No description available.

The principal substrate used by the normal adult human heart is free fatty acids, the remainder being, predominantly,carbohydrate. During failure, the heart becomes less reliant on fatty acid metabolism, possibly as a result of tissue hypoxia. Therefore, understanding hypoxic adaptation may explain the metabolic changes that occur during the development of heart failure.As peroxisome proliferator activated receptor alpha (PPARα) modulates cardiac fatty acid metabolism, the work in this thesis focused on the role of PPARα in cardiac metabolic adaptation to chronic hypoxia. It was found that isolated hearts from chronically hypoxic (11% O₂ for 3 weeks)mice were more glycolytic, had reduced PPARα expression and decreased fatty acid metabolism,but had normal function, determined using in vivocine-MRI. ³¹P MRS of isolated perfused mouse hearts showed a drop in PCr with hypoxia, but ΔG_ATP was not altered, indicating that metabolic reprogramming was sufficient to maintain ATP production and contractile function. Increased or decreasedPPARα expression, using a high fat diet or PPARα null mice, respectively, prevented metabolic adaptation to hypoxia and caused cardiac dysfunction. Hypoxia with high fat feeding was particularly deleterious, reducing ejection fraction by 9%,possibly due to increased mitochondrial uncoupling. PPARβ/δ and γ were not involved in the adaptation to hypoxia, and none were modified by PPARα stimulation or ablation. Cardiac VEGF and PDK1, prominent hypoxia-inducible factor (HIF) targets, were increased by hypoxia, indicating that HIF may have been involved in metabolic adaption. However, high fat feeding prevented VEGF accumulation during hypoxia, suggesting that impaired HIF signalling may have contributed to the maladaptive response to hypoxia. In order to determine the relationship between HIF and PPARα, HIFwas stabilised pharmacologically using FG2216/BIC in HL-1 cardiomyocytes, to show decreased PPARα expression and caused similar metabolic changes to those seen in the in vivo hypoxic heart. In conclusion, this study demonstrated that HIF downregulation of PPARα is crucial for metabolic adaptation and maintenance of cardiac function during chronic hypoxia. Similar metabolic changes that occur in end-stage heart failure may also be a response to increasing hypoxia.

No description available.

The incidence of cardiovascular diseases including cardiac hypertrophy and failure in pre-menopausal women is lower compared to age-matched men but the risk of heart disease increases substantially after the onset of menopause. It has been postulated that female sex hormones play an important role in cardiovascular health in pre-menopausal women. In animal studies including spontaneously hypertensive (SHR) rats, the development of cardiac hypertrophy is attenuated by 17β-estradiol treatment. Cardiac energy metabolism is crucial for normal function of the heart. In cardiac hypertrophy and heart failure, the myocardium undergoes a metabolic shift from fatty acid as primary cardiac energy source to glucose, which re-introduces the fetal type of metabolism that representing the glucose as a major source of energy. Many studies have reported that the disruption of the balance between glucose and fatty acid metabolism plays an important role in cardiac pathologies including hypertrophy, heart failure, diabetes, dilative cardiomyopathy and myocardial infarction. Glucose enters cardiomyocytes via GLUT1 and GLUT4 glucose transporters and GLUT4 is the major glucose transporter which is insulin-dependent. Cardiac-selective GLUT4 deficiency leads to cardiac hypertrophy. This shows that the decrease in cardiac glucose uptake may play a direct role in the pathogenesis of cardiac hypertrophy. Estrogens modulate glucose homeostasis in the liver and the skeletal muscle. But it is not known whether estrogens affect also cardiac glucose uptake which could provide another mechanism to explain the prevention of cardiac hypertrophy by female sex hormones. In the present study, SHR Rats were ovariectomized (OVX), not ovariectomized (sham) or ovariectomized and treated with subcutaneous 17β-estradiol. After 6 weeks of treatment, body weight, the serum levels of estrogen, insulin, intra-peritoneal glucose tolerance test (IP-GTT), myocardial glucose uptake by FDG-PET (2-(18F)-fluoro-deoxyglucose (18FDG) and Positron Emission Tomography), cardiac glucose transporter expression and localization and cardiac hexokinase activity were analyzed. As results of this study, PET analysis of female SHR revealed decreased cardiac glucose uptake in OVX animals compared to intact that was normalized by estrogen supplementation. Interestingly, there was no change in global glucose tolerance among the treatment groups. Serum insulin levels and cardiac hexokinase activity were elevated by E2 substitution. The protein