Binary transgenic (BT) mice with doxycycline (DOX)-suppressible cardiac-specific over-expression of endothelin 1 (ET 1) exhibit progressive heart failure, QRS prolongation, and death following DOX withdrawal. However, the molecular basis and reversibility of the electrophysiological abnormalities in this model were not known. Here we assess the mechanisms underlying ET 1 mediated electrical remodelling, and its role in heart failure. Prior attempts to prevent this model of ET-1 induced cardiomyopathy with ET receptor antagonism were not beneficial. We now propose to evaluate the effectiveness of blocking the synthesis of ET-1 with CGS 26303, a dual inhibitor of endothelin converting enzyme (ECE) and neutral endopeptidase. BT vs. littermate control mice were withdrawn from DOX and serially studied with ultrasound biomicroscopy, octapolar catheters, multi-electrode epicardial mapping, histopathology, Western blot, immunohistochemistry and qRT-PCR. Prolonged ventricular activation and depressed rate of ventricular activation were detected as early as 4 wks after transgene activation, when structure and function of the heart remained unaffected. By 8 wks of ET-1 over-expression, biventricular systolic and diastolic dysfunction, myocardial fibrosis, cardiomyocyte hypertrophy, prolonged ventricular activation and repolarization, depressed rate of ventricular activation, and abnormal atrioventricular nodal function were observed. Within 4 wks of ET-1 induction, reduction were observed in connexin-43 mRNA, protein, and phosphorylation, Nav1.5 mRNA and protein, Na+ conductance, K+ channel interacting protein-2 mRNA and Kv4.2 mRNA. Chromatin immunoprecipitation revealed that nuclear factor κB preferentially binds to Cx43 and Nav1.5 promoters. Importantly, the associated electrophysiological abnormalities at this time point were reversible upon suppression of ET 1 over-expression and completely prevented the development of structural and functional remodelling. Treatment with CGS-26303 (5 mg/kg/day) failed to improve survival, or hemodynamic and contractile decline. ET-1-mediated ventricular conduction delays correlates with gap junction and ion channel remodelling, and precedes heart failure. The sequence and reversibility of this phenotype suggest that a primary abnormality in electrical remodelling may contribute to the pathogenesis of heart failure. CGS 26303 failed to prevent this cardiomyopathic phenotype. These data suggest that chronically high levels of bigET-1, as seen in heart failure, may induce increased ECE activity and/or non-ECE ET-1 synthesis, thus circumventing the efficacy of ECE blockade in this model.

Binary transgenic (BT) mice with doxycycline (DOX)-suppressible cardiac-specific over-expression of endothelin 1 (ET 1) exhibit progressive heart failure, QRS prolongation, and death following DOX withdrawal. However, the molecular basis and reversibility of the electrophysiological abnormalities in this model were not known. Here we assess the mechanisms underlying ET 1 mediated electrical remodelling, and its role in heart failure. Prior attempts to prevent this model of ET-1 induced cardiomyopathy with ET receptor antagonism were not beneficial. We now propose to evaluate the effectiveness of blocking the synthesis of ET-1 with CGS 26303, a dual inhibitor of endothelin converting enzyme (ECE) and neutral endopeptidase. BT vs. littermate control mice were withdrawn from DOX and serially studied with ultrasound biomicroscopy, octapolar catheters, multi-electrode epicardial mapping, histopathology, Western blot, immunohistochemistry and qRT-PCR. Prolonged ventricular activation and depressed rate of ventricular activation were detected as early as 4 wks after transgene activation, when structure and function of the heart remained unaffected. By 8 wks of ET-1 over-expression, biventricular systolic and diastolic dysfunction, myocardial fibrosis, cardiomyocyte hypertrophy, prolonged ventricular activation and repolarization, depressed rate of ventricular activation, and abnormal atrioventricular nodal function were observed. Within 4 wks of ET-1 induction, reduction were observed in connexin-43 mRNA, protein, and phosphorylation, Nav1.5 mRNA and protein, Na+ conductance, K+ channel interacting protein-2 mRNA and Kv4.2 mRNA. Chromatin immunoprecipitation revealed that nuclear factor κB preferentially binds to Cx43 and Nav1.5 promoters. Importantly, the associated electrophysiological abnormalities at this time point were reversible upon suppression of ET 1 over-expression and completely prevented the development of structural and functional remodelling. Treatment with CGS-26303 (5 mg/kg/day) failed to improve survival, or hemodynamic and contractile decline. ET-1-mediated ventricular conduction delays correlates with gap junction and ion channel remodelling, and precedes heart failure. The sequence and reversibility of this phenotype suggest that a primary abnormality in electrical remodelling may contribute to the pathogenesis of heart failure. CGS 26303 failed to prevent this cardiomyopathic phenotype. These data suggest that chronically high levels of bigET-1, as seen in heart failure, may induce increased ECE activity and/or non-ECE ET-1 synthesis, thus circumventing the efficacy of ECE blockade in this model.

