BACKGROUND: Cardiovascular disease, principally manifest as myocardial infarction or stroke, is the dominant cause of death worldwide and despite therapeutic advances, the global burden of these conditions continues to increase. In order to address this ongoing disease burden, there is a clear need to more effectively target the use of existing and novel diagnostic investigations and medical therapies. Emerging cardiovascular biomarkers include the biochemical, such as high-sensitivity cardiac troponin, and the radiological, such as computed tomography coronary angiography (CTCA) and 18Ffluoride positron emission tomography (PET). Cardiac troponins can now be reliably quantified in clinically stable or asymptomatic populations and provide information about myocardial pathophysiology, whilst CTCA can non-invasively quantify atherosclerotic burden and 18F-fluoride PET imaging offers insight into plaque vulnerability. Improved targeting of diagnostic investigations requires more reliable estimation of pre-test probability of coronary disease whilst optimizing the use of pharmacological or interventional treatments requires more accurate prognostic stratification. Achieving both objectives in an equitable manner across all population groups will depend upon updated clinical guidelines containing improved risk models and enhanced management pathways. The objective of this thesis was to investigate the potential clinical benefit of novel approaches to the diagnostic and prognostic assessment of coronary heart disease. EVALUATION OF THE 2016 NATIONAL INSTITUTE FOR HEALTH AND CARE EXCELLENCE (NICE) GUIDANCE ON THE ASSESSMENT OF SUSPECTED STABLE ANGINA. A post-hoc analysis was undertaken of the Scottish COmputed Tomography of the HEART (SCOT-HEART) trial of 4,146 participants with suspected angina randomised to assessment with computed tomography coronary angiography or standard care. Patients were dichotomised according to guideline definitions into groups representing possible angina and non-anginal presentations. The primary (diagnostic) endpoint was diagnostic certainty of angina at 6 weeks and the prognostic endpoint comprised fatal and non-fatal myocardial infarction. In 3,770 eligible participants, CTCA increased diagnostic certainty more in those with possible angina (relative risk [RR] 2.22 (95% CI 1.91-2.60), p < 0.001) than those with non-anginal symptoms (RR 1.30 (1.11-1.53), p=0.002; pinteraction < 0.001). In the possible angina cohort, CTCA did not change rates of invasive angiography (p=0.481) but markedly reduced rates of normal coronary angiography (hazard ratio [HR] 0.32 (0.19-0.52), p < 0.001). In the non-anginal cohort, rates of invasive angiography increased (HR 1.82 (1.13-2.92), p=0.014) without reducing rates of normal coronary angiography (HR 0.78 (0.30-2.05), p=0.622). At 3.2 years of follow-up, fatal or nonfatal MI was reduced in patients with possible angina (3.2% to 1.9%; HR 0.58 (0.34- 0.99), p=0.045) but not in those with non-anginal symptoms (HR 0.65 (0.25-1.69), p=0.379). Overall the updated NICE guidance on patient assessment maximises the benefits of CTCA with respect to diagnostic certainty, the use of invasive coronary angiography, and reductions in fatal and non-fatal myocardial infarction. Patients with non-anginal chest pain derive minimal benefit from CTCA, which instead increases rates of invasive investigation. EXTERNAL VALIDATION OF THE PROSPECTIVE MULTICENTER IMAGING STUDY FOR EVALUATION OF CHEST PAIN (PROMISE) TOOL FOR DETERMINING MINIMAL-RISK OF CORONARY ARTERY DISEASE. The PROspective Multicenter Imaging Study for Evaluation of chest pain (PROMISE) minimal-risk tool was recently developed to identify patients with suspected stable angina at very low risk of coronary artery disease and clinical events. The external validity of this tool was investigated within the context of the Scottish Computed Tomography of the HEART multicenter randomised controlled trial of patients with suspected stable angina due to coronary artery disease. Model discrimination and calibration was determined amongst 1,764 patients in whom complete CCTA data were available and compared with the European Society of Cardiology guideline-endorsed Coronary Artery Disease Consortium (CADC) risk score. The PROMISE minimal-risk tool improved discrimination compared with the CADC model (c-statistic 0.785 vs 0.730, p < 0.001) and was improved further following re-estimation of covariate coefficients (c-statistic 0.805, p < 0.001). Model calibration