content of cardiac glucose transporters GLUT-4 and GLUT-1, and their translocation as determined by fractionation studies and immuno-staining did not show any significant change by ovariectomy and estrogen replacement. Also levels of insulin receptor substrate-1 (IRS-1) and its tyrosine phosphorylation, which is required for activation and translocation of GLUT4, was un-affected in all groups of SHR. Cardiac gene expression analysis in SHR heart showed that ei4Ebp1 and Frap1 genes which are involved in the mTOR signaling pathway, were differentially expressed upon estrogen treatment. These genes are known to be activated in presence of glucose in the heart. As a conclusion of this study, reduced myocardial FDG uptake in ovariectomized spontaneously hypertensive rat is normalized by 17β-estradiol treatment. Increased myocardial hexokinase appears as a potential mechanism to explain increased myocardial glucose uptake by 17β-estradiol. Increased cardiac glucose uptake in response to 17β-estradiol in ovariectomized SHR may provide a novel mechanism to explain the reduction of cardiac hypertrophy in E2 treated SHR. Therefore, 17β-estradiol improves cardiac glucose utilization in ovariectomized SHR which may give rise to possible mechanism for its protective effects against cardiac hypertrophy. / Erkrankungen des kardiovaskulären Systems, wie beispielsweise Herzhypertrophie oder Herzinsuffizienz treten bei Frauen vor der Menopause im Vergleich zu gleichaltrigen Männern seltener auf. Das Risiko für eine solche kardiovaskuläre Erkrankung steigt jedoch drastisch mit dem Beginn der Menopause an. Aus diesem Grund wird angenommen, dass weibliche Geschlechtshormone kardioprotektive Wirkungen besitzen. Tierstudien an spontan hypertensiven Ratten (SHR) haben belegt, dass eine Herzhypertrophie durch die Behandlung der Tiere mit 17β-Estradiol abgemildert werden kann. Entscheidend für die Funktion des Myokards ist sein Energiemetabolimus, der sich im Verlauf einer Hypertrophie oder Herzinsuffizienz vom primären Fettsäurestoffwechsel auf Glucosemetabolismus umschaltet. Diese Situation entspricht der des fetalen Herzens. Viele Studien haben belegt, dass eine Störung der Balance zwischen Glucose- und Fettsäurestoffwechsel oftmals ein erstes Anzeichen für einen pathologischen Zustand des Herzens, wie z.B. Hypertrophie, Herzinsuffizienz, Diabetes, dilative Kardiomyopathie und Myokardinfarkt ist. Im gesunden Herzen gelangt Glucose über die zwei Glucosetransporter GLUT1 und GLUT4 in die Zellen des Myokards, wobei der insulinabhängige Glut4-Transporter der Hauptglucosetransporter ist. Eine GLUT4-Defizienz führt daher ebenfalls zu einer Herzhypertrophie was wiederum zeigt, dass eine verminderte Glucoseaufnahme im direkten Zusammenhang mit pathologischen Zuständen des Herzens steht. Bisherige Studien haben gezeigt, dass Östrogen an der Glucosehomöostase in Leber und Skelettmuskeln beteiligt ist. Jedoch ist wenig darüber bekannt, ob Östrogen ebenfalls in die kardiale Glucosehomöostase eingreift und inwiefern die kardioprotektive Wirkung des Östrogens in diesem Zusammenhang steht.In der vorliegenden Arbeit wurden weibliche SH-Ratten ovariektomiert (OVX), nicht ovariektomiert (sham) oder ovariektomiert und zusätzlich subkutan mit 17β-Estradiol behandelt. Nach einer Behandlungszeit von 6 Wochen wurden dann das Körpergewicht, die Serumspiegel von Östrogen, Insulin und IPGTT bestimmt, und die Glucoseaufnahme des Myokards mittels FDG-PET analysiert. Zusätzlich wurden Expression und zelluläre Lokalisation der kardialen Glucosetransporter sowie die kardiale Hexokinaseaktivität untersucht. Es konnte gezeigt werden, dass sich eine verminderte Glucoseaufnahme des Herzens bei ovariektomierten Tieren durch Östrogen-Supplementation normalisieren lässt. Eine Abweichung bezüglich der Glucosetoleranz der einzelnen Gruppen konnte nicht beobachtet werden. Jedoch konnte ein erhöhter Insulinspiegel des Serums und eine erhöhte kardiale Aktivität des Enzyms Hexokinase durch die Behandlung mit Östrogen bei den ovariektomierten Tieren beschrieben werden. Durch Fraktionierungen und immunhistologische Untersuchungen konnte kein signifikanter Unterschied in Bezug auf die Menge sowie die Translokation der Glucosetransporter GLUT1 und GLUT4 im Myokard zwischen den einzelnen Behandlungen der Tiere beschrieben werden. Ferner konnte zwischen den einzelnen Tiergruppen auch kein Unterschied zwischen dem Insulin Rezeptor Substrat-1 (IRS-1) und seiner Tyrosin-phosphorylierten Form festgestellt werden, die für die Aktivierung und Translokation des GLUT4 benötigt werden. Analysen der Genexpression in den Herzen der SH-Ratten konnten allerdings zeigen, dass die Gene ei4Ebp1 und Frap1, die im mTOR Signalweg involviert sind, bei den Östrogen-supplementierten Tieren ein abweichendes Expressionsmuster aufweisen. Über diese Gene ist bekannt, dass sie in der Gegenwart von Glucose im Herzen