com do uso de camundongos nocaute para os receptores b1- ou b2-AR e selvagens tratados por 7 dias com isoproterenol; 2) a expressão gênica e protéica de mediadores pró-inflamatórios na aorta de ratos tratados por 7 dias com isoproterenol. Os dados demonstram que: em aorta de camundongos selvagens o receptor b1-AR é mais importante em mediar vasodilatação, enquanto o b2AR modula negativamente a contração vascular de maneira dependente do endotélio. A hiperativação dos receptores b-AR aumenta a vasoconstrição à fenilefrina via desequilíbrio NO/ ânion superóxido em aorta de camundongos e o b2-AR parece estar envolvido no desencadeamento destes efeitos. Sugere-se que a ativação crônica dos receptores b-AR aumenta a síntese de fatores pró-inflamatórios e induz maior ativação do NF-kB no tecido vascular, efeitos que parecem estar associados ao aumento da reatividade vascular à fenilefrina. / In this study, it was investigated: 1) the b-adrenoceptor (AR) subtypes involved in the altered vascular reactivity induced by overactivation of b-AR using b1- or b2-adrenoceptor knock-out mice and respective wild-types treated for 7 days with isoproterenol or with vehicle; 2) gene and protein expression of vascular inflammatory mediators in aorta from 7-day isoproterenol-treated rats. In conclusion, the data suggest that b1-AR is more important in the modulation of vasodilator mechanisms while b2 down regulates mice vascular contraction in an endothelium-dependent manner. Overactivation of b-AR increased vasoconstrictor response to phenylephrine, by NO/ superoxide anion unbalance in mice aortic rings, and b2-AR seems to be involved in the triggering of these chronic isoproterenol effects. The results also suggest that chronic activation of b-AR enhances the synthesis of proinflammatory factors and induces higher NF-kB activation on vascular tissue, and these effects seem to be related to hiperreactivity to phenylephrine induced by chronic isoproterenol.

Este estudo teve como objetivo avaliar as alterações do complexo de glicoproteínas associadas à distrofina que conferem estabilidade estrutural aos cardiomiócitos na isquemia miocárdica induzida pelo isoproterenol. Materiais e Métodos: Ratos Wistar machos foram divididos em dois grupos: grupo controle (SAL), injeção subcutânea de salina, e grupo isoproterenol (ISO), injeção subcutânea de isoproterenol (85mg/kg) diluído em água destilada, em dois dias consecutivos separados por intervalo de 24hs. Os ratos foram mortos 24 horas após a segunda injeção de salina ou isoproterenol. Os corações foram rapidamente excisados, lavados em salina gelada, pesados e colocados em formol PBS por 24hs a 4ºC e incluídos em parafina ou Historesina. Para análise morfométrica, os corações foram cortados transversalmente na porção medioventricular, equidistante entre o ápice e a base, e incluídos em parafina. Áreas dos ventrículos direito e esquerdo, espessura de parede livre dos ventrículos e septo interventricular foram medidas. Os corações cortados frontalmente nas metades anterior e posterior e incluídos em Historesina foram utilizados para avaliação das áreas de miocitólise. Porções hemiventriculares foram congeladas para as reações de imunofluorescência com os seguintes marcadores: distrofina, ?-1 integrina, ?-actina sarcomérica, ?-sarcoglicana, ?-distroglicana, merosina laminina, albumina, CD68, CD45, CD4 e eNOS. A apoptose foi avaliada através do método de TUNEL. A função cardíaca, as dimensões das cavidades ventriculares e a mobilidade de parede foram analisadas através da ecocardiografia. A análise estatística foi realizada através do teste t de Student, com nível de significância de 5%. Resultados e Conclusão: Houve diferença significativa no peso do coração, na taxa de crescimento corporal, na área do ventrículo esquerdo e na espessura de parede do ventrículo direito entre os grupos. Não houve diferença estatística significativa na espessura da parede do ventrículo esquerdo e septo, mas observou-se tendência à diminuição. As áreas de miocitólise representaram 26,89%, 36,12%, 28,15% no ventrículo direito, septo e ventrículo esquerdo, respectivamente. A imunofluorescência mostrou que a distrofina foi a estrutura mais sensível ao dano provocado pelo isoproterenol, seguida pela perda completa da actina. A redução na expressão de ?-sarcoglicana, ?-distroglicana, ?