Cyclic 3',5'-guanosine monophosphate (cGMP) is a key signaling molecule involved in a myriad of physiological processes, including vascular smooth muscle (VSM) tone, water- and electrolyte homeostasis, platelet aggregation, airway smooth muscle tone, smooth muscle proliferation and bone formation. Increased occurrence of vascular disorders including erectile dysfunction, hypertension, stroke and coronary artery disease, have made it increasingly important to study the dynamic interplay between cGMP synthesis and hydrolysis in VSM cells. This dissertation examines the spatial distribution of intracellular cGMP, [cGMP]i, in response to NO and atrial natriuretic peptide (ANP) in VSM cells. To investigate the spatial patterning of [cGMP]i, we have developed a new generation of non-FRET (fluorescence resonance energy transfer) cGMP biosensors that are suitable to monitor [cGMP]i in response to physiological (low-nanomolar) NO and ANP concentrations and that qualify for real-time, confocal imaging techniques. We have termed these indicators FlincGs, for green fluorescent indicators of cGMP. For the development of FlincGs, we made use of the specific cGMP binding characteristics of PKG. We utilized site-specific mutagenesis, kinetic cGMP binding, dissociation and kinase assays, as well as crystallography, in order to investigate PKG activation and cGMP binding dynamics in greater detail. Based on these studies, our novel, non-FRET cGMP biosensors were designed by attaching cGMP binding fragments of PKG to the N-terminus of circular permutated green fluorescent protein. We applied FlincGs in cultured VSM cells as well as in intact tissue to determine whether two spatially distinct populations of guanlylyl cyclase (cytosolic versus membrane bound) underlie the generation of spatiotemporally-specific patterns of [cGMP]i formation.

No description available.

A obesidade é uma doença nutricional crescente em humanos e de alta prevalência nos cães. Este distúrbio nutricional e decorrente, principalmente, de um balanço energético inadequado. O excesso de peso pode exacerbar uma doença preexistente e predispor ao desenvolvimento de outros problemas de saúde, como Diabetes mellitus, aumento dos níveis de colesterol e triglicérides, doenças hepáticas, lesões osteoarticulares, hipertensão, alterações cardíacas e respiratórias. Em medicina humana, a associação entre hiperinsulinemia, hiperlipidemia, hiperleptinemia, hipertensão arterial, hipertrofia ventricular e disfunção cardíaca demonstra que a obesidade e um fator de risco para a doença cardiovascular. O aminoterminal peptídeo natriurético tipo B (Nt- proBNP) é um marcador útil de disfunção ventricular nas doenças cardíacas em humanos, bem como em cães e gatos. No entanto, ainda não está claro como interpretar os níveis de Nt- proBNP em pacientes obesos, visto que os níveis deste peptídeo parecem ter correlação negativa com o índice de massa corpórea. A disfunção diastólica é comumente observada em indivíduos obesos e pode ser detectada precocemente por meio de ecocardiograma Doppler tecidual pulsado. Assim, considerando que os diversos estudos corn Nt- proBNP em humanos obesos são controversos e a inexistência de estudos em animais obesos, o objetivo deste estudo foi avaliar o possível papel dos peptídeos natriuréticos como marcadores pré-clínicos de doença cardíaca na obesidade canina e sua relação com os parâmetros de função diastólica avaliados pelo Doppler tecidual pulsado. Para isto, selecionaram-se 49 cães com escore de condição corporal normal (ECC 5) e 48 obesos (ECC 8 e 9), pareados com relação ao gênero, raça e idade. Os cães obesos apresentaram concentração de Nt- proBNP major que os cães com ECC5/9, e alterações em algumas variáveis ecocardiográficas de função diastólica de VE, avaliadas pelo Doppler tecidual pulsado. Também foi observado aumento na relação Ef/Em associado a alta concentração de Nt-proBNP, sugerindo aumento na pressão de enchimento do ventrículo esquerdo em cães obesos. / Obesity is an increasing nutritional disease in humans, and highly prevalent in dogs. This nutritional disturbance is a consequence of an inadequate energetic balance. Weight excess may exacerbate a pre-existing disease and predisposes to the development of other health problems as diabetes mellitus, increased colesterol and triglyceride