aktiviert werden und bei der Entstehung einer Herzhypertrophie mitwirken. Basierend auf den PET-Analysen und der Hexokinaseaktivität lässt sich als Resultat dieser Arbeit aussagen, dass Östrogen die kardiale Glucoseaufnahme in SH-Ratten fördert. Diese Ergebnisse könnten einen Hinweis auf einen noch unbekannten Mechanismus geben, um die protektive Wirkung des Östrogens im Hinblick auf die Herzhypertrophie zu erklären. Hinsichtlich der Tatsache, dass keine Veränderungen in der Translokation der GLUT4-Transporter in der Plasmamembran bei den einzelnen Behandlungen der Tiere zu verzeichnen sind, jedoch Veränderungen der Glucoseaufnahme durch die PET-Analysen dargestellt werden konnten, besteht jedoch noch Erklärungsbedarf. Es liegen diverse Studien vor, die diesen Unterschied damit erklären könnten, dass der GLUT4-Transporter in einer inaktiven Form in der Plasmamembran vorliegt bis die Glucoseaufnahme durch den GLUT4-Transporter mittels der Insulin Signaltransduktionskaskade reguliert wird.

Hypoxia inducible factor-1α (HIF-1α) plays a critical role in the oxygen homeostasis of all metazoans. HIF-1α is a master transcriptional regulator which coordinates the adaptive response to low oxygen tension. Through activation of a plethora of downstream target genes, HIF-1α facilitates oxygenation by promoting angiogenesis and blood vessel dilation, in addition to modulating metabolic pathways to inhibit oxidative phosphorylation and promote glycolytic energy production. Given the critical roles of hypoxia, insufficient blood supply and perturbed energetics in the pathogenesis of cardiovascular disorders, notably ischaemic heart disease, therapeutic modulation of HIF-1α is of significant clinical interest. Previous studies have demonstrated an acute cardioprotective role for both endogenous and supraphysiological HIF-1α signalling in the context of myocardial ischaemia. In contrast, chronic supraphysiological HIF-1α activation in the unstressed heart has been shown to induce cardiac dysfunction. To address the effect of chronic endogenous HIF-1α activation post-myocardial infarction (MI), the present work employed a murine coronary artery ligation (CAL) model in conjunction with temporally-inducible, cardiac-specific deletion of Hif-1α. While CAL surgery successfully modelled myocardial infarction – eliciting substantial adverse cardiac remodelling and contractile dysfunction – there was no evidence of chronic HIF-1α activation by CAL in HIF knockout or control left ventricular samples. In keeping with this, chronic ablation of Hif-1α (from 2 weeks post-CAL) had no discernible additional effect upon cardiac function. Overall, these findings do not support a potential therapeutic role for inhibition of HIF-1α signalling in the chronic phase post-MI. The fundamental tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) converts fumarate to malate. FH deficiency is associated with smooth muscle and kidney tumours which exhibit normoxic HIF signalling due to fumarate accumulation. To investigate the potential for fumarate accumulation to elicit protective HIF signalling, a cardiac-specific Fh1 null mouse was developed through Cre-loxP recombination. Strikingly, despite interruption of the TCA cycle in a highly metabolically demanding organ, cardiac Fh1 null mice were viable, fertile and survived into adulthood, demonstrating the remarkable metabolic plasticity of the heart. However, by 3-4 months Fh1 null mice develop a lethal cardiomyopathy characterised by cardiac hypertrophy, ventricular dilatation and contractile dysfunction. Despite lack of a pseudohypoxic response, Fh1 null hearts did exhibit another phenomenon observed in FH-deficient cancers and also attributed to fumarate accumulation – activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) antioxidant pathway. Heterozygous, but not homozygous, somatic deletion of Nrf2 extended the life expectancy of cardiac Fh1 null mice. Exploration of redox status revealed a more reductive environment in Fh1 null hearts than controls. As a corollary, inhibition of the rate limiting enzyme of the pentose phosphate pathway – a major source of cellular reducing equivalents – with dehydroepiandrosterone conferred striking amelioration of the Fh1 null cardiomyopathy, suggesting a possible pathogenic role for reductive stress. While loss of mitochondrial Fh1 activity and subsequent TCA cycle dysfunction likely contribute to the Fh1 null phenotype, the importance of cytosolic FH was unclear. To clarify this, FH was expressed specifically in the cytosol in vivo. This was sufficient to substantially rescue the Fh1 null cardiomyopathy, supporting a role for cytosolic FH disruption in its pathogenesis. Taken together, these findings highlight the potential for reductive stress to contribute to cardiac dysfunction and suggest a function for cytosolic FH in cardiac metabolic homeostasis.

