Avaliação de setup para teste de elevação de pós-carga em pacientes com insuficiência cardíaca com fração de ejeção preservada: um estudo experimental / Evaluation of afterload elevation test setup in heart failure patients with preserved ejection fraction: an experimental study

INTRODUÇÃO: A Insuficiência Cardíaca com Fração de Ejeção Preservada (ICFEP) é uma doença de alta prevalência e de difícil diagnóstico devido ao fato que parte dos pacientes apresenta alterações apenas quando submetidos a determinadas condições de estresse hemodinâmico como elevação da pré-carga e da pós-carga do ventrículo esquerdo. OBJETIVOS: Avaliar a efetividade de um setup para elevação da pós-carga em pacientes com ICFEP. MÉTODOS: Realizou-se medida da pressão arterial sistólica (PAS), pressão arterial diastólica (PAD) e frequência cardíaca (FC) em repouso no membro superior direito com o paciente em decúbito supino seguindo as diretrizes da Sociedade Brasileira de Cardiologia. Em seguida, realizou-se um teste de elevação de pós-carga através da contração isométrica da mão esquerda com 60% da força máxima com um dinamômetro combinada com compressão pneumática de ambos os membros inferiores 20 mmHg acima da pressão sistólica utilizando dois esfigmomanômetros. Uma nova medida da pressão e frequência cardíaca foi realizada após 30s de início da manobra. Para comparação das métricas entre repouso e estresse, utilizou-se o teste-t pareado com intervalo de confiança de 95%. RESULTADOS: A população do estudo foi de 37 pacientes: 17 controles e 20 com ICFEP. A idade foi 58 ± 15 anos e 19 (51%) eram do sexo feminino. Houve diferença significativa para PAS: 128,2 ± 17,7 mmHg e 146,7 ± 19,9 mmHg (p<0,001) e FC 66 ± 11 bpm x 77 ± 14 bpm (p<0,001) repouso e estresse, respectivamente (Figura 1). CONCLUSÃO: O setup utilizado foi efetivo em elevar a pós-carga na população estudada. Os dados desta pesquisa têm grande potencial de aplicação para protocolos alternativos ao exercício padrão em cicloergômetro como teste provocativo para diagnóstico de ICFEP. Palavras-chaves: Insuficiência cardíaca com fração de ejeção preservada, Aumento de pós-carga, Teste de Estresse.

Ferramentas para otimização de digitalização, armazenamento e visualização de dados do teste cardiopulmonar de exercício / Tools for optimizing the digitization, storage and visualization of cardiopulmonary exercise test data

INTRODUÇÃO: O teste cardiopulmonar de exercício (TCPE) é o padrão ouro para função cardiorrespiratória de pacientes. Por conta disso, os dados obtidos por meio deste são extremamente relevantes em pesquisas clínicas. No entanto, há situações em que, por conta de limitações dos equipamentos, a obtenção e armazenamento dos resultados destes exames se torna um processo complicado e dispendioso. OBJETIVO: Desenvolver ferramentas capazes de: 1) Digitalizar os dados nos formatos disponibilizados pelos equipamentos e montar um banco de dados; 2) Permitam uma análise, visualização e comparação otimizadas desses dados. MÉTODOS: Inicialmente, os laudos obtidos diretamente do equipamento são lidos por um programa, os valores dos parâmetros são armazenados durante os tempos mais relevantes do teste (limiares aeróbico e anaeróbico, VO2 máximo etc.) em um arquivo que servirá como banco de dados relacional. Esta base de dados é então utilizada para montar um Dashboard onde se pode selecionar a variável e o intervalo de tempo que se deseja comparar entre os grupos. Nele são mostrados histogramas e gráficos violinos para se comparar visualmente os grupos (figuras 1 e 2) e estatísticas dos dados coletados junto a: 1) Resultado de um teste de normalidade para o parâmetro escolhido; 2) Resultado (p-valor) de um teste de hipótese (paramétrico caso a distribuição seja considerada normal, ou não paramétrico caso contrário); 3) Os tamanhos mínimos que as amostras devem possuir para que os resultados dos testes possam ser considerados estatisticamente significativos. Por fim, é possível analisar, em uma segunda página desta ferramenta os valores individuais de cada paciente, sendo possível filtrar e visualizar apenas os valores dentro de um intervalo desejado. (Figura 4) RESULTADOS: As ferramentas desenvolvidas foram utilizadas na base de dados dos pacientes do Ambulatório de Medicina Cardiovascular Personalizada com os resultados de 102 exames. O Dashboard foi utilizado para realizar um estudo comparativo entre os pacientes homens e mulheres do ambulatório com resultados coerentes com a literatura. CONCLUSÕES: Com estas ferramentas foi possível, rapidamente, digitalizar e armazenar os dados montando um arquivo com as informações de todos pacientes. Ademais, também permitiu realizar um estudo exemplo comparando todos pacientes gerando imagens que auxiliam a visualizar os resultados e os respectivos p-valores para cada comparação.