levels, hepatic diseases, osteoarticular diseases, hypertension, cardiac and respiratory problems. In human medicine, the association between hyperinsulinemia, hyperlipidemia, hyperleptinemia, arterial hypertension, ventricular hypertrophy and cardiac disfunction demonstrates that obesity is a risk factor for cardiovascular disease. Type B aminoterminal natriuretic peptide (Nt- proBNP) is a useful marker of ventricular dysfunction in heart disease in humans, as well as in dogs and cats. Nonetheless, it is still unclear how to interpret the levels of Nt-proBNP in obese patients, because it has been negatively correlated to body mass index. Diastolic dysfunction is frequently observed in obese patients and may be detected early by pulsed tissue Doppler imaging (TDI). Therefore, considering that controversial results in several studies on Nt-proBNP in obese humans, and the lack of studies in obese animals, the purpose of this study was to evaluate the possible role of Nt-proBNP as preclinical marker of cardiac disease in obese dogs, and the relationship with diastolic function parameters assessed by pulsed TDI. Foully-nine dogs with ideal body score condition (BCS5/9) and 48 obese dogs (BCS 8 and 9/9), were selected, paired for gender, breed/size and age. A higher Nt-proBNP concentration was found in obese dogs in relation to BCS 5/9 dogs, and some changes in echocardiographic variables of diastolic function, assessed by pulsed TDI. There was also increase Ef/Em ratio associated with a higher Nt-proBNP levels, suggesting an increased filling pressure of left ventricle in obese dogs.

A degeneração valvar crônica mitral (DVCM) é uma cardiopatia de alta prevalência na clínica médica de pequenos animais e acomete principalmente cães idosos de raças de pequeno porte. A fim de se acompanhar a evolução da insuficiência cardíaca (IC), a galectina-3 (Gal-3) vem sendo utilizada como um biomarcador na identificação de doenças cardíacas pré-clínicas, progressão e descompensação em pacientes humanos. O objetivo deste estudo clínico foi estabelecer intervalos de referência da Gal-3 na população canina estudada e determinar a utilidade desse novo biomarcador sérico, isoladamente ou em associação com o pró-peptídeo natriurético tipo B (NT-proBNP) e a troponina cardíaca I (cTnI), para estimativa de prognóstico em curto prazo em cães com IC decorrente de DVCM. O delineamento fundamentou-se em um estudo clínico observacional transversal prospectivo com braço longitudinal. A amostra foi composta por 139 cães distribuídos em cinco grupos criteriosamente selecionados de acordo com o estadiamento da DVCM (Grupo controle: estágio A- composto por 60 cães hígidos de raças de pequeno porte com predisposição à DVCM, 28 cães em estágio B1, 20 cães em B2, 20 cães em estágio C e 11 cães em estágio D), advindos da rotina do Serviço de Cardiologia do VCM, Hospital Veterinário da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo. Os grupos B1, B2, C e D foram avaliados em um segundo momento, aos 60 dias. Foram dosados Gal-3 humana e canina, NTproBNP e cTnI. Os valores de referência mensurados no grupo A para Gal-3 humana foram de 7,548 ng/mL (P25%-75%=8,933-10,960). A recuperação da concentração de Gal-3 em cães clinicamente saudáveis, obtida com kit canino, foi significativamente mais baixa, com baixa repetibilidade e reprodutibilidade, em comparação com o kit humano, sugerindo assim menor sensibilidade do kit canino utilizado. Ainda, a magnitude e a variação nas concentrações de Gal-3 humana e canina não permitiram a detecção de diferenças entre os estágios da DVCM e também não foram capazes de identificar pacientes em IC. Conclui-se, portanto, que diferentemente dos demais marcadores avaliados, NT-proBNP e cTnI, já consagrados na IC para a espécie canina, a Gal-3 não se constitui em um biomarcador adequado para avaliar a IC secundária à DVCM em cães / Chronic mitral valve degeneration (CMVD) is a highly prevalent heart disease in small animal internal medicine seen mainly in older small breed dogs. In order to follow the progression of heart failure (HF), galectin-3 (Gal-3) has been applied as a biomarker to identify pre-clinical cardiac diseases, progression and decompensation in human patients. This study aimed to establish reference intervals for Gal-3 in a canine population, and to determine the utility of