O coração é um órgão notável por sua flexibilidade estrutural e metabólica em resposta a variações de demanda. Na dormência sazonal, a interrupção da alimentação, associada à inatividade física e à acentuada redução da frequência cardíaca, ocasiona uma inibição da demanda sobre a função do órgão e, provavelmente, uma reorganização estrutural e metabólica do tecido cardíaco. Estes aspectos foram investigados ao longo do ciclo anual de atividades em lagartos teiú Tupinambis merianae, com o objetivo de examinar as alterações de capacidade funcional cardíaca dadas por ajustes da massa, estrutura e composição do tecido, por regulação do fluxo de substratos energéticos em vias de produção de energia e por mudanças da composição de ácidos graxos dos fosfolipídios das membranas. Grupos de animais jovens foram mortos em diferentes fases do primeiro ciclo anual e após 20 dias de jejum na fase ativa e o ventrículo cardíaco foi removido e pesado. Um fragmento da parede ventricular foi retirado, transferido para fixador e utilizado posteriormente para a confecção de cortes histológicos de 10 μm de espessura que foram analisados utilizando-se método estereológico. O restante do tecido ventricular foi congelado em N2 líquido e conservado em freezer -80 ºC. Os teores de água, proteína total e solúvel e lipídio total foram medidos por meio de ensaios padrão; as atividades máximas de enzimas foram medidas por espectrofotometria em condições saturantes de substratos e cofatores; e o perfil de ácidos graxos dos lipídios neutros e polares foi determinado por cromatografia gasosa. No início do outono, a massa ventricular relativa é 0,16% e aumenta 31% até o final desta fase, quando o miocárdio esponjoso possui aspecto denso e poucos espaços lacunares que ocupam cerca de 8% da área total do corte. Este arranjo é mantido na dormência, quando a massa ventricular relativa aumenta 29% em relação ao final do outono, e no início do despertar, quando a massa ventricular relativa diminui para valores semelhantes aos do final do outono. Após a retomada da alimentação, a massa ventricular relativa volta a exibir uma porcentagem comparável a da dormência, juntamente com um pequeno aumento da área de lacunas no miocárdio esponjoso. Na primavera, a massa ventricular relativa é de 0,24% e o miocárdio esponjoso possui aspecto extremamente reticulado, com 29% da área total do corte ocupada por espaços lacunares. Animais ativos submetidos a jejum apresentam redução de 19% da massa ventricular relativa em relação a animais alimentados. A densidade numérica de cardiomiócitos na camada esponjosa é 37% menor na dormência em relação à atividade de primavera, resultando em um volume calculado de um cardiomiócito nesta fase 52% maior em relação à atividade de primavera. A análise do teor de água, proteínas totais e solúveis não indica variação ao longo do ciclo anual, com exceção de uma tendência ao aumento do teor de água na dormência e de uma tendência à redução do teor de proteínas solúveis após o despertar e ingestão de água e no grupo de animais ativos submetidos ao jejum. Na atividade de outono e dormência de inverno a concentração de proteínas miofibrilares é reduzida em relação à atividade de primavera e aumenta no início do despertar após a ingestão de água. A concentração de lipídios totais é menor na dormência e despertar em relação à atividade de outono e no grupo de animais submetidos a jejum em relação a animais alimentados. As enzimas glicolíticas PK e LDH não variam ao longo do ciclo anual, enquanto a CS, indicadora da capacidade aeróbia, exibe forte tendência ao aumento na dormência, e a HOAD, enzima da β-oxidação lipídica, encontra-se inibida na dormência e no despertar em relação ao outono. Em contraste, com exceção da LDH que também não varia, a PK e a CS diminuem, enquanto que a HOAD é mantida constante após jejum na fase ativa. As variações do perfil de ácidos graxos da fração lipídica neutra sugerem que ácidos graxos insaturados são preferencialmente mobilizados das reservas do miocárdio durante a dormência e início do despertar, enquanto que no jejum durante a fase ativa as diferentes classes de ácidos graxos são equitativamente mobilizadas. A composição de ácidos graxos da fração lipídica polar exibe uma notável constância ao longo do ciclo anual, sugerindo que os ajustes à dormência sazonal não afetam de modo abrangente os fosfolipídios do tecido cardíaco e, portanto, não sugerem um papel preponderante de mudanças da composição lipídica das membranas na regulação metabólica sazonal nos teiús. Além disso, o contraste em relação às alterações observadas em mamíferos hibernantes