Ferramenta para processamento e armazenamento de curvas de strain para estudo da mecânica miocárdica / Tool for processing and storing strain curves for studying myocardial mechanics

INTRODUÇÃO: As curvas de deformação cardíacas derivadas de Speckle Tracking, permitem avaliação detalhada de diversos aspectos da mecânica miocárdica, entretanto o estudo de novos parâmetros é limitado por restrições impostas pelos softwares proprietários dos diferentes fabricantes. OBJETIVO: Desenvolver um programa com interface gráfica que permita visualizar e processar as curvas de Strain e Strain Rate facilitando o desenvolvimento de pesquisas. MÉTODOS: Inicia-se com a exportação das curvas de Strain do programa do fabricante. Elas são a entrada do software que as converte em variáveis do Matlab. Pode-se então, selecionar o exame de interesse, ou seja, paciente e janela de aquisição. As curvas de Strain e Strain Rate são exibidas e é possível selecionar os segmentos de interesse e, caso exista, outros exames do mesmo paciente com a mesma janela de aquisição. As curvas de deformação são exibidas sincronizadas com o Eletrocardiograma possibilitando ter em uma única tela o sinal elétrico e o sinal de deformação. Além disso, também é possível anotar o tempo de abertura e fechamento das válvulas com relação ao pico do QRS, que é encontrado automaticamente e pode ser editado caso necessário. As demais abas do programa apresentam as seguintes funcionalidades: em "Curvas Separadas" é possível visualizar as porções das curvas correspondentes à fase do ciclo cardíaco desejada, na aba "Plot Simul" é possível visualizar, simultaneamente, uma janela do ventrículo esquerdo e uma de átrio esquerdo sincronizados pelos respectivos ECGs, e na aba "Doppler" é possível verificar a imagem de um doppler e utilizá-lo para demarcar os tempos de válvula. RESULTADOS: O software funcional foi implementado em um ambulatório, sendo responsável por converter e processar curvas de mais de 2000 arquivos exportados, segmentando-os nas fases específicas do ciclo cardíaco (relaxamento e contração isovolumétricos, períodos de ejeção, enchimento rápido e enchimento lento) de exames utilizados para desenvolvimento de uma linha de pesquisa. CONCLUSÕES: O software livre desenvolvido no IDPC permite explorar as curvas de deformação cardíaca de maneira personalizada sendo adequado ao estudo do ecocardiograma em diversas situações.