Motivation: The prevalence of obesity is the highest among African women in South Africa. Since obesity is a major cardiovascular risk factor, African women in South Africa could be regarded as a high risk group. However, investigations on obesity-related hypertension are limited in this population group. The associations of body fat distribution and hormones such as leptin and endothelin-1 with cardiovascular function have not yet been determined in these women. It has been determined that endothelin-1 is a role player in the development and/or maintenance of hypertension in various population groups, especially African Americans. Endothelin-1 has also been found to be involved in obesity-related hypertension in non-African population groups. It has been indicated that the obesity-related hormone, leptin, also plays a role in obesity-related hypertension, especially in African Americans. Leptin levels have been found to be higher in obese hypertensive African American women compared to an obese normotensive control group. Since the above-mentioned two hormones playa prominent role in obesity and hypertension in African American and non-African population groups, the lack of data on African women in South Africa serves as motivation to conduct this investigation. Aim: To investigate obesity-related hypertension in African women through the determination of associations between various anthropometric and endocrinological variables with cardiovascular, especially vascular function. Methodology: Manuscripts presented in Chapters 2, 3 and 4 made use of data from the POWIRS (Profiles of Obese Women suffering from the Insulin Resistance Syndrome) I project where African women were selected from a government institution in the North West Province. A group of 98 women were divided into lean normotensive, overweight/obese normotensive and overweight/obese hypertensive groups. Anthropometric and cardiovascular measurements were taken and the lipid profile, leptin and endothelin-1 levels determined. The analysis of covariance (ANCOVA) was used to show significant differences between groups while adjusting for age. Partial correlation coefficients were used to show associations between various variables while adjusting for age. Stepwise linear regression analysis was also used to show associations between variables. The study presented in Chapter 5 made use of both POWIRS I and II, which are studies including Africans and Caucasians, respectively. The methodology of the two studies was the same. All subjects gave informed consent in writing and the Ethics Committee of the North-West University approved the study. The reader is referred to the "Materials and Methods" section of Chapters 2-5 for a more elaborate description of the subjects, study design and analytical methods used in each article. vii Results and conclusions of the individual manuscripts > Results from Chapter 2 showed that the volume loading effect associated with obesity was present in both overweight/obese normotensive and overweight/obese hypertensive groups, however, the accommodating effect observed in the overweight/obese normotensive group was absent in the overweight/obese hypertensive group due to decreased vascular function. This was confirmed by a high pulse pressure. Decreased vascular functioning was associated with the abdominal skin fold. This suggests that abdominal subcutaneous fat may either be a marker of visceral fat, or may in itself contribute to increased cardiovascular risk in Africans. > Results from Chapter 3 showed a negative result. Plasma endothelin-1 levels were similar for the lean normotensive, overweight/obese normotensive and overweight/obese hypertensive groups. After re-dividing the groups into normotensive and hypertensive, and then into lean and overweight/obese, still no differences could be obtained. Additionally, no correlations could be obtained between endothelin-1 and cardiovascular function in any of the groups. These findings suggest that endothelin-1 is not implicated in obesity-related hypertension in African women. > In Chapter 4, leptin levels were elevated in both overweight/obese normotensive and hypertensive groups compared to the lean normotensive group. However, leptin levels did not differ between the two overweight/obese groups. Even though leptin levels were the same, leptin was directly and positively associated with systolic blood pressure and pulse pressure and negatively with arterial compliance only in the overweight/obese hypertensive group, independent of obesity, insulin resistance, hyperinsulinemia and age. > In Chapter 5 the volume loading, as well as the accommodating effect, that is, decreased total peripheral resistance and increased arterial compliance, was present in both African and Caucasian obese groups compared to their lean controls. Even though leptin levels, body mass index and age were similar for both African and Caucasian obese groups, the accommodating effect seemed to be more prominent in the obese Caucasian group, explaining a lower diastolic blood pressure compared to the obese African group. Leptin showed a favourable negative association with diastolic blood pressure and total peripheral resistance in the obese Caucasian group, but not in the obese African group. This may indicate that leptin predominantly exerts pathological influences on obese African women, as determined previously in Chapter 4. / Thesis (Ph.D. (Physiology))--North-West University, Potchefstroom Campus, 2005.