this new biomarker, isolated or in association with Type B natriuretic pro-peptide (NT-proBNP) and cardiac troponin I to estimate short term prognosis in dogs with HF caused by CMVD. It was designed as an observational prospective cross-sectional clinical study with a longitudinal arm. One hundred thirty nine dogs were distributed among five groups with rigorous selection criteria, according to ACVIM CMVD staging (Control group: stage A- 60 healthy small breed dogs, predisposed to CMVD; 28 dogs in stage B1, 20 dogs in stage B2, 20 dogs in stage C and 11 dogs in stage D), recruited from the Cardiology Service from the Veterinary Teaching Hospital, School of Veterinary Medicine, University of São Paulo. Groups B1, B2, C and D had a second blood sampling at day 60. Measurements were obtained for human and canine Gal-3, NT-proBNP and cTnI. Reference values obtained for group A for human Gal-3 were 7.548 ng/mL (P25%-75%=8.933-10.960). Gal-3 concentration recovery for healthy dogs obtained with canine kit was significantly lower, with low repeatability and reproducibility, compared to the human kit, suggesting lower sensitivity of the canine Gal-3 kit used. We concluded that the magnitude and variation observed in human and canine Gal-3 did not allow for detection of differences between stages of CMVD nor were capable of identifying patients in HF, compared to the other measured biomarkers, NT-proBNP and cTnI, already established for canine HF evaluation

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Estudo dos efeitos de uma dieta rica em sal e / ou gordura saturada em grânulos de peptídeo natriurético atrial (ANP), hipertensão, expressão da renina e ultraestrutura cardíaca em camundongos C57Bl / 6. Camundongos machos adultos jovens foram separados em quatro grupos (n = 12) e alimentados com uma das seguintes dietas por 9 semanas: dieta padrão para roedores (Grupo P), dieta hiperlipídica (Grupo HL), dieta hipersódica (Grupo HS) e dieta hiperlipídica e hipersódica simultaneamente (HL-HS). Foram examinados: alterações no ANP sérico, ultra-estrutura dos cardiomiócitos produtores de ANP, estrutura do ventrículo esquerdo, pressão arterial sanguínea, expressão da renina no rim, taxa de filtração glomerular (TFG), eficiência alimentar, parâmetros lipídicos e glicídicos. Os animais alimentados com dieta hiperlipídica mostraram um pequeno aumento na produção de ANP, discreta hipertrofia ventricular esquerda, aumento da eficiência alimentar, dislipidemia e hiperglicemia. Animais alimentados com dieta hipersódica tiveram um grande aumento na produção de grânulos de ANP e correspondente elevação do seu nível sérico, hipertrofia ventricular esquerda, hipertensão arterial, diminuição dos níveis de renina e aumento da TFG. A combinação das duas dietas (HL-HS) teve um efeito aditivo. A ingestão de uma dieta com alto teor de sal e lipídeos induz alterações ultraestruturais dos cardiomiócitos, aumento da produção de ANP e elevação de seu nível sérico e reduz a quantidade de renina no rim. / To study the effects of a diet rich in salt and/or saturated fat on atrial natriuretic peptide (ANP)-granules, hypertension, renin expression and cardiac structure in C57Bl/6 mice. Young adult male mice were separated into four groups (n=12) and fed one of the following for 9 weeks: standard chow/normal-salt (SC-NS), high-fat chow/normal salt (HF-NS), standard chow/high-salt (SC-HS) and high-fat chow/high-salt (HF-HS). Alterations in the serum ANP, ultrastructural analysis of cardiomyocytes that produce ANP, structural analysis of the left ventricle, blood pressure, renin expression, glomerular filtration rate (GFR), feed efficiency and lipid and glucose parameters were examined. The HF-NS diet showed a small increase in ANP production and left ventricular hypertrophy, increased food efficiency and abnormal lipid and glucose parameters. The SC-HS diet showed a large increase in ANP granules in myocytes and corresponding elevation in ANP serum levels, left ventricular hypertrophy, hypertension, decrease of renin levels and increase in GFR. The combination of the two diets (HF-HS) had an additive effect. The incorporation of a high-fat high-salt diet induced ultrastructural changes in cardiomyocytes, increased the production of ANP and increased its serum level, and reduced the amount of renin in the kidney.