Variations in regional strain under different afterload conditions may help to identify is-chemic myocardium

INTRODUCTION: Acute coronary syndromes are a major health problem worldwide. The Speckle Tracking Imaging has been proposed as ad-junctive for diagnosis and prognosis purposes in the context of NSTE-ACS. Purpose: We sought to determine the regional myocardial strain in ischemic versus non-ischemic segments in subjects with NSTE-ACS, before and during acute pressure overload. METHODS: Data from 2D-echocardiography were obtained from 21 patients (mean age, 50 ± 10 years) undergoing cinecoronariography for NSTE-ACS indications. A blinded observer performed an offline analysis to obtain the Systolic Positive Peak (P), Systolic Peak Strain (S), Peak Strain (G) and Time to Peak Strain (TTP) of each segment acquired before and during pressure overload-induced by left hand-grip (42± 8 Kg) combined with simultaneous pneumatic constriction of the right arm and both lower limbs. Moreover, LVEF and GLS were also calculated. A second blinded observer labelled the ischemic regions according to AHA-16 Bull's eye using data from cinecoronariography. RESULTS: The handgrip manuever caused mild reductions in the median LVEF (56 %; IQR 48 -58% vs 54%; IQR 51-56%) as well as the median GLS (-17%; IQR-19%-15% vs -16%; IQR -19-15%). For the analysis of regional myocardial function, we obtained 360 segments (29% labelled as ischemic) with optimal tracking quality. Mean regional strain changes during pressure overload were significantly different in ischemic vs non-ischemic segments for Peak S (1,07% x -0.5%; p-value <0,01) and Peak G (0.85% x -0.65%; p-value < 0.01) (Table). Notably, however, we found marked increases in the systolic deformation, restricted to the apical ischemic segments during pressure overload (Figure). CONCLUSIONS: Our data point to a distinct increase in the systolic deformation of the apical segments in contrast to the overall reductions in the GLS and EF in the ischemic left ventricle.

Evidence of macrophage modulation in the mouse pubic symphysis remodeling during the end of first pregnancy and postpartum.

In mouse pregnancy, pubic symphysis (PS) remodels into an elastic interpubic ligament (IpL) in a temporally regulated process to provide safe delivery. It restores at postpartum to assure reproductive tract homeostasis. Recently, macrophage localization in the IpL and dynamic changes in the expression of inflammatory mediators observed from the end of pregnancy (D18, D19) to early days postpartum (1dpp, 3dpp) highlighted the necessity of the identification of the key molecules involved in innate immune processes in PS remodeling. Therefore, this study uses morphological and high-sensitivity molecular techniques to identify both macrophage association with extracellular matrix (ECM) remodeling and the immunological processes involved in PS changes from D18 to 3dpp. Results showed macrophage association with active gelatinases and ECM components and 25 differentially expressed genes (DEGs) related to macrophage activities in interpubic tissues from D18 to 3dpp. Additionally, microarray and proteomic analysis showed a significant association of interpubic tissue DEGs with complement system activation and differentially expressed proteins (DEPs) with phagocytosis, highlighting the involvement of macrophage-related activities in mouse PS remodeling. Therefore, the findings suggest that PS ECM remodeling is associated with evidence of macrophage modulation that ensures both IpL relaxation and fast PS recovery postpartum for first labor.

Nuclear inositol 1,4,5-trisphosphate is a necessary and conserved signal for the induction of both pathological and physiological cardiomyocyte hypertrophy.

It is well established that inositol 1,4,5-trisphosphate (IP3) dependent Ca(2+) signaling plays a crucial role in cardiomyocyte hypertrophy. However, it is not yet known whether nuclear IP3 represents a Ca(2+) mobilizing pathway involved in this process. The goal of the current work was to investigate the specific role of nuclear IP3 in cardiomyocyte hypertrophic response. In this work, we used an adenovirus construct that selectively buffers IP3 in the nuclear region of neonatal cardiomyocytes. We showed for the first time that nuclear IP3 mediates endothelin-1 (ET-1) induced hypertrophy. We also found that both calcineurin (Cn)/nuclear factor of activated T Cells (NFAT) and histone deacetylase-5 (HDAC5) pathways require nuclear IP3 to mediate pathological cardiomyocyte growth. Additionally, we found that nuclear IP3 buffering inhibited insulin-like growth factor-1 (IGF-1) induced hypertrophy and prevented reexpression of fetal gene program. Together, these results demonstrated that nuclear IP3 is an essential and a conserved signal for both pathological and physiological forms of cardiomyocyte hypertrophy.