-1 integrina e laminina, foram considerados como epifenômenos. A expressão de eNOS estava praticamente ausente nas áreas de miocitólise. A expressão aumentada de eNOS nos pequenos vasos ao redor das áreas de miocitólise sugere uma resposta compensatória à isquemia provocada pelo isoproterenol na tentativa de melhora do fluxo sangüíneo para as áreas de lesão. Foi observada alteração na permeabilidade sarcolemal nos cardiomiócitos dos animais tratados com isoproterenol com acúmulo de albumina no espaço intracelular. Observou-se que os cardiomiócitos e os macrófagos estavam constante e claramente marcados para apoptose nas áreas de miocitólise. Na ecocardiografia, os diâmetros sistólico e diastólico do ventrículo esquerdo foram significativemente maiores no grupo ISO em comparação com os controles. A fração de ejeção não foi diferente entre os grupos. O escore de mobilidade de parede mostrou hipocinesia ou acinesia nos segmentos apicais nos corações do grupo ISO. Essas mudanças, relacionadas à isquemia, podem explicar as graves alterações na integridade estrutural do sarcolema dos cardiomiócitos e a lesão induzida pelo isoproterenol. Mecanismos compensatórios no curto período de nosso experimento poderiam manter a função cardíaca normal apesar das graves alterações morfológicas encontradas. / This study tested the hypothesis that the dystrophin-glycoprotein complex that confers structural stability in cardiomyocytes was affected in the isoproterenol-induced myocardial ischemia. Materials and Methods: Male Wistar rats were divided in control group (SAL), injected subcutaneously with physiological saline, and isoproterenol-treated group (ISO), injected with isoproterenol (85mg/Kg) diluted in distilled water, in two consecutive days, separated by a 24-hour interval. These rats were killed 24 hours after the second injection of isoproterenol or physiological saline. The hearts were rapidly removed, rinsed in ice-cold 0.9% saline solution, weighed, and fixed as a whole in phosphate-buffered for 24 hours at 4oC. For morphometric analysis, the hearts cut into two fragments by a midventricular coronal section and embedded in paraffin. The absolute thicknesses of the septum and left and right ventricular walls and the areas of each ventricular chamber were measured. The hearts for Historesin embedding were frontally cut into anterior and posterior halves for analysis of myocytolytic areas. Hearts frontally cut were frozen for immunofluorescence study using primary antibodies against dystrophin, ?-1 integrin, ?- sarcomeric actin, ?-sarcoglycan, ?-dystroglycan, merosin laminin, albumin, CD68, CD45, CD4 e eNOS. The occurrence of apoptotic cells was evaluated by TUNEL method. The cardiac function, LV dimensions and wall motion segmented score were analyzed by echocardiography. For analysis of differences between the two groups the Student\'s t-test was performed and the level of significance of 5% was chosen to denote difference between means. Results and Conclusion: There was significant difference in the heart weight, in the heart ratio, in the LV area and right ventricular (RV) thicknesses between the two groups. No statistical difference was observed in the thicknesses of the free wall of the LV and septum, although tended to be lower in isoproterenol-treated myocardium. The percentage of myocytolysis in the LV, septum, and RV with myocytolysis in isoproterenol treated rats was: 26.89%, 36.12%, 28.15%, respectively. Immunofluorescence demonstrated that loss of dystrophin was the primary event in the myocytolytic process. Decreased expression of ?-dystroglycan, ?-sarcoglycan, ?-1 integrin and laminin occurred, appearing as epiphenomena. The eNOS expression was almost completely absent in the myocytolytic foci. eNOS expression was enhanced in blood vessels of cardiomyocytes through the entire myocardium of rats given isoproterenol. This is likely a compensatory response to the ischemic insult elicited by isoproterenol administration. In the myocytolytic foci a positive reaction for apoptosis was constantly and clearly noted in cardiomyocytes and macrophages. The echocardiography showed that diastolic and systolic LV dimensions in ISO-group were significantly higher in comparison with control group. The ejection fraction was not different between groups. The wall motion segmented score showed hypokinesis or akinesis in the apical segments in the hearts of ISO-group as compared with controls. These changes, related to ischemic injury, can explain the severe alterations in the structural integrity of the sarcolemma of cardiomyocytes and hence severe and irreversible injury induced by isoproterenol. Compensatory mechanisms in the short time of our experiment could maintain the normal cardiac function in spite of severe myocardial morphological changes.