sugere que, nestes, os ajustes seriam mais relacionados com a adaptação às baixas temperaturas corpóreas típicas da hibernação. A análise de regressão indica uma variação do conteúdo dos ácidos graxos C18:1n-9, C22:5n-6 e C22:6n-3 em função da massa corpórea dos jovens teiús e as mudanças do padrão alométrico sugerem uma relação entre o conteúdo destes ácidos graxos e as diferenças de taxa metabólica em animais de diferentes massas corpóreas, observadas em determinadas fases do ciclo anual de atividades e após o jejum durante a fase ativa. / The structural and metabolic flexibility of cardiac response to a variable physiological demand is notable. During seasonal dormancy, interruption of feeding together with inactivity and reduced heart beating, cause a large decrease of demand which probably brings about structural and metabolic heart tissue reorganization. These aspects were studied during the annual cycle in young tegu lizards Tupinambis merianae to investigate the hypothesis of seasonal changes of the heart capacities given by adjustments of tissue mass, structure and composition, by regulation of flux of substrates in the pathways of energy production, and by changes in the composition of fatty acids of tissue membranes. Groups of animals were killed in selected phases during the first year cycle of young tegus and after a 20 days fasting period during spring activity. Heart ventricle was removed and weighed and a tissue sample was collected and transfered to fixative solution, being used to obtain tissue slices of 10μm width for histological analysis with stereological tools. The remaining tissue was cut and split into aliquots, frozen in liquid N2 and stored at -80ºC. Later, the aliquots were used to assess the content of water, total and soluble proteins, and total lipids, by standard assays, the maximum activity of enzymes by spectrophotometry, and neutral and polar fatty acids profiles by gas chromatography. In early fall, the relative mass ventricle is 0.16%, and 31% increased in late fall, when the spongy myocardium appears dense and with few lacunar spaces which area corresponds to 8% of slice total área. During dormancy, the ventricle mass increases further 29%, decreasing to values of late fall during early arousal. After food intake, mass ventricle is again increased together with a small increase of the lacunar spaces, which appear highly expanded later in spring (29% of the total area), when tissue mass is 0,24% increased in relation to early fall. Unlike dormancy, fasting during spring caused a decrease of 19% of the ventricle mass. The cardiomyocytes density in the spongy layer is 37% decreased during dormancy while estimated cell volume is 52% increased, in relation to spring activity. There was no seasonal changes in the content of water and proteins in the groups analysed, except to a tendency to increase in the water content during dormancy, and to decrease in the soluble proteins in early arousal and in fasted animals. Myofibrillar protein is lower during fall and dormancy in relation to spring, increasing soon in early arousal after water intake. Total lipids decrease in the tissue during dormancy in relation to late fall by similar proportion than after fasting during spring. The glycolytic enzymes PK e LDH are unchanged during the year cycle, whereas the mitochondrial CS shows a tendency to increase, and HOAD, a β-oxidation enzyme, is decreased during dormancy and early arousal, in relation to fall. Unlike, PK and CS are decreased, while HOAD is unchanged after a period of fasting during spring. Fatty acids (FA) profiles of neutral lipids suggest that unsaturated FA are preferentially mobilized during dormancy and arousal, whereas all FA would be equally used during spring fasting. FA of polar lipids are remarkably constant during the year, suggesting that membrane FA in the heart tissue are not generally affected by season, and thus, results do not support a predominant role played by compositional changes of membranes in metabolic depression in the tegu. In addition, the otherwise distinct findings with hibernating mammals suggest that changes of FA composition in these animals would be an adaptation to the low body temperature of torpor, rather than mechanism of metabolic inhibition. Regression analysis indicate significant relationships of C18:1n-9, C22:5n-6, and C22:6n-3 contents as a function of body mass in young tegus, and changes in the allometric patterns are consistent with a putative relationship between these FA levels and the scaling of mass specific metabolic rates of young tegus during the year cycle.