INTRODUÇÃO: A rejeição constitui-se em uma das principais causas de mortalidade após o transplante cardíaco pediátrico. O peptídeo natriurético tipo B (BNP), tem sido estudado como método no diagnóstico de rejeição aguda principalmente em pacientes adultos submetidos ao transplante cardíaco. OBJETIVOS: Avaliar o peptídeo natriurético tipo B no diagnóstico de rejeição aguda em crianças submetidas ao transplante cardíaco ortotópico, avaliar o papel do BNP como método adicional não invasivo na elucidação diagnóstica da doença coronariana após transplante e comparar parâmetros clínicos, ecocardiográficos e hemodinâmicos em relação à biópsia endomiocárdica no diagnóstico de rejeição cardíaca aguda. MÉTODOS: Foram coletadas 50 amostras de BNP de 33 crianças em pós-operatório de transplante cardíaco e analisados dados de idade, sexo, cor, grupo sangüíneo, painel imunológico, tempo de evolução após o transplante, sintomatologia, imunossupressão utilizada, número de rejeições, dados ecocardiográficos e parâmetros hemodinâmicos. Os grupos foram divididos em pacientes com rejeição e pacientes sem rejeição. RESULTADOS: Foram analisadas 50 amostras consecutivas de 33 crianças, durante período de 17 meses. A idade mediana foi de 10,1 anos, com predomínio do sexo feminino (54%) e da cor branca (85%). No momento da dosagem de BNP o tempo médio pós-transplante foi 4,3 anos. A biópsia endomiocárdica diagnosticou nove rejeições em oito pacientes (27%), sendo três com grau 3 A, cinco com grau 2 e um com rejeição humoral. No momento da biópsia, a maioria dos pacientes encontrava-se assintomática. O nível sérico de BNP teve mediana de 77,2 pg/ml, sendo 144,2 pg/ml no grupo com rejeição e 62,5 pg/ml no grupo sem rejeição, com p = 0,02. Análise de curva ROC mostra que níveis sangüíneos de BNP maiores que 38 pg/ml apresentam sensibilidade de 100% e especificidade de 56% na detecção de rejeição cardíaca. Os níveis de BNP foram maiores que 100 pg/ml nos pacientes com doença coronariana, com mediana de 167,5 pg/ml, em relação à 15 mediana de 40,5 pg/ml dos pacientes que não apresentaram doença coronariana. A curva ROC mostra ponto de corte de 90 pg/ml como ideal para diagnóstico de doença coronariana, com p = 0,01. Os parâmetros hemodinâmicos não foram diferentes entre os grupos com rejeição e sem rejeição. A sensibilidade do ecocardiograma para detecção de rejeição foi de 44% e especificidade de 90%, com p= 0,02. CONCLUSÕES: Pacientes podem apresentar-se assintomáticos durante episódio de rejeição aguda. O nível sérico de BNP apresentou diferença estatisticamente significante no grupo com rejeição, podendo ser método adicional no diagnóstico de rejeição cardíaca. A doença coronariana esteve associada com níveis elevados de BNP, independente da presença de rejeição aguda. O ecocardiograma mostrou baixa sensibilidade para o diagnóstico de rejeição cardíaca, mas alta especificidade. A avaliação dos parâmetros hemodinâmicos não apresentou neste estudo correlação com os resultados de biópsia. / INTRODUCTION: The rejection is one of the main causes of mortality after pediatric heart transplant. B natriuretic peptide has been used as a diagnostic method for rejection mainly in adult patients after heart transplantation. OBJECTIVE: To correlate BNP levels collected at the moment of endomyocardial biopsy with rejection, to evaluate BNP as an additional method for coronary artery disease and to compare clinical, echocardiograph assessment and hemodynamic parameters with endomyocardial biopsy findings. METHODS: There were 50 BNP blood samples from 33 children submitted to orthotopic cardiac transplantation. Analyzed parameters included: age, gender, race, blood type, reactive panel, functional class, immunosuppressive regimens, number of rejection episodes, echocardiography findings and hemodynamic parameters. The patients were divided in two groups: with rejection and without rejection. RESULTS: Thirty three children with a median age of 10.3 years (54% female) were studied at median