CYBA C242T polymorphism is associated with obesity and diabetes mellitus in Brazilian hypertensive patients.

AIMS: The CYBA C242T polymorphism has been associated with cardiovascular phenotypes such as hypertension and atherosclerosis, but available data are conflicting. This report investigated the impact of this variant on hypertension and metabolic determinants of cardiovascular risk in a large Brazilian sample. METHODS: We cross-sectionally evaluated 1856 subjects (826 normotensive subjects and 1030 hypertensive patients) by clinical history, anthropometry, laboratory analysis and genotyping of the CYBA C242T polymorphism. RESULTS: Genotype frequencies in the whole population were consistent with the Hardy-Weinberg equilibrium and genotype distributions were not different between hypertensive and normotensive subjects. Hypertensive patients with the CC genotype presented lower fasting plasma glucose levels (5.9 ± 0.1 vs. 6.2 ± 0.1 mmol/l, P = 0.020) and waist circumference (94.5 ± 0.6 vs. 96.3 ± 0.6 cm, P = 0.028) than CT + TT ones. Similarly, the prevalence of diabetes mellitus and obesity was also lower in hypertensive patients carrying the CC genotype (16% vs. 21%, P = 0.041; 36% vs. 43%, P = 0.029, respectively). In addition, multiple and logistic regression analysis demonstrated that the CYBA C242T polymorphism was associated with glucose levels, waist circumference, obesity and diabetes mellitus in hypertensive patients independently of potential confounders. Conversely, in normotensive subjects, no significant difference in studied variables was detected between the genotype groups. CONCLUSIONS: These data suggest that the T allele of the CYBA C242T polymorphism may be used as a marker for adverse metabolic features in Brazilian subjects with systemic hypertension.

Focal adhesion kinase -- the basis of local hypertrophic signaling domains.

Focal adhesion kinase (FAK), a broadly expressed non-receptor tyrosine kinase which transduces signals from integrins, growth and hormonal factors, is a key player in many fundamental biological processes and functions, including cell adhesion, migration, proliferation and survival. The involvement of FAK in this range of functions supports its role in important aspects of organismal development and disease, such as central nervous system and cardiovascular development, cancer, cardiac hypertrophy and tissue fibrosis. Many functions of FAK are correlated with its tyrosine kinase activity, which is temporally and spatially controlled by complex intra-molecular autoinhibitory conformation and inter-molecular interactions with protein and lipid partners. The inactivation of FAK in mice results in embryonic lethality attributed to the lack of proper development and function of the heart. Accordingly, embryonic FAK myocyte-specific knockout mice display lethal cardiac defects such as thin ventricle wall and ventricular septum defects. Emerging data also support a role for FAK in the reactive hypertrophy and failure of adult hearts. Moreover, the mechanisms that regulate FAK in differentiated cardiac myocytes to biomechanical stress and soluble factors are beginning to be revealed and are discussed here together with data that connect FAK to its downstream effectors. This article is part of a Special Issue entitled "Local Signaling in Myocytes".

Focal adhesion kinase governs cardiac concentric hypertrophic growth by activating the AKT and mTOR pathways.

The heart responds to sustained overload by hypertrophic growth in which the myocytes distinctly thicken or elongate on increases in systolic or diastolic stress. Though potentially adaptive, hypertrophy itself may predispose to cardiac dysfunction in pathological settings. The mechanisms underlying the diverse morphology and outcomes of hypertrophy are uncertain. Here we used a focal adhesion kinase (FAK) cardiac-specific transgenic mice model (FAK-Tg) to explore the function of this non-receptor tyrosine kinase on the regulation of myocyte growth. FAK-Tg mice displayed a phenocopy of concentric cardiac hypertrophy, reflecting the relative thickening of the individual myocytes. Moreover, FAK-Tg mice showed structural, functional and molecular features of a compensated hypertrophic growth, and preserved responses to chronic pressure overload. Mechanistically, FAK overexpression resulted in enhanced myocardial FAK activity, which was proven by treatment with a selective FAK inhibitor to be required for the cardiac hypertrophy in this model. Our results indicate that upregulation of FAK does not affect the activity of Src/ERK1/2 pathway, but stimulated signaling by a cascade that encompasses PI3K, AKT, mTOR, S6K and rpS6. Moreover, inhibition of the mTOR complex by rapamycin extinguished the cardiac hypertrophy of the transgenic FAK mice. These findings uncover a unique role for FAK in regulating the signaling mechanisms that governs the selective myocyte growth in width, likely controlling the activity of PI3K/AKT/mTOR pathway, and suggest that FAK activation could be important for the adaptive response to increases in cardiac afterload. This article is part of a Special Issue entitled "Local Signaling in Myocytes".