Cyclic guanosine 3', 5'-monophosphate (cGMP) has recently emerged as an endogenous regulator for controlling or reversing cardiac hypertrophy. Increased protein accumulation is a key feature of cardiac hypertrophy; thus, our study investigates the effects of a cGMP analog on protein accumulation in primary culture of adult rat cardiomyocytes and dissects out the mechanisms involved. We confirmed that a cGMP analog, 8-bromo-cGMP, inhibits phenylephrine (PE)-increased accumulation of newly synthesized proteins in cultured adult rat ventricular cardiomyocytes. Firstly, we have obtained data showing that 8-bromo-cGMP does not inhibit phosphorylation of S6K1 by PE during short time treatment (10 min to 2 h), but blocks phosphorylation of S6K1 by PE at 6 h; moreover this blocking effect is completely abolished by phosphatase inhibitor Tautomycin. Then, we have demonstrated that PE and cGMP induce sustained and transient increased phosphorylation of ERK, respectively. Moreover, cGMP inhibits PE-induced phosphorylation of ERK during long term treatment (3 and 6h). We have also shown that 8-bromo-cGMP inhibits ROS generation induced by PE. Other effects of PE that could be related to hypertrophy (i.e. increased concentration of upstream binding factor mRNA and decreased concentration of the mRNAs of Atrogin and muscle specific RING finger) were not abolished by 8-bromo-cGMP. We conclude that cGMP analog blocks protein accumulation by inhibiting the sustained phosphorylation of S6K1 via the activation of phosphatases.

Heart failure represents an important cause of death in Western Countries. The pathophysiology of heart failure is mainly associated with abnormalities in intracellular calcium control. We previously showed that Egr-1 negatively regulates expression of sodium-calcium exchanger (NCX) in vivo and in vitro. Here we tested the hypothesis that Egr-1 regulates expression of calcium storage proteins in the sarco-endoplasmic reticulum (SER), calsequestrin (CSQ) and/or ER, calreticulin (CRT) directly or indirectly via Egr-1:NFAT (nuclear factor of activated T-cells) formation. Secondarily, we hypothesized that this will reduce calcium mobilization. We found that undifferentiated 1293F cells, overexpressing Egr-1, have reduced CSQ compared to control H9c2 cells. We demonstrated that Egr-1 negatively regulates CSQ but not CRT expression. The Egr-1 mediated decrease in CSQ is linked to decreased calcium availability. Repression is by a novel NAB-independent (NGFI-A binding protein) activity localized to a.a. region 1-307. We conclude that Egr-1-mediated reductions in calcium storage protein expression alter calcium availability for cardiac contraction/relaxation.

O coração é um órgão notável por sua flexibilidade estrutural e metabólica em resposta a variações de demanda. Na dormência sazonal, a interrupção da alimentação, associada à inatividade física e à acentuada redução da frequência cardíaca, ocasiona uma inibição da demanda sobre a função do órgão e, provavelmente, uma reorganização estrutural e metabólica do tecido cardíaco. Estes aspectos foram investigados ao longo do ciclo anual de atividades em lagartos teiú Tupinambis merianae, com o objetivo de examinar as alterações de capacidade funcional cardíaca dadas por ajustes da massa, estrutura e composição do tecido, por regulação do fluxo de substratos energéticos em vias de produção de energia e por mudanças da composição de ácidos graxos dos fosfolipídios das membranas. Grupos de animais jovens foram mortos em diferentes fases do primeiro ciclo anual e após 20 dias de jejum na fase ativa e o ventrículo cardíaco foi removido e pesado. Um fragmento da parede ventricular foi retirado, transferido para fixador e utilizado posteriormente para a confecção de cortes histológicos de 10 μm de espessura que foram analisados utilizando-se método estereológico. O restante do tecido ventricular foi congelado em N2 líquido e conservado em freezer -80 ºC. Os teores de água, proteína total e solúvel e lipídio total foram medidos por meio de ensaios padrão; as atividades máximas de enzimas foram medidas por espectrofotometria em condições saturantes de substratos e cofatores; e o perfil de ácidos graxos dos lipídios neutros e polares foi determinado por cromatografia gasosa. No início do outono, a massa ventricular relativa é 0,16% e aumenta 31% até o final desta fase, quando o miocárdio esponjoso possui aspecto denso e poucos espaços lacunares que ocupam cerca de 8% da área total do corte. Este arranjo é mantido na dormência, quando a massa ventricular relativa aumenta 29% em relação ao final do outono, e no início do despertar, quando a massa ventricular relativa diminui para valores semelhantes aos do final do outono. Após a retomada da alimentação, a massa ventricular relativa volta a exibir uma porcentagem comparável a da dormência, juntamente com um pequeno aumento da área de lacunas no miocárdio esponjoso. Na primavera, a massa ventricular relativa é de 0,24% e o miocárdio esponjoso possui aspecto extremamente reticulado, com 29% da área total do corte ocupada por espaços lacunares. Animais ativos submetidos a jejum apresentam