was initially poor (c2 197.6, Hosmer-Lemeshow [HL] p < 0.001), with significant overestimation of probability of minimal risk, but improved significantly following revision of the PROMISE minimal-risk intercept and covariate coefficients (c2 5.6, HL p=0.692). HIGH-SENSITIVITY CARDIAC TROPONIN I IN THE DIAGNOSIS OF STABLE CORONARY ARTERY DISEASE In a pre-specified sub-study of the Scottish COmputed Tomography of the Heart trial, plasma cardiac troponin was measured using a high-sensitivity single molecule counting assay in 943 adults with suspected stable angina who had undergone coronary computed tomography angiography. Rates of obstructive coronary artery disease were compared with the pre-test probability determined by the European Society of Cardiology Coronary Artery Disease Consortium risk model with and without cardiac troponin concentrations. External validation was undertaken in an independent study population from Denmark comprising 487 patients with suspected stable angina. Higher cardiac troponin concentrations were associated with obstructive coronary artery disease with a 5-fold increase across quintiles (9 to 48%, p < 0.001) independent of known cardiovascular risk factors (odds ratio [OR] 1.35 [95% confidence interval (CI) 1.25-1.46] per doubling of troponin). Cardiac troponin concentrations improved the discrimination of the ESC model for identifying obstructive coronary artery disease (c-statistic 0.785 to 0.800, p=0.003) and improved classification into ESCrecommended categories of clinical risk (net reclassification improvement 0.143 [95% CI, 0.093-0.193]). The revised model achieved similar improvements in discrimination and net reclassification when applied in the external validation cohort. HIGH-SENSITIVITY CARDIAC TROPONIN I IN CARDIOVASCULAR RISK STRATIFICATION OF PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE AND HEIGHTENED CARDIOVASCULAR RISK. The association between plasma high-sensitivity cardiac troponin I concentration and cardiovascular events in patients with chronic obstructive pulmonary disease and heightened cardiovascular risk was examined within the context of a double-blind randomised controlled trial of inhaled corticosteroids and bronchodilators (1 placebo arm and 3 different treatment arms). Plasma cardiac troponin I concentrations were measured with a high-sensitivity assay in a subgroup of 1,599 patients. The cardiovascular endpoint was a composite of cardiovascular death, myocardial infarction, stroke, unstable angina and transient ischaemic attack during follow-up of 1.5 years. Baseline plasma cardiac troponin I concentrations were above the lower limit of detection (1.0 ng/L) in 1,559 (97.5%) patients and were unaffected by inhaled therapies at 3 months (p > 0.05 for all). Compared with the lowest tertile (cardiac troponin I ≤3.0 ng/L), patients in the highest tertile (≥ 5.5 ng/L) were at greater risk of cardiovascular events (hazard ratio 3.0, 95% confidence interval 1.5 to 6.2, p=0.002) and cardiovascular death (hazard ratio 9.6, 95% confidence interval 2.6 to 35.6, p < 0.001) after adjustment for cardiovascular risk factors. There were no differences in COPD exacerbations between tertiles even after adjustment (p > 0.05). / REPRODUCIBILITY OF CORONARY 18F-FLUORIDE PET-CT IMAGING The inter-observer and scan-rescan reproducibility of coronary 18F-fluoride PET-CT imaging was investigated in 20 patients with clinically stable but high risk multi-vessel coronary artery disease who underwent repeated 18F-fluoride PET-CT scans 11.5±4.5 days apart. Scan analysis using the currently accepted approach of normalisation to a referent coronary segment (TBRREFERENT) identified 10 (50%) patients with evidence of focal coronary 18F-fluoride uptake and demonstrated moderate agreement across observers on a per-patient level (k = 0.56). This was similar to the level of agreement achieved with visual assessment alone (k = 0.64). Reproducibility was improved by semi-quantitative reporting combining visual assessment with a threshold uptake value for determining the presence of tracer uptake (k = 0.84). Using the optimised approach achieved excellent agreement on overall segmental uptake counts (intra-class correlation = 0.97). CONCLUSION: Cardiovascular diagnostic and prognostic assessments represent a complex endeavour and established tools for risk prediction can demonstrate suboptimal predictive accuracy when evaluated in patient cohorts that are independent of the population used for model derivation.