time of 4.2 years after heart transplantation. Endomyocardial biopsy diagnosed nine rejection episodes (27%): three were grade 3A; five were grade 2 and one was humoral rejection. At the moment of biopsy most patients were asymptomatic. Average BNP level was 77.2 pg/ml (144.2 pg/ml in the patients with rejection and 65.8 pg/ml in the group without rejection, p=0.02). BNP level was increased in humoral rejection and in patients with coronary artery disease. ROC curve demonstrates BNP levels over 38 pg/ml to present 100% sensibility and 56% specificity to detect acute rejection. The levels of BNP were higher than 100 pg/ml in most of the patients with coronary artery disease (median of 167.5 pg/ml compared with a 40.5 pg/ml in patients without coronary artery disease). The curve ROC shows a critical cut off value for the diagnosis of coronary artery disease at the level of 90 pg/ml in, with p = 0.01. The hemodynamic parameters did not show significant differences between the patients with rejection and the group without rejection. The echocardiogram presented 44% sensibility and a 17 90% specificity to detect the rejection episode (p = 0.02). CONCLUSIONS: Children could be asymptomatic at allograft rejection episodes. BNP level was significantly elevated in children with the allograft rejection episode and may add a valuable information for the rejection assessment. Also, the higher BNP levels associated with coronary artery disease may contribute for its surveillance. Although the echocardiography presented low sensibility to screen for acute rejection episodes, its high specificity enhances its role to structural and functional alterations. The hemodynamic parameters did not contribute for the diagnosis nor presented correlation with the biopsy findings.

<p>Reduced performance of the left ventricle to eject blood – left ventricular systolic dysfunction (LVSD) – is a common predecessor of the heart failure syndrome. With or without symptoms, LVSD is associated with a poor prognosis. However, with adequate treatment, the development or progression of symptoms, the need for hospitalisation and mortality can all be reduced. In the present work, the occurrence of LVSD was evaluated by echocardiography in a community-based sample of 75-year-old men and women (n = 433). LVSD was a common condition, with a prevalence rate of 6.8%. In nearly half the participants with LVSD, there was no clinical evidence of heart failure.</p><p>Community-based screening for asymptomatic LVSD has been proposed as a strategy to reduce the incidence of heart failure. Because of the high costs and low availability, echocardiography is not a suitable screening tool. The plasma concentration of B-type natriuretic peptide (BNP) has been the most advocated screening tool. Another alternative is the standard 12-lead electrocardiogram (ECG). Both the ECG and BNP were effective in excluding LVSD in our 75-year-old community-based sample. However, compared with BNP, the ECG had considerably better specificity. In screening for LVSD, BNP had a diagnostic value in addition to the ECG, but only in individuals with abnormal ECGs.</p><p>The left ventricular ejection fraction (LVEF) measured by echocardiography is a well-established index for describing left ventricular systolic function. The wall motion index (WMI) and the amplitude of mitral annulus motion (MAM) are suggested as alternative echocardiographic methods. Compared with MAM, the WMI had a more favourable agreement with the LVEF in our 75-year-old participants. Nonetheless, MAM was a strong predictor of mortality. MAM predicted the risk of all-cause and cardiac mortality independently of other risk factors. In addition, when it came to cardiac mortality, the predictive ability of MAM was independent of the LV function measured as the WMI.</p>