redução de 19% da massa ventricular relativa em relação a animais alimentados. A densidade numérica de cardiomiócitos na camada esponjosa é 37% menor na dormência em relação à atividade de primavera, resultando em um volume calculado de um cardiomiócito nesta fase 52% maior em relação à atividade de primavera. A análise do teor de água, proteínas totais e solúveis não indica variação ao longo do ciclo anual, com exceção de uma tendência ao aumento do teor de água na dormência e de uma tendência à redução do teor de proteínas solúveis após o despertar e ingestão de água e no grupo de animais ativos submetidos ao jejum. Na atividade de outono e dormência de inverno a concentração de proteínas miofibrilares é reduzida em relação à atividade de primavera e aumenta no início do despertar após a ingestão de água. A concentração de lipídios totais é menor na dormência e despertar em relação à atividade de outono e no grupo de animais submetidos a jejum em relação a animais alimentados. As enzimas glicolíticas PK e LDH não variam ao longo do ciclo anual, enquanto a CS, indicadora da capacidade aeróbia, exibe forte tendência ao aumento na dormência, e a HOAD, enzima da β-oxidação lipídica, encontra-se inibida na dormência e no despertar em relação ao outono. Em contraste, com exceção da LDH que também não varia, a PK e a CS diminuem, enquanto que a HOAD é mantida constante após jejum na fase ativa. As variações do perfil de ácidos graxos da fração lipídica neutra sugerem que ácidos graxos insaturados são preferencialmente mobilizados das reservas do miocárdio durante a dormência e início do despertar, enquanto que no jejum durante a fase ativa as diferentes classes de ácidos graxos são equitativamente mobilizadas. A composição de ácidos graxos da fração lipídica polar exibe uma notável constância ao longo do ciclo anual, sugerindo que os ajustes à dormência sazonal não afetam de modo abrangente os fosfolipídios do tecido cardíaco e, portanto, não sugerem um papel preponderante de mudanças da composição lipídica das membranas na regulação metabólica sazonal nos teiús. Além disso, o contraste em relação às alterações observadas em mamíferos hibernantes sugere que, nestes, os ajustes seriam mais relacionados com a adaptação às baixas temperaturas corpóreas típicas da hibernação. A análise de regressão indica uma variação do conteúdo dos ácidos graxos C18:1n-9, C22:5n-6 e C22:6n-3 em função da massa corpórea dos jovens teiús e as mudanças do padrão alométrico sugerem uma relação entre o conteúdo destes ácidos graxos e as diferenças de taxa metabólica em animais de diferentes massas corpóreas, observadas em determinadas fases do ciclo anual de atividades e após o jejum durante a fase ativa. / The structural and metabolic flexibility of cardiac response to a variable physiological demand is notable. During seasonal dormancy, interruption of feeding together with inactivity and reduced heart beating, cause a large decrease of demand which probably brings about structural and metabolic heart tissue reorganization. These aspects were studied during the annual cycle in young tegu lizards Tupinambis merianae to investigate the hypothesis of seasonal changes of the heart capacities given by adjustments of tissue mass, structure and composition, by regulation of flux of substrates in the pathways of energy production, and by changes in the composition of fatty acids of tissue membranes. Groups of animals were killed in selected phases during the first year cycle of young tegus and after a 20 days fasting period during spring activity. Heart ventricle was removed and weighed and a tissue sample was collected and transfered to fixative solution, being used to obtain tissue slices of 10μm width for histological analysis with stereological tools. The remaining tissue was cut and split into aliquots, frozen in liquid N2 and stored at -80ºC. Later, the aliquots were used to assess the content of water, total and soluble proteins, and total lipids, by standard assays, the maximum activity of enzymes by spectrophotometry, and neutral and polar fatty acids profiles by gas chromatography. In early fall, the relative mass ventricle is 0.16%, and 31% increased in late fall, when the spongy myocardium appears dense and with few lacunar spaces which area corresponds to 8% of slice total área. During dormancy, the ventricle mass increases further 29%, decreasing to values of late fall during early arousal. After food intake, mass ventricle is again increased together with a small increase of the lacunar spaces, which appear highly expanded later in spring (29% of the total area), when tissue mass is 0,24% increased in relation to early fall. Unlike dormancy, fasting during spring caused a decrease of 19% of the ventricle mass. The cardiomyocytes density in the spongy layer is 37% decreased during dormancy while estimated cell volume is 52% increased, in relation to spring activity. There was no seasonal changes in the content of water and proteins in the groups analysed, except to a tendency to increase in the water content during dormancy, and to decrease in the soluble proteins in early arousal and in