A insuficiência cardíaca é a doença crônica com maior impacto na sobrevida e qualidade de vida dos pacientes. Apesar do constante desenvolvimento de novas estratégias de tratamento, esta doença continua atingindo altos índices de mortalidade. O coração adulto tem capacidade de regeneração limitada e há grande evidência experimental de que os transplantes de células-tronco poderiam ser uma abordagem eficiente na recuperação do miocárdio lesado. Contudo, a maioria dos estudos são realizados em cardiomiopatias isquêmicas, existindo poucos estudos na cardiomiopatia dilatada (CMD). Em função disto, este trabalho foi realizado com o objetivo de avaliar a regeneração do miocárdio em coelhos com CMD induzida pela doxorrubicina, por meio do uso de células-tronco mesenquimais (MSC) obtidas de tecido adiposo, associadas ou não com plasma rico em plaquetas (PRP). Foram utilizadas 40 coelhas, Nova Zelândia, e um coelho macho doador das MSCs derivadas do tecido adiposo. As coelhas foram divididas em dois grupos: CMD induzida pela doxorrubicina e o grupo saudável. Cada grupo foi subdividido conforme o tratamento recebido: solução fisiológica, MCSs, PRP e MSCs associadas ao PRP. Os subgrupos receberam o tratamento por injeção diretamente no miocárdio no ventrículo esquerdo mediante toracoscopia vídeo assistida. Os coelhos foram avaliados por exames de ecocardiograma, eletrocardiograma, troponina I, no dia da chegada, após a indução da CMD e 15 dias após o recebimento das terapias. Nesta última avaliação, foi realizada a eutanásia e coletado o coração para análise histológica. Foi observado que após a indução, a troponina I se elevou, o segmento QRS visto no eletrocardiograma, aumentou e, no ecocardiograma, as frações de ejeção (FE) e encurtamento (FS) diminuíram e o diâmetro sistólico do ventrículo esquerdo (VEs) aumentou, em todos os animais avaliados. Após os tratamentos, o subgrupo MSCs obtiveram os melhores resultados em todas as análises citadas. Houve menor elevação da troponina I, o segmento QRS diminuiu, as FS e FE aumentaram e o VEs diminuiu. No exame histopatógico, analisado pela coloração de hematoxicilina-eosina, constatou-se que o subgrupo MSCs apresentou menos lesões, e nos subgrupos MSCs associadas com PRP, solução fisiológica e PRP as lesões aumentaram gradualmente, respectivamente. Os resultados sugerem que o uso das MSCs melhoraram a função cardíaca em coelhos com cardiomiopatia dilatada e que há necessidade de mais estudos no uso de PRP no miocárdio. / Heart failure is a chronic disease with major impact on survival and quality of patient’s life. Despite the constant development of new treatment strategies, this disease still affects high mortality rates. The adult heart has limited ability to regenerate and there is experimental evidence that large transplants of stem cells could be an effective approach in the recovery of injured myocardium. However, most studies are performed in ischemic cardiomyopathy, there are few studies in dilated cardiomyopathy. Because of this, this study aimed at evaluating the regeneration of the myocardium in rabbits with dilated cardiomyopathy induced by doxorubicin through the use of mesenchymal stem cell (MSC) derived from adipose tissue, associated or not with platelet-rich plasma (PRP). 40 New Zealand rabbits were utilized and a male rabbit donor MSCs derived from adipose tissue. The rabbits were divided into two groups: dilated cardiomyopathy doxorubicin-induced and the healthy group. Each group was divided according to treatment received: saline, MSCs, PRP and MSCs associated with PRP. The subgroups receiving treatment through an injection directly into the myocardium of the left ventricle through video-assisted thoracoscopy. The rabbits were evaluated by echocardiogram, electrocardiogram, troponin I, on the day of arrival, after induction of dilated cardiomyopathy and 15 days after receipt of therapies. This last evaluation, euthanasia was performed and the hearts collected for histological analysis. It was observed that after induction the troponin I increased, the QRS segment, seen on the electrocardiogram, increased, and, in echocardiography, the ejection and shortening fractions decreased, and left ventricular systolic diameter increased in all animals evaluated. After treatments, the subgroup MSCs have the best results in all tests cited. There was a lower elevation of troponin I, decreased QRS segment, the ejection and shortening fractions increased and left ventricular systolic diameter decreased. On examination histologic, analyzed by hematoxylin-eosin staining, the subgroup found that MSCs had fewer injuries, and in the subgroups MSCs associated with PRP, PRP and saline lesions gradually increased, respectively. The results suggest that the use of MSCs improved cardiac function in rabbits with dilated cardiomyopathy and that there is need for more studies on the use of PRP in the myocardium.