Hip circumference is associated with high density lipoprotein cholesterol response following statin therapy in hypertensive subjects.

AIM: This report investigated the relationship between anthropometric measurements of body fat distribution and lipid response to statins in hypercholesterolemic hypertensive patients. METHODS: We prospectively examined 129 subjects who used either simvastatin 20 mg/day (no.=83) or atorvastatin 10 mg/day (no.=46) for 3 months. Anthropometry included evaluation of body mass index, waist and hip circumferences, and waist-to-hip-ratio. RESULTS: Significant decreases in LDL (p<0.001), total cholesterol (p<0.001), and triglycerides (p=0.04) levels were detected after 3 months of therapy in the whole sample. At baseline, only an inverse correlation between waist circumference and HDLcholesterol levels was detected (r=-0.18; p=0.04). Conversely, a direct relationship between hip circumference and HDLcholesterol response to statins was found in the whole sample (r=0.24; p=0.006), while no other anthropometric measurement displayed significant correlation with lipid changes. The association between HDL-cholesterol response and hip circumference was further confirmed by stepwise regression analysis adjusted for baseline HDL-cholesterol levels, metabolic syndrome, body mass index, and waist circumference. CONCLUSIONS: Hip circumference, a surrogate marker of peripheral adiposity, is associated with HDL-cholesterol changes following statin therapy in hypertensive patients.

Focal adhesion kinase signaling in cardiac hypertrophy and failure.

Focal adhesion kinase (FAK) is a broadly expressed tyrosine kinase implicated in cellular functions such as migration, growth and survival. Emerging data support a role for FAK in cardiac development, reactive hypertrophy and failure. Data reviewed here indicate that FAK plays a critical role at the cellular level in the responses of cardiomyocytes and cardiac fibroblasts to biomechanical stress and to hypertrophic agonists such as angiotensin II and endothelin. The signaling mechanisms regulated by FAK are discussed to provide insight into its role in the pathophysiology of cardiac hypertrophy and failure.

Focal adhesion kinase signaling in cardiac hypertrophy and failure

Focal adhesion kinase (FAK) is a broadly expressed tyrosine kinase implicated in cellular functions such as migration, growth and survival. Emerging data support a role for FAK in cardiac development, reactive hypertrophy and failure. Data reviewed here indicate that FAK plays a critical role at the cellular level in the responses of cardiomyocytes and cardiac fibroblasts to biomechanical stress and to hypertrophic agonists such as angiotensin II and endothelin. The signaling mechanisms regulated by FAK are discussed to provide insight into its role in the pathophysiology of cardiac hypertrophy and failure.

Mandibular appliance modulates condylar growth through integrins.

Functional orthopedic therapy corrects growth discrepancies between the maxilla and mandible, possibly through postural changes in the musculature and modulation of the mandibular condylar cartilage growth. Using Wistar rats, we tested the hypothesis that chondrocytes respond to forces generated by a mandibular propulsor appliance by changes in gene expression, and that integrins are important mediators in this response. Immunohistochemical analyses demonstrated that the use of the appliance for different periods of time modulated the expression of fibronectin, alpha5 and alphav integrin subunits, as well as cell proliferation in the cartilage. In vitro, cyclic distension of condylar cartilage-derived cells increased fibronectin mRNA, as well as Insulin-like Growth Factor-I and II mRNA and cell proliferation. A peptide containing the Arginine-Glycine-Asparagine sequence (RGD), the main cell-binding sequence in fibronectin, blocked almost all these effects, confirming that force itself modulates the growth of the rat condylar cartilage, and that RGD-binding integrins participate in mechanotransduction.