fasted animals. Myofibrillar protein is lower during fall and dormancy in relation to spring, increasing soon in early arousal after water intake. Total lipids decrease in the tissue during dormancy in relation to late fall by similar proportion than after fasting during spring. The glycolytic enzymes PK e LDH are unchanged during the year cycle, whereas the mitochondrial CS shows a tendency to increase, and HOAD, a β-oxidation enzyme, is decreased during dormancy and early arousal, in relation to fall. Unlike, PK and CS are decreased, while HOAD is unchanged after a period of fasting during spring. Fatty acids (FA) profiles of neutral lipids suggest that unsaturated FA are preferentially mobilized during dormancy and arousal, whereas all FA would be equally used during spring fasting. FA of polar lipids are remarkably constant during the year, suggesting that membrane FA in the heart tissue are not generally affected by season, and thus, results do not support a predominant role played by compositional changes of membranes in metabolic depression in the tegu. In addition, the otherwise distinct findings with hibernating mammals suggest that changes of FA composition in these animals would be an adaptation to the low body temperature of torpor, rather than mechanism of metabolic inhibition. Regression analysis indicate significant relationships of C18:1n-9, C22:5n-6, and C22:6n-3 contents as a function of body mass in young tegus, and changes in the allometric patterns are consistent with a putative relationship between these FA levels and the scaling of mass specific metabolic rates of young tegus during the year cycle.

Diabetes Mellitus (DM) is one of the most common metabolic disorders in the world with an estimated prevalence of over 415 million patients. Heart failure (HF) is the most common cardiovascular complication of diabetes. The prevalence of diabetes in patients with HF is reported at approximately 30%. However, the molecular mechanisms that contribute to the development of heart failure in diabetic patients remain uncertain. To study this, a genetic mouse model of diabetes (GENA348) with a point mutation in the glucokinase gene was used. Glucokinase is a glucose sensor that controls insulin release. This mutation in the glucokinase is similar to that found in Maturity Onset Diabetes of the Young Type 2 (MODY2) in humans. Our group has previously shown that GENA348 mice exhibit a diabetic phenotype. At 6 months, the mice developed a diabetic cardiomyopathy analogous to that seen in clinical practice with the development of cardiac hypertrophy and diastolic dysfunction, which progressed to dilatation of the left ventricle and systolic dysfunction at 12 months. The aim of the project was to examine the molecular and pathophysiological mechanisms that contribute to development of this cardiac phenotype in diabetic GENA348 mice in the setting of hypertension and at baseline. To study the mice under hypertensive stress conditions, 6 month old-GENA348 HO and WT mice were infused with angiotensin II (ANG II) via minipump. After ANG II treatment, HO and WT GENA348 mice showed a significantly greater increase in systolic and diastolic blood pressure compared to untreated controls. It was evident that ANG II treatment resulted in cardiac hypertrophy with the same level observed in both HO and WT mice. The diastolic function was generally preserved in the WT and HO mice following the ANG II treatment. Our data indicates that the HO mice have had a blunted hypertrophic response to the hypertension induced by ANG II. At baseline, two hypothesis-generating methods were used. Firstly, gas chromatography-mass spectrometry (GC-MS) and ultra performance liquid chromatography-mass spectrometry (UPLC-MS) were used on 12-month-old GENA348 mice heart and serum samples. Secondly, diabetes PCR array plates were used on 6- and 12-month-old GENA348 mice heart samples. For the GCMS and UPLC-MS, there were 43 differences in metabolites from tissue samples and 93 from serum samples. The main altered metabolites from tissue samples were sugars and fatty acids. However, fatty acids, phospholipids and sphingolipids were the main altered metabolites from serum samples. After the validation of the array plates the most apparent observation was that only two up-regulated genes, Phosphoenolpyruvate carboxykinase 1 (Pck1)and Glucose-6-Phosphatase, Catalytic Subunit (G6pc) showed a comparable pattern as the array results. Pck1 and G6pc are the main enzymes that play a key role in gluconeogenesis regulation. We also looked at the expression level of one of the main transcriptional regulators of gluconeogenesis, Forkhead boxprotein O1 (FoxO1). It was found that the expression was altered at 12 months. In conclusion, it was clear that hyperglycaemia altered gene expression and the metabolites profiles in 12 month old HO mice, with evident alterations detected in genes involved in the metabolic regulation of the heart. In addition, this study may provide preliminary insight into pathophysiological alterations in the cardiac metabolism that may contribute to the development of diabetic cardiomyopathy.