Em todo o mundo, milhões de pacientes são atendidos em emergências por apresentarem dor torácica de início aguda, mas apenas uma parcela deve-se a síndrome coronariana aguda (SCA). Em situações como essa é de extrema importância distinguir quando a dor torácica é devido à isquemia do miocárdio, pois esta é de alto risco e o início do tratamento deve ser imediato. Novos biomarcadores são necessários para auxiliar no diagnóstico e conduta clínica a ser tomada diante de situações de emergência como esta. Recentemente a quantificação de troponinas através de ensaios ultrassensíveis tem sido amplamente utilizado para diagnósticos e prognóstico de isquemia cardíaca, porém esses ensaios não tiveram seus valores de referências estabelecidos e validados para diversas situações clínicas. O presente estudo identificou a troponina I cardíaca nitrada como um novo biomarcador para isquemia cardíaca. Através de experimentos de imunoluorecência, foi possível colocalizar a marcação de troponina I cardíaca e nitrotirosina em modelos celulares e murinos de isquemia cardíaca, sugerindo assim que a troponina I cardíaca é nitrada. A partir do soro de modelos porcinos de isquemia, foi realizado o enriquecimento de proteínas nitradas por imunoprecipitação seguido da identificação da troponina I cardíaca por western blot. Dessa maneira foi possível identificar a troponina I cardíaca nitrada no soro poucos minutos após o evento x isquêmico, a qual permaneceu circulante por até 24 horas. Nessas mesmas amostras outros biomarcadores de isquemia como CKMB, Troponina I e Troponina T ultrassensível foram dosados e nenhum marcador de elevou após a isquemia cardíaca seguida de reperfusão. A troponina I cardíaca nitrada foi caracterizada por espectrometria de massas. Esse proteína é um potencial marcador circulante sensível para o diagnóstico e prognóstico precoce de isquemia cardíaca com ou sem necrose do miocárdio / Worldwide, millions of patients are treated in emergencies because they had acute-onset chest pain, but only a portion is due to coronary syndrome. In situations like this is extremely important to distinguish when the chest pain is due to myocardial ischemia, as this is high risk and initiation of treatment should be immediate. New biomarkers are needed to assist clinical decision-making in ACS. Recently, the quantification of ultra-sensitive tests for troponins has been widely used for diagnosis and prognosis of myocardial ischemia, however the reference values was not well validated and established for different subjects groups. The present study identified the nitrated cardiac troponin I as a novel biomarker of cardiac ischemia. We performed immunofluorescence colocalization marking of cardiac troponin I and nitrotyrosine in cell and rat model of cardiac ischemia, suggesting that cardiac troponin I is a nitrated protein. From serum of porcine models cardiac ischemia was made enrichment of nitrated proteins by immunoprecipitation with anti-nitrotyrosine followed by detection of cardiac troponin I by western blot. It was possible to identify the cardiac troponin I in serum nitrated few minutes after the ischemic event, which remains current for up to 24 hours. In these samples, other markers of cardiac ischemia such as CK-MB, troponin I and ultra-sensitive troponin T did not increase after ischemia followed by reperfusion. Nitrated cardiac troponin I was characterized by MS/MS. The xii nitrated cardiac troponin I is a potential circulating marker sensitive for the diagnosis and prognosis for early cardiac ischemia with or without myocardial necrosis

A insuficiência cardíaca é a doença crônica com maior impacto na sobrevida e qualidade de vida dos pacientes. Apesar do constante desenvolvimento de novas estratégias de tratamento, esta doença continua atingindo altos índices de mortalidade. O coração adulto tem capacidade de regeneração limitada e há grande evidência experimental de que os transplantes de células-tronco poderiam ser uma abordagem eficiente na recuperação do miocárdio lesado. Contudo, a maioria dos estudos são realizados em cardiomiopatias isquêmicas, existindo poucos estudos na cardiomiopatia dilatada (CMD). Em função disto, este trabalho foi realizado com o objetivo de avaliar a regeneração