Diss. (sammanfattning) Göteborg : Göteborgs universitet , 2007. / Härtill 5 uppsatser.

Charité - Universiẗat, Diss--Berlin, 2005.

Freie Universiẗat, Diss., 2007--Berlin.

Zugl.: Giessen, Univ., Diss., 2007

Os relógios biológicos são fundamentais para a sincronização do comportamento dos organismos a mudanças no fotoperíodo. Todas as alterações rítmicas são determinantes para a sobrevivência da espécie uma vez que elas prevêem que os ajustes internos coincidam com a fase mais propícia do ciclo ambiental, permitindo aos organismos a capacidade de sincronizar esses eventos internos com os ciclos ambientais. Muitos desses ritmos biológicos são claramente associados ao ciclo claro-escuro, sendo este ciclo de grande importância para as espécies que possuem algum tipo de pigmento fotossensível. Os melanóforos de Xenopus laevis são fotossensíveis, respondendo à luz com dispersão dos grânulos de melanina, devido à presença de duas melanopsinas, Opn4x e Opn4m. As células pigmentares dos vertebrados heterotérmicos respondem com migração pigmentar a uma variedade de agentes, incluindo as endotelinas. Em peixes teleósteos, ETs induzem a agregação pigmentar em melanóforos, enquanto que em anfíbios, ET-3 induz a dispersão de grânulos de pigmentos em melanóforos de Xenopus laevis e de Rana catesbeiana, através da ativação de receptores ETc. Propusemos determinar o padrão temporal de expressão dos genes das melanopsinas e do receptor ETc em melanóforos dérmicos de X. laevis em cultura, bem como os efeitos temporais e dose- dependentes da endotelina sobre essa expressão. Demonstramos, através de ensaios de PCR quantitativo, que o tratamento de 12C:12E , somado a uma troca de meio, assim como o de endotelina-3 10-9 e 10-8M em escuro constante, foi capaz de sincronizar a expressão de Opn4x e Opn4m. Entretanto, o receptor ETc parece não ser sincronizado pelo ciclo claro-escuro, ou pelo tratamento hormonal. Dependendo da dose utilizada e do ZT analisado, ET-3 pode promover um aumento ou inibição da expressão gênica de Opn4x, Opn4m e ETc, indicando uma modulação de forma dose-dependente. Além disso, pode atuar como um agente sincronizador da expressão dos transcritos das melanopsinas. / The biological clocks are critical for synchronizing the behavior of organisms to changes in photoperiod. All rhythmic changes are crucial to the survival of the species since they provide for internal adjustments to coincide with the phase of the cycle most favorable. Many of these biological rhythms are clearly associated with the light-dark cycle, of major importance for species that have some type of photosensitive pigment. Melanophores of Xenopus laevis are photosensitive, responding to light with dispersion of melanin granules, due to the presence of two melanopsins, Opn4x and Opn4m. The pigment cells of ectothermic vertebrates respond with pigment migration to a variety of agents including the endothelins. In teleost fish, ETs induce pigment aggregation in melanophores, whereas in amphibians, ET-3 induces the dispersion of pigment granules in melanophores of Xenopus laevis and Rana catesbeiana, by activation of ETc. We proposed to determine the temporal pattern of gene expression of the ETc receptor and melanopsins in dermal melanophores of X. laevis in culture as well as the effects of endothelin-3 on the temporal expression of the 3 genes. Using quantitative PCR, we demonstrated that 12L: 12D regimen, combined with medium changes, as well as the treatment with 10-9 and 10-8M endothelin-3, was able to synchronize the expression of Opn4x and Opn4m. However, ETc receptor seems not to be synchronized by light-dark cycle, or hormone treatment. Depending on the dose and the ZT, ET-3 may promote an increase or inhibition of gene expression of Opn4x, Opn4m and ETc, indicating a dose-dependent modulatory effect. In addition, endothelin-3 may also act as a synchronizing agent of the melanopsins transcripts.