Non-effect of p22-phox -930A/G polymorphism on end-organ damage in Brazilian hypertensive patients.

The p22-phox subunit is an essential component of NAD(P)H oxidase enzymatic complex, which is considered the major source of oxidative stress products in the cardiovascular system. The -930G allele of p22-phox has been associated with higher promoter activity, increased NAD(P)H oxidase-mediated oxidative stress and hypertension. We recently reported that left ventricular hypertrophy is accompanied by increased myocardial p22-phox expression in aortic-banded rats, suggesting that this protein might be involved in hypertensive cardiac hypertrophy.

Antiatherogenic effects of S-nitroso-N-acetylcysteine in hypercholesterolemic LDL receptor knockout mice.

BACKGROUND: The pathophysiology of the NO/NO synthase system and dysfunctional changes in the endothelium in the early phases of the atherogenic process are incompletely understood. In this study, we investigated the effects of the nitrosothiol NO donor S-nitroso-N-acetylcysteine (SNAC) in the early prevention of plaque development in the hypercholesterolemic LDLr-/- mice as well as the changes in endothelium-dependent relaxation and NO synthase expression. METHODS AND RESULTS: LDLr-/- mice were fed a 1.25% cholesterol-enriched diet for 15 days. Plasma cholesterol/triglyceride levels increased and this increase was accompanied by the development of aortic root lesions. Aortic vasorelaxation to acetylcholine was increased, although endothelium-independent relaxation in response to sodium nitroprusside did not change, which suggest stimulated NO release enhanced. This dysfunction was associated with enhanced aortic superoxide production and with increased levels of constitutive NOS isoform expression, particularly neuronal NOS. SNAC (S-nitroso-N-acetylcysteine) administration (0.51 micromol/kg/day i.p. for 15 days) decreased the extent of the plaque by 55% in hypercholesterolemic mice, but had no effects on vasomotor changes. It did, however, lead to a decrease in constitutive NOS expression. The SNAC induced only minor changes in plasma lipid profile. CONCLUSION: The present study has shown that, in early stages of plaque development in LDLr-/- mice, specific changes in NO/NO synthase system develop, that are characterized by increased endothelium-dependent vasorelaxation and increased constitutive NOS expression. Since the development of plaque and the indicator of endothelial cell dysfunction were prevented by SNAC, such treatment may constitute a novel strategy for the halting of progression of early plaque.

Regulation of insulin signalling by hyperinsulinaemia: role of IRS-1/2 serine phosphorylation and the mTOR/p70 S6K pathway.

AIM/HYPOTHESIS: Several epidemiological studies have suggested an association between chronic hyperinsulinaemia and insulin resistance. However, the causality of this relationship remains uncertain. METHODS: We performed chronic hyperinsulinaemic-euglycaemic clamps and delineated, by western blotting, an IR/IRSs/phosphatidylinositol 3-kinase(PI[3]K)/Akt pathway in insulin-responsive tissues of hyperinsulinaemic rats. IRS-1/2 serine phosphorylation, IR/protein tyrosine phosphatase 1B (PTP1B) association, and mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70 S6K) activity were also evaluated in the liver, skeletal muscle and white adipose tissue of hyperinsulinaemic animals. RESULTS: We found that chronic hyperinsulinaemic rats have insulin resistance and reduced levels of glycogen content in liver and muscle. In addition, we demonstrated an impairment of the insulin-induced IR/IRSs/PI3K/Akt pathway in liver and muscle of chronic hyperinsulinaemic rats that parallels increases in IRS1/2 serine phosphorylation, IR/PTP1B association and mTOR activity. Despite a higher association of IR/PTP1B, there was an increase in white adipose tissue of chronic hyperinsulinaemic rats in IRS-1/2 protein levels, tyrosine phosphorylation and IRSs/PI3K association, which led to an increase in basal Akt serine phosphorylation. No increases in IRS-1/2 serine phosphorylation and mTOR activity were observed in white adipose tissue. Rapamycin reversed the insulin resistance and the changes induced by hyperinsulinaemia in the three tissues studied. CONCLUSIONS/INTERPRETATION: Our data provide evidence that chronic hyperinsulinaemia itself, imposed on normal rats, appears to have a dual effect, stimulating insulin signalling in white adipose tissue, whilst decreasing it in liver and muscle. The underlying mechanism of these differential effects may be related to the ability of hyperinsulinaemia to increase mTOR/p70 S6K pathway activity and IRS-1/2 serine phosphorylation in a tissue-specific fashion. In addition, we demonstrated that inhibition of the mTOR pathway with rapamycin can prevent insulin resistance caused by chronic hyperinsulinaemia in liver and muscle. These findings support the hypothesis that defective and tissue-selective insulin action contributes to the insulin resistance observed in hyperinsulinaemic states.