do miocárdio em coelhos com CMD induzida pela doxorrubicina, por meio do uso de células-tronco mesenquimais (MSC) obtidas de tecido adiposo, associadas ou não com plasma rico em plaquetas (PRP). Foram utilizadas 40 coelhas, Nova Zelândia, e um coelho macho doador das MSCs derivadas do tecido adiposo. As coelhas foram divididas em dois grupos: CMD induzida pela doxorrubicina e o grupo saudável. Cada grupo foi subdividido conforme o tratamento recebido: solução fisiológica, MCSs, PRP e MSCs associadas ao PRP. Os subgrupos receberam o tratamento por injeção diretamente no miocárdio no ventrículo esquerdo mediante toracoscopia vídeo assistida. Os coelhos foram avaliados por exames de ecocardiograma, eletrocardiograma, troponina I, no dia da chegada, após a indução da CMD e 15 dias após o recebimento das terapias. Nesta última avaliação, foi realizada a eutanásia e coletado o coração para análise histológica. Foi observado que após a indução, a troponina I se elevou, o segmento QRS visto no eletrocardiograma, aumentou e, no ecocardiograma, as frações de ejeção (FE) e encurtamento (FS) diminuíram e o diâmetro sistólico do ventrículo esquerdo (VEs) aumentou, em todos os animais avaliados. Após os tratamentos, o subgrupo MSCs obtiveram os melhores resultados em todas as análises citadas. Houve menor elevação da troponina I, o segmento QRS diminuiu, as FS e FE aumentaram e o VEs diminuiu. No exame histopatógico, analisado pela coloração de hematoxicilina-eosina, constatou-se que o subgrupo MSCs apresentou menos lesões, e nos subgrupos MSCs associadas com PRP, solução fisiológica e PRP as lesões aumentaram gradualmente, respectivamente. Os resultados sugerem que o uso das MSCs melhoraram a função cardíaca em coelhos com cardiomiopatia dilatada e que há necessidade de mais estudos no uso de PRP no miocárdio. / Heart failure is a chronic disease with major impact on survival and quality of patient’s life. Despite the constant development of new treatment strategies, this disease still affects high mortality rates. The adult heart has limited ability to regenerate and there is experimental evidence that large transplants of stem cells could be an effective approach in the recovery of injured myocardium. However, most studies are performed in ischemic cardiomyopathy, there are few studies in dilated cardiomyopathy. Because of this, this study aimed at evaluating the regeneration of the myocardium in rabbits with dilated cardiomyopathy induced by doxorubicin through the use of mesenchymal stem cell (MSC) derived from adipose tissue, associated or not with platelet-rich plasma (PRP). 40 New Zealand rabbits were utilized and a male rabbit donor MSCs derived from adipose tissue. The rabbits were divided into two groups: dilated cardiomyopathy doxorubicin-induced and the healthy group. Each group was divided according to treatment received: saline, MSCs, PRP and MSCs associated with PRP. The subgroups receiving treatment through an injection directly into the myocardium of the left ventricle through video-assisted thoracoscopy. The rabbits were evaluated by echocardiogram, electrocardiogram, troponin I, on the day of arrival, after induction of dilated cardiomyopathy and 15 days after receipt of therapies. This last evaluation, euthanasia was performed and the hearts collected for histological analysis. It was observed that after induction the troponin I increased, the QRS segment, seen on the electrocardiogram, increased, and, in echocardiography, the ejection and shortening fractions decreased, and left ventricular systolic diameter increased in all animals evaluated. After treatments, the subgroup MSCs have the best results in all tests cited. There was a lower elevation of troponin I, decreased QRS segment, the ejection and shortening fractions increased and left ventricular systolic diameter decreased. On examination histologic, analyzed by hematoxylin-eosin staining, the subgroup found that MSCs had fewer injuries, and in the subgroups MSCs associated with PRP, PRP and saline lesions gradually increased, respectively. The results suggest that the use of MSCs improved cardiac function in rabbits with dilated cardiomyopathy and that there is need for more studies on the use of PRP in the myocardium.