Abstract The heart responds to increased hemodynamic stress by increased cardiac myocyte size, enhanced protein synthesis and altered gene expression. Regulation of hypertrophic adaptation involves a number of neural and hormonal factors, which act on the cardiovascular system. The aim of the present study was to elucidate the regulation of gene expression of natriuretic peptides and adrenomedullin (AM) in cardiac overload in vivo. Furthermore, the interactions of AM and angiotensin II (Ang II) in cardiac function and development of left ventricular hypertrophy were studied both in vivo and in vitro. The effects of cardiac hypertrophy on the regulation of natriuretic peptides (atrial natriuretic peptide, ANP and B-type natriuretic peptide, BNP) and AM gene expression were studied during pressure overload in the hearts of two hypertensive rat strains, angiotensinogen-renin transgenic rats and spontaneously hypertensive rats as well as their normotensive control strains. Increased workload resulted in rapid upregulation of both BNP and AM gene expression in all rat strains; the response of AM was, however, augmented in hypertensive rats. Direct left ventricular wall stretch induced AM gene expression in isolated, perfused rat hearts, whereas stretching of cultured cardiac myocytes downregulated AM mRNA levels. In cultured cardiac cells exposed to Ang II, endothelin-1 or the α-agonist phenylephrine, Ang II-induced myocyte hypertrophy was selectively inhibited by AM. In vivo, AM interacted with Ang II in circulation by attenuating the hypertensive effects of Ang II, and in the heart by augmenting the Ang II-induced improvement in cardiac systolic function. However, AM had no direct modulatory effects on Ang II-induced left ventricular hypertrophy. These results show that cardiac wall stretch is a major stimulus for the early induction of AM gene expression in both normal and hypertrophied ventricle, and the response in hypertrophied myocardium is augmented. Furthermore, cardiac non-muscle cells may be involved in mediating effects of direct stretch. In vitro, AM acts as a selective inhibitor of Ang II-induced myocyte hypertrophy, suggesting a cardioprotective role for AM to counteract the local renin-angiotensin system and Ang II in cardiac hypertrophy and heart failure. Circulating AM appears to act mainly as a regulator of vascular tone and cardiac function.

Abstract The natriuretic peptides are a family of hormones secreted by the heart. They play a fundamental role in salt and water balance and blood pressure regulation. Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are the known members of the mammalian natriuretic peptide family. A major stimulus for the secretion of cardiac natriuretic peptides is myocyte stretch. Therefore, the secretion of natriuretic peptides is increased in response to elevated blood volume. Natriuretic peptide production and release is also affected by several other factors, such as endothelin-1 (ET-1), acting in paracrine fashion. The aim of this study was to elucidate factors regulating the novel cardiac peptide hormone, salmon cardiac peptide (sCP), belonging to the family of natriuretic peptides. The role of mechanical load, temperature and ET-1 in sCP secretion and production was studied using in vitro (isolated perfused ventricle preparation) and in vivo methods. Comparisons between the natriuretic peptide systems in fish and mammals were done to clarify functional evolution of this hormone family. Salmon (Salmo salar) was selected as a model, since it has an outstanding adaptability to wide variations in environmental salinity and has developed defence mechanisms against volume or salt load. The results showed that salmon ventricle stores large amounts of the prohormone of sCP, whereas the secreted form is the mature 29-amino acid form. The N-terminal fragment of pro-sCP is co-secreted with sCP in equimolar amounts. sCP is released rapidly in response to appropriate stimulus, whereas induction of its gene expression is slower. Mechanical load is an important regulator of sCP secretion. Temperature also plays a major role in regulating sCP plasma concentration by affecting its elimination from circulation. Additionally, ET-1 is a potent secretagogue of the sCP system and an inotropic agent in salmon heart. Furthermore, the present results reveal remarkable synergism between the cardiac effects of ET-1 and β-adrenergic stimulation. In conclusion, the sCP system in salmon ventricle largely resembles the ANP system in mammalian atrium, while also having specific characteristics, such as a regulated ventricular natriuretic peptide secretion pathway. Therefore, the sCP system offers a unique model for studying mechanisms of natriuretic peptide biology.