Insulin signalling pathways in aorta and muscle from two animal models of insulin resistance--the obese middle-aged and the spontaneously hypertensive rats.

AIMS/HYPOTHESIS: The aim of this study was to investigate insulin signalling pathways directly in vivo in skeletal muscle and thoracic aorta from obese middle-aged (12-month-old) rats, which have insulin resistance but not cardiovascular disease, and from spontaneously hypertensive rats (SHR), an experimental model of insulin resistance and cardiovascular disease. METHODS: We have used in vivo insulin infusion, followed by tissue extraction, immunoprecipitation and immunoblotting. RESULTS: Obese middle-aged rats and the SHR showed marked insulin resistance, which parallels the reduced effects of this hormone in the insulin signalling cascade in muscle. In aortae from obese middle-aged rats, the PI 3-kinase/Akt pathway is preserved, leading to a normal activation of endothelial nitric oxide synthase. In SHR this pathway is severely blunted, with reductions in eNOS protein concentration and activation. Both animals, however, showed higher concentrations and higher tyrosine phosphorylation of mitogen-activated protein (MAP) kinase isoforms in aortae. CONCLUSIONS/INTERPRETATION: Alterations in the IRS/PI 3-K/Akt pathway in muscle of 12-month-old rats and SHR could be involved in the insulin resistance of these animals. The preservation of this pathway in aorta of 12-month-old rats, apart from increases in MAP kinase protein concentration and activation, could be a factor that contributes to explaining the absence of cardiovascular disease in this animal model. However, in aortae of SHR, the reduced insulin signalling through IRS/PI 3-kinase/Akt/eNOS pathway could contribute to the endothelial dysfunction of this animal.

Expression and distribution of NOS1 and NOS3 in the myocardium of angiotensin II-infused rats.

Studies have indicated a complex functional interaction between angiotensin (Ang) II and NO in the heart. The purpose of the present study was to examine the protein expression and tissue distribution of NO synthases 1 (NOS1) and 3 (NOS3) in the myocardium of rats that underwent continuous infusion of Ang II at 2 different rates (10 and 40 ng. kg(-1). min(-1)) for 6 days. Mean arterial pressure increased by approximately 15 mm Hg in rats infused with Ang II at 40 ng. kg(-1). min(-1), but it remained close to the values observed in saline-infused rats ( approximately 110 mm Hg) when Ang II was infused at 10 ng. kg(-1). min(-1). The protein expression of a 160-kDa NOS1 and a 135-kDa NOS3 were found to increase ( approximately 200%) in the myocardium of rats infused with both subpressor and pressor doses of Ang II. Immunohistochemistry studies showed that NOS1 and NOS3 are differentially expressed in myocardial cells. NOS1 was detected in cardiac myocytes and in smooth muscle cells of small and large coronary arteries, whereas NOS3 was detected in the endothelium and in perivascular and interstitial tissues, but NOS3 was not detected in cardiac or smooth muscle cells. Ang II infusion enhanced the tissue immunoreactivity of both isoforms in their specific locations but did not change the distribution throughout the myocardium. Myocardium staining with anti-angiotensin type 1 (AT(1)) receptor antibody indicated that AT(1) receptor is expressed in cardiac myocytes, coronary smooth muscle cells, and interstitial and perivascular tissues. Ang II infusion did not change the protein expression and distribution of AT(1) receptor in the myocardium. These results indicate that long-term increases in the circulating levels of Ang II modulate the protein expression of NOS1 and NOS3 and, consequently, the function of the local myocardial NO system.