Ao contrário do homem, as cardiopatias de natureza hipóxica/isquêmica são pouco relatadas nos cães. Raros são os relatos de infarto agudo do miocárdio (IAM) nesses espécimes; entretanto, existem achados eletrocardiográficos que indicam hipóxia/isquemia miocárdica, como os desníveis (infra e supra) do segmento ST. Com o intuito de constatar algum dano nas células do miocárdio em condições de má perfusão, utilizaram-se 38 cães, dos quais 20 com traçados eletrocardiográficos normais e 18 com desníveis (infra e supra) do segmento ST, na derivação DII, velocidade de 50 mm/s e sensibilidade N. Nos animais normais (grupo 1), a dosagem sérica da enzima creatinafosfoquinase isoenzima MB (CK-MB) e da troponina I (cTnI) destinou-se à obtenção dos valores de referência (em ng/mL). Estes valores de referência foram confrontados com os obtidos de cães portadores de desnível (grupo 2), permitindo confirmar ou não a injúria miocárdica. Em relação à CK-MB, os cães do grupo 1 apresentaram média de 0,54 ng/mL e desvio-padrão de 0,89 ng/mL e os do grupo 2 apresentaram média de 0,44 ng/mL e desvio-padrão de 1,106 ng/mL. A média e o desvio-padrão foram, respectivamente, de 0,16 ng/mL e 0,110 ng/mL e de 0,20 ng/mL e 0,111 ng/mL, nos grupos 1 e 2. Houve 18 valores nulos de CK-MB, igualmente distribuídos entre ambos os grupos. O grupo 1 apresentou três valores nulos para cTnI. Houve diferença marcante em relação à idade, sendo o grupo 1 constituído por animais, preponderantemente, abaixo de 7 anos; o contrário ocorreu no grupo 2. São significativas, ao nível de significância de 5%, as associações da variável CK-MB com as variáveis idade, massa e CK-T (creatinafosfoquinase total) no grupo 1, e com a variável CK-T no grupo 2. A variável cTnI não apresentou evidências de associação, ao nível de significância de 5% , com as variáveis idade, massa, CK-T e nível sérico de potássio, para cada um dos níveis da variável grupo. Tanto para a variável CK-MB quanto para a cTnI, não houve diferenças significativas, ao nível de 5%, entre os grupos 1 e 2. Conclui-se que é possível a utilização do \"kit\" de ensaio imunométrico quimioluminescente humano para a espécie canina e que a hipóxia/isquemia, revelada pelo desnível do segmento ST, não acarreta dano miocárdico, ou que este é mínimo. / Although very often in men, hypoxic and ischemic heart diseases are poorly documented in dogs. There are few reports of acute myocardial infarction (AMI) in this species. However, some electrocardiographic findings may suggest myocardium hypoxia/ischemia, like ST segment elevation or depression. In order to investigate myocardial cells injury in poor perfusion conditions, 38 dogs, being 20 with normal electrocardiogram and 18 with ST segment elevation or depression in lead II, at a paper speed of 50 mm/sec and N sensibility (1 mV = 1cm), were included. Serum measurement of creatinephosphokinase isoenzyme MB (CK-MB) enzym and troponin I (cTnI) in normal dogs (group 1) determined reference values (in ng/ml). These values were compared to those obtained in dogs with deviation (group 2), which allowed confirmation or not of myocardial injury. CK-MB mean values obtained from dogs in groups 1 and 2 was 0,54 ng/mL (SD±0,54 ng/mL) and 0,44 ng/mL (SD±1,106), respectively. Mean cTnI values in groups 1 and 2 was 0,16 ng/mL (SD±0,110 ng/mL) and 0,20 ng/mL (SD±0,111 ng/mL) respectively. Three cTnI null values were found in group 1. There was a marked difference concerning to age, being group 1 composed, mainly, by animals ageing under 7 years, on the contrary of group 2. At a significance level of 5%, was significant the relation of CK-MB with age, mass and total creatinephosphokinase (CK-T) in group 1 and with CK-T in group 2. There is no relation, at a significance level of 5%, of cTnI with age, mass, CK-T or serum potassium concentration, for each level of group variable. Both CK-MB and cTnI variables showed no difference, at 5% level, between groups 1 and 2. In conclusion, it is possible to use the human chemiluminescent immunometric assay kit in canine species and that hypoxia/ischemia revealed by ST segment deviation does not mean significant myocardium injury.