Made available in DSpace on 2016-04-28T20:18:34Z (GMT). No. of bitstreams: 1 2009 - Camilla Pedreira da Silva.pdf: 3646522 bytes, checksum: 1265bedbb6032df689d55556830004da (MD5) Previous issue date: 2009-10-26 / The role of estrogen in isoproterenol-induced cardiac hypertrophy model is not often studied so far. Therefore, the main purpose of this study was to investigate the best protocolo of isoproterenol-induced cardiac hypertrophy and assess the role the 17 &#946;- estradiol in this model. In the first part female Wistar rats were treated with different doses of D-L isoproterenol chloride: 0,5 (Iso 0,5), 5 (Iso 5), 10 ( Iso 10) mg/kg/day, or vehicle (saline 0.9%) s.c., during 8 or 16 days (n = 10/group). The electrocardiography (ECG), echocardiogram (ECO) and histology were performed in the end of the experiments (8 or 16 days). In second part, female Wistar rats were ovariectomized (Ovx) or false operated (sham) and 48 hours after were treated with isoproterenol (5mg/kg/day), s.c. or vehicle (saline 0.9%) during 16 days (n=10/ group) comprising the groups as follow: sham+saline, Ovx+saline, sham+iso e Ovx+iso. Four additional groups were treated with estradiol benzoate: 7 &#956;g/kg/day (Ovx+E2.7+iso e Ovx+E2.7) or 140 &#956;g/kg/day (Ovx+E2.140+iso) during 16 days (n = 10/group). The dose of isoproterenol was divided into twice a day while estrogen administration once a day. Two weeks before the beginning of the experiments,the rats underwent estrous cycle assessment and those which did not present three consecutive regular cycles were excluded from the study. ECG, and post- mortem studies were performed in the end of the experiments. The groups ISO-0.5, ISO-5 and ISO-10 showed a significant increase in cardiac index when compared to control groups (p < 0,01). The groups treated with isoproterenol during 16 days showed cardiac indexes higher than those treated with the same doses but during 8 days (p < 0,05). ECG showed increase QRS index only in the ISO-5 and Iso -10 groups. The groups treated with isoproterenol showed subendocardic fibrosis, especially in the left ventricle. In second part of this study, the groups treated with isoproterenol showed a significant increase in cardiac index (IC) when compared to control groups. O Ovx+E2.140+iso showed significant increase in IC when compared to Ovx+iso (p < 0,05).The cardiac hypertrophy induced by isoproterenol was time but not dose-dependent, at least in the dose used herein. The fibrosis seems be dose-dependent from the use of bigger doses. High dose of 18-beta estradiol enhanced isoproterenol-induced cardiac hypertrophy which was not detected by conventional ECG assessment. / Um estudo pouco frequente no modelo de hipertrofia card?aca induzida pelo isoproterenol, ? aquele referente ao papel dos estr?genos no desenvolvimento da hipertrofia mioc?rdica. Portanto, neste trabalho, buscou-se a padroniza??o do modelo de hipertrofia card?aca induzida pelo isoproterenol, bem como, a avalia??o do papel do17 &#946;- estradiol neste modelo. Na primeira etapa foram utilizadas ratas Wistar (200 250g) que foram tratadas com diferentes doses de D-L cloridrato de isoproterenol (Iso): 0,5 (Iso 0,5), 5 (Iso 5), 10 (Iso 10) mg/kg/dia, s.c., durante 8 ou 16 dias (n=10/grupo).O Eletrocardiograma (ECG), ecocardiograma (ECO) e an?lise histol?gica foram realizados ao final do experimento (8 ou 16 dias). Na segunda etapa ratas Wistar foram ovariectomiazadas bilateralmente (Ovx) ou falso- operadas (sham) e 48 horas ap?s foram tratadas com Iso (5mg/kg/dia) s.c ou ve?culo (salina 0,9%) s.c, durante 16 dias, formando os seguintes grupos: sham+salina, Ovx+salina, sham+Iso e Ovx+Iso. Quatro grupos adicionais foram tratados com benzoato de estradiol nas doses de 7&#956;g/kg/dia (Ovx+E2.7+Iso e Ovx+E2.7) ou 140&#956;g/kg/dia (Ovx+E2.140+Iso e Ovx+E2.140) durante 16 dias (n=10/grupo). A dose de Iso foi dividida duas vezes ao dia e a administra??o hormonal foi feita uma vez ao dia. Duas semanas antes do in?cio do experimento foi realizado o acompanhamento do ciclo estral das ratas sendo descartadas do experimento aquelas que n?o apresentavam tr?s ciclos regulares consecutivos. Ao final do experimento foram realizados o ECG e os estudos post mortem. Os grupos Iso 0,5 , Iso 5 e Iso 10 apresentaram um aumento significativo do ?ndice card?aco (IC) quando comparado com o controle (p < 0,001). O grupo tratado com Iso durante 16 dias (Iso 5) apresentou um aumento no ?ndice card?aco quando comparado com o mesmo grupo durante 8 dias. No ECG houve aumento no ?ndice QRS somente nos grupos Iso 5 e Iso 10. Os grupos tratados com Iso mostraram uma fibrose subendoc?rdica ventricular. Na segunda etapa todos os grupos tratados com Iso apresentaram um aumento no IC quando comparado com os seus respectivos controles. O Ovx+E2.140+Iso apresentou aumento significativo do IC quando comparado ao Ovx+Iso. Ao contr?rio do ?ndice card?aco, n?o houve diferen?a estat?stica entre a amplitude do complexo QRS do Ovx+E2.140+Iso e Ovx+Iso. A hipertrofia card?aca induzida pelo Iso parece ser tempo e n?o dose dependente, pelo menos no tempo e dose utilizada.A fibrose parece ser dose dependente a partir da utiliza??o de doses maiores de Iso. O estr?geno na dose de 140 &#956;g/kg/dia potencializou a hipertrofia card?aca induzida pelo isoproterenol, por?m o ECG n?o foi sens?vel em detectar essa diferen?a e o estr?geno, no protocolo utilizado, n?o reduziu a porcentagem de fibrose no modelo de hipertrofia card?aca induzida pelo isoproterenol.