Hemodilution mediates hemodynamic changes during acute expansion in unanesthetized rats.

Studies were carried out to determine the relative importance of volume and hemodilution on hemodynamic adjustments to acute volume expansion. Systemic and renal hemodynamics were monitored in unanesthetized and unrestrained rats during progressive and equivalent blood volume expansion with saline (Sal; 1, 2, and 4% body wt), 7% BSA solution (0.35, 0.7, and 1.4% body wt), and reconstituted whole blood from donor rats (WBL; 0.35, 0.7, and 1.4% body wt). Mean arterial pressure remained unchanged in Sal and BSA but increased progressively in WBL-expanded rats (from 92 to 106 mmHg after maximal expansion). In Sal and BSA-expanded rats, cardiac output (CO) and renal blood flow (RBF) increased (CO: Sal from 19 to 20, 22, and 25; BSA from 21 to 23, 27, and 31; RBF: Sal from 1.6 to 1.8, 2.2, and 2.5; BSA from 2 to 2.4, 2.7, and 3.1 ml. min(-1). 100 g body wt(-1)), whereas total peripheral (TPR) and renal vascular (RVR) resistance decreased in parallel with the expansions. After expansion with WBL, CO increased progressively but less extensively than in cell-free expanded rats (21 to 22, 24, and 26 ml. min(-1). 100 g body wt(-1)), whereas TPR and RVR remained unchanged. Systemic hematocrit (Hct) decreased approximately the same after expansion with Sal or BSA solutions but remained unchanged after expansion with WBL. Isovolemic hemodilution to Hct levels comparable to those seen after maximal expansion with cell-free solutions also reduced SVR and RVR, although less extensively. These findings suggest that in unanesthetized rats hemodilution plays a major role in the systemic and renal hemodynamics during expansion.

Early activation of the multicomponent signaling complex associated with focal adhesion kinase induced by pressure overload in the rat heart.

Mechanical overload elicits functional and structural adaptive mechanisms in cardiac muscle. Signaling pathways linked to integrin/cytoskeleton complexes may have a function in mediation of the effects of mechanical stimulus in myocardial cells. We investigated the tyrosine phosphorylation and the assembly of the multicomponent signaling complex associated with focal adhesion kinase (Fak) and the actin cytoskeleton in the overloaded myocardium of rats. Pressure overload induced a 3-fold increase in Fak tyrosine phosphorylation within 3 minutes after a 60-mm Hg rise in aortic pressure. A pressure stimulus that lasted for 60 minutes was accompanied by a 5-fold increase in the amount of tyrosine-phosphorylated Fak, and a stimulus as low as 10 mm Hg doubled the amount of tyrosine-phosphorylated Fak in the myocardium within 10 minutes. Pressure overload also induced a time-dependent association of actin with Fak and an increase in the amount of Fak detected in the cytoskeletal fraction of the myocardium. These events were paralleled by c-Src activation and binding to Fak and by an association of Grb2 and p85 subunit of phosphatidylinositol 3-kinase with Fak. Erk1/2 and Akt, two possible downstream effectors of Fak via Grb2 and phosphatidylinositol 3-kinase, were also shown to be activated in parallel with Fak. These findings show that pressure overload induced a rapid activation of the Fak multiple signaling complex in the myocardium of rats, which suggests that this mechanism may have a role in mechanotransduction in the myocardium.