Reduced performance of the left ventricle to eject blood – left ventricular systolic dysfunction (LVSD) – is a common predecessor of the heart failure syndrome. With or without symptoms, LVSD is associated with a poor prognosis. However, with adequate treatment, the development or progression of symptoms, the need for hospitalisation and mortality can all be reduced. In the present work, the occurrence of LVSD was evaluated by echocardiography in a community-based sample of 75-year-old men and women (n = 433). LVSD was a common condition, with a prevalence rate of 6.8%. In nearly half the participants with LVSD, there was no clinical evidence of heart failure. Community-based screening for asymptomatic LVSD has been proposed as a strategy to reduce the incidence of heart failure. Because of the high costs and low availability, echocardiography is not a suitable screening tool. The plasma concentration of B-type natriuretic peptide (BNP) has been the most advocated screening tool. Another alternative is the standard 12-lead electrocardiogram (ECG). Both the ECG and BNP were effective in excluding LVSD in our 75-year-old community-based sample. However, compared with BNP, the ECG had considerably better specificity. In screening for LVSD, BNP had a diagnostic value in addition to the ECG, but only in individuals with abnormal ECGs. The left ventricular ejection fraction (LVEF) measured by echocardiography is a well-established index for describing left ventricular systolic function. The wall motion index (WMI) and the amplitude of mitral annulus motion (MAM) are suggested as alternative echocardiographic methods. Compared with MAM, the WMI had a more favourable agreement with the LVEF in our 75-year-old participants. Nonetheless, MAM was a strong predictor of mortality. MAM predicted the risk of all-cause and cardiac mortality independently of other risk factors. In addition, when it came to cardiac mortality, the predictive ability of MAM was independent of the LV function measured as the WMI.

Calcific aortic valve disease (CAVD) occurs through multiple mutually non-exclusive mechanisms that are mediated by valvular interstitial cells (VICs). VICs undergo pathological differentiation during the progression of valve calcification; however the factors that regulate cellular differentiation are not well defined. Most commonly recognized are biochemical factors that induce pathological differentiation, but little is known regarding the biochemical factors that may suppress this process. Further, the contribution of matrix mechanics in valve pathology has been overlooked, despite increasing evidence of close relationships between changes in tissue mechanics, disease progression and the regulation of cellular response. In this thesis, the effect of matrix stiffness on the differentiation of and calcification by VICs in response to pro-calcific and anti-calcific biochemical factors was investigated. Matrix stiffness modulated the response of VICs to pro-calcific factors, leading to two distinct calcification processes. VICs cultured on the more compliant matrices underwent calcification via osteoblast differentiation, whereas those cultured on the stiffer matrices were prone to myofibroblast differentiation. The transition of fibroblastic VICs to myofibroblasts increased cellular contractility, which led to contraction-mediated, apoptosis-dependent calcification. In addition, C-type natriuretic peptide (CNP), a putative protective molecule against CAVD, was identified. CNP supressed myofibroblast and osteoblast differentiation of VICs, and thereby inhibited calcification in vitro. Matrix stiffness modulated the expression of CNP-regulated transcripts, with only a small number of CNP-regulated transcripts not being sensitive to matrix mechanics. These data demonstrate the combined effects of mechanical and biochemical cues in defining VIC phenotype and responses, with implications for the interpretation of in vitro models of VIC calcification and possibly disease devleopment. The findings from this thesis emphasize the necessity to consider both biochemical and mechanical factors in order to improve fundamental understanding of VIC biology.