A insuficiência cardíaca é a doença crônica com maior impacto na sobrevida e qualidade de vida dos pacientes. Apesar do constante desenvolvimento de novas estratégias de tratamento, esta doença continua atingindo altos índices de mortalidade. O coração adulto tem capacidade de regeneração limitada e há grande evidência experimental de que os transplantes de células-tronco poderiam ser uma abordagem eficiente na recuperação do miocárdio lesado. Contudo, a maioria dos estudos são realizados em cardiomiopatias isquêmicas, existindo poucos estudos na cardiomiopatia dilatada (CMD). Em função disto, este trabalho foi realizado com o objetivo de avaliar a regeneração do miocárdio em coelhos com CMD induzida pela doxorrubicina, por meio do uso de células-tronco mesenquimais (MSC) obtidas de tecido adiposo, associadas ou não com plasma rico em plaquetas (PRP). Foram utilizadas 40 coelhas, Nova Zelândia, e um coelho macho doador das MSCs derivadas do tecido adiposo. As coelhas foram divididas em dois grupos: CMD induzida pela doxorrubicina e o grupo saudável. Cada grupo foi subdividido conforme o tratamento recebido: solução fisiológica, MCSs, PRP e MSCs associadas ao PRP. Os subgrupos receberam o tratamento por injeção diretamente no miocárdio no ventrículo esquerdo mediante toracoscopia vídeo assistida. Os coelhos foram avaliados por exames de ecocardiograma, eletrocardiograma, troponina I, no dia da chegada, após a indução da CMD e 15 dias após o recebimento das terapias. Nesta última avaliação, foi realizada a eutanásia e coletado o coração para análise histológica. Foi observado que após a indução, a troponina I se elevou, o segmento QRS visto no eletrocardiograma, aumentou e, no ecocardiograma, as frações de ejeção (FE) e encurtamento (FS) diminuíram e o diâmetro sistólico do ventrículo esquerdo (VEs) aumentou, em todos os animais avaliados. Após os tratamentos, o subgrupo MSCs obtiveram os melhores resultados em todas as análises citadas. Houve menor elevação da troponina I, o segmento QRS diminuiu, as FS e FE aumentaram e o VEs diminuiu. No exame histopatógico, analisado pela coloração de hematoxicilina-eosina, constatou-se que o subgrupo MSCs apresentou menos lesões, e nos subgrupos MSCs associadas com PRP, solução fisiológica e PRP as lesões aumentaram gradualmente, respectivamente. Os resultados sugerem que o uso das MSCs melhoraram a função cardíaca em coelhos com cardiomiopatia dilatada e que há necessidade de mais estudos no uso de PRP no miocárdio. / Heart failure is a chronic disease with major impact on survival and quality of patient’s life. Despite the constant development of new treatment strategies, this disease still affects high mortality rates. The adult heart has limited ability to regenerate and there is experimental evidence that large transplants of stem cells could be an effective approach in the recovery of injured myocardium. However, most studies are performed in ischemic cardiomyopathy, there are few studies in dilated cardiomyopathy. Because of this, this study aimed at evaluating the regeneration of the myocardium in rabbits with dilated cardiomyopathy induced by doxorubicin through the use of mesenchymal stem cell (MSC) derived from adipose tissue, associated or not with platelet-rich plasma (PRP). 40 New Zealand rabbits were utilized and a male rabbit donor MSCs derived from adipose tissue. The rabbits were divided into two groups: dilated cardiomyopathy doxorubicin-induced and the healthy group. Each group was divided according to treatment received: saline, MSCs, PRP and MSCs associated with PRP. The subgroups receiving treatment through an injection directly into the myocardium of the left ventricle through video-assisted thoracoscopy. The rabbits were evaluated by echocardiogram, electrocardiogram, troponin I, on the day of arrival, after induction of dilated cardiomyopathy and 15 days after receipt of therapies. This last evaluation, euthanasia was performed and the hearts collected for histological analysis. It was observed that after induction the troponin I increased, the QRS segment, seen on the electrocardiogram, increased, and, in echocardiography, the ejection and shortening fractions decreased, and left ventricular systolic diameter increased in all animals evaluated. After treatments, the subgroup MSCs have the best results in all tests cited. There was a lower elevation of troponin I, decreased QRS segment, the ejection and shortening fractions increased and left ventricular systolic diameter decreased. On examination histologic, analyzed by hematoxylin-eosin staining, the subgroup found that MSCs had fewer injuries, and in the subgroups MSCs associated with PRP, PRP and saline lesions gradually increased, respectively. The results suggest that the use of MSCs improved cardiac function in rabbits with dilated cardiomyopathy and that there is need for more studies on the use of PRP in the myocardium.

Com o objetivo de avaliar se o exercício físico prolongado causa alterações miocárdicas em eqüinos de enduro e se estas alterações cardíacas têm influência no desempenho dos animais durante as provas, avaliaram-se 30 cavalos, divididos em três grupos de dez animais cada, sendo G1 composto por animais que percorreram distâncias acima de 100 km, G2 por animais que percorreram distâncias menores de 100 km e G3 por animais desqualificados por alterações metabólicas. Os animais foram avaliados em três momentos distintos, T0 (pré-exercício, em repouso), T1 (entre 30 e 60 minutos após o exercício) e T2 (entre 90 e 120 minutos pós-exercício). Realizaram-se o exame físico e o exame ecocardiográfico, além de colheita de amostras de sangue para determinação de troponina I cardíaca sérica e outras provas bioquímicas. Não houve diferença nos valores de troponina I cardíaca entre os diversos grupos nem nos diferentes tempos. Observou-se diminuição dos valores do diâmetro interno do ventrículo esquerdo em diástole e aumento de espessura de septo interventricular pós-exercício. Não houve diferença nos índices funcionais cardíacos e houve manutenção do débito cardíaco por aumento da freqüência cardíaca. Estas alterações ecocardiográficas de pequena magnitude foram mais evidentes nos animais desqualificados por alterações metabólicas e não parecem estar relacionadas a injúria miocárdica e sim secundárias a outras condições orgânicas. Conclui-se que o exercício físico prolongado não leva a injúrias cardíacas severas em cavalos de enduro. / With the purpose of evaluating myocardial alterations caused by prolonged physical exercise and whether these alterations influence the endurance horses\' performance during the races, 30 horses were evaluated, divided into three groups of ten horses each, being G1 composed by animals that performed distances of more than 100 km, G2 by animals that performed distances of less than 100 km and G3 by animals disqualified by metabolic alterations. The horses were evaluated in three distinct moments, T0 (pre-exercise, at rest), T1 (between 30 and 60 minutes post-exercise) and T2 (between 90 and 120 minutes post-exercise). Physical and echocardiographic examinations and else blood sample collection for the determination of cardiac troponin I and other biochemical tests were done. There was no difference in cardiac troponin I values neither between the various groups, nor between the moments. There was a decrease in the post-exercise values of the diastolic left ventricle internal diameter and an increase in the post-exercise values of the interventricular septal thickness. There was no difference in the cardiac functional indexes and the cardiac output was maintainded by augmentation of the heart rate. These minor echocardiographic alterations were more evident in the animals that were disqualified by metabolic alterations, and they don\'t seem to be related to myocardial injury, but secondary to other organic conditions. Based on these results, prolonged physical exercise doesn\'t seem to cause severe cardiac injuries in endurance horses.

O diagnóstico da tromboembolia pulmonar aguda (EPA) e a avaliação da gravidade desta condição é desafiador. Enquanto as concentrações de troponina I cardíaca (TI) já estão bem estabelecidas quanto ao risco de estratificação, não há estudos prévios que tenham examinado se há alguma relação linear entre as concentrações de TI cardíaca e a gravidade da EPA. Além disso, as metaloproteinases (MMPs) da matriz extracelular estão envolvidas na fisiopatologia da EPA. Entretanto, é desconhecido se o aumento da atividade gelatinolítica das MMPs após a EPA reflete a gravidade desta condição. Nós examinamos se as concentrações circulantes destes biomarcadores aumentam em proporção à gravidade da EPA experimental induzida em cães anestesiados. A EPA foi induzida com coágulos de sangue autólogo (salina, 1, 3 ou 5 mL/Kg) injetados no átrio direito. As avaliações hemodinâmicas foram realizadas no momento basal e 120 minutos após a EPA. Da mesma forma, foram realizadas as quantificações de troponina I no soro e a zimografia das MMPs 2 e 9 no plasma. Nossos resultados sugerem não haver aumento significativo da atividade gelatinolítica da pró-MMP-2 no plasma após a EPA, enquanto que a atividade da pró-MMP-9 aumenta em 80% apenas no grupo que recebeu 5 mL/Kg de coágulos. A TI cardíaca no soro e a atividade da pró-MMP-9 no plasma tiveram uma correlação positiva com o índice de resistência vascular pulmonar (p=0,007 e rs=0,833 para a TI, e p=0,034 e rs=0,684 para a pró-MMP-9) e com a pressão média na artéria pulmonar (p=0,005 e rs=0,610 para a TI, e p=0,022 e rs=0,720 para a pró-MMP-9). Concluímos que a TI cardíaca e a pró-MMP-9 circulantes aumentam em proporção à gravidade da EPA, embora o aumento da pró-MMP-9 não seja muito evidente em graus menos severos da EPA. Estes achados podem ser relevantes para a clínica da EPA. / Making the diagnosis of acute pulmonary thromboembolism (APT) and assessing its severity is very challenging. While cardiac troponin I (CTI) levels are promising in risk stratification, no previous study has examined whether there is a linear relation between CTI levels and the severity of APT. Moreover, matrix metalloproteinases (MMPs) are involved in the pathophysiology of APT. However, it is unknown