BACKGROUND: Sepsis is a clinical syndrome characterised by the systemic response to infection. It is a common problem in modern intensive care units and is associated with significant morbidity and mortality. Though the underlying cause of death is often multifactorial, refractory hypotension and cardiovascular collapse are frequently observed in the terminal phases of the condition. The aetiology of these cardiovascular abnormalities is complex but appears to be mediated by a circulating factor(s). The impact of sepsis upon left ventricular systolic function has been studied extensively. This may be because it is more readily assessed than diastolic function. Despite being increasingly appreciated as a contributor to morbidity and mortality in other clinical settings, there are scant data regarding the evaluation of left ventricular diastolic function in sepsis. Review of the haemodynamic monitoring literature reveals that many conventional measures of left ventricular filling, intravascular volume status and fluid responsiveness are influenced by ventricular diastolic (dys)function, such that interpretation can be challenging in critical care settings. In addition, many available techniques, such as pulmonary artery catheterisation, are invasive and potentially associated with risk to the patient. More robust and less invasive measures of left ventricular diastolic function and filling that can be applied within the intensive care unit (ICU) must be developed. The use of cardiac biomarkers, such as B-type natriuretic peptide (BNP), might represent a novel approach to evaluating left ventricular diastolic function and filling. BNP is released by the myocardium in response to wall stretch/tension. It has demonstrated value in the emergency department diagnosis of heart failure but interpretation of plasma BNP concentrations in critical care remains problematic. At least in part, this appears to relate to the significant number of potential confounders in patients with critical illness. Associations between BNP concentration and diastolic function have not previously been evaluated in severe sepsis and septic shock. The overall aim of this thesis is to investigate the usefulness of plasma BNP concentration in the evaluation of left ventricular diastolic function (including ventricular filling) in severe sepsis and septic shock. DIASTOLIC (DYS)FUNCTION IN SEPSIS: Review of the literature reveals that sepsis is associated with a spectrum of diastolic dysfunction. Characterisation of diastolic function in sepsis is challenging. In this regard, tissue Doppler imaging (TDI), offers promise. TDI is an echocardiographic technique that measures myocardial velocities, which are low frequency, high-amplitude signals filtered from conventional Doppler imaging. TDI has gained acceptance amongst cardiologists in the evaluation of diastolic function, particularly as a measure of ventricular relaxation and ventricular filling pressure; however, there are scant data regarding its use in critical care. We analysed echocardiographs from a large heterogeneous cohort of consecutive ICU patients (n=94) who had TDI as part of their clinically requested echocardiography. As well as supporting the feasibility of TDI in critically ill and mechanically ventilated patients, we demonstrated a wide range of TDI variables and a high prevalence of diastolic dysfunction using this modality. RODENT MODELS OF SEPSIS: We also sought to adapt, refine and evaluate rodent models of sepsis. Such models would allow control for a multitude of potential confounders commonly encountered in clinical sepsis. Two commonly employed rodent models of sepsis include caecal ligation and perforation (CLP) and endotoxin infusion. Comparison between CLP, sham and control groups demonstrated no difference in TDI or BNP. The observed changes in echocardiographic diastolic variables did not reflect those expected in sepsis and may be best explained by increases in heart rate rather than diastolic dysfunction per se. Endotoxaemia was associated with changes consistent with impaired myocardial relaxation (TDI) and reversible myocardial injury (histopathology), as expected in sepsis. BNP did not change significantly from baseline. This might be explained by the potential influence of fluid management upon BNP secretion. CLINICAL RESEARCH: The prediction of fluid responsiveness potentially prevents ineffective, excessive or deleterious intravenous fluid administration. Prospective evaluation of plasma BNP concentration in patients with septic shock found that it was not a predictor of a fluid responsive state. Furthermore, elevated BNP did not rule out a favourable response and therefore does not contraindicate a fluid challenge. Both impaired diastolic dysfunction, especially E/e’, and elevated BNP, have been associated with excess mortality in a range of cardiovascular diseases. These have not previously been compared in septic shock. In a cohort of patients with septic shock, E/e’ was a stronger predictor of mortality than cardiac biomarkers, including BNP. Fluid balance was an independent predictor of BNP in septic shock. OVERALL CONCLUSION: BNP appears not to be clinically useful in the evaluation of ventricular filling or diastolic function in sepsis. The association with fluid balance is a new finding and should be evaluated in a wider range of critically ill patients. In contrast to BNP, TDI appears to be a promising bedside tool in the evaluation of diastolic function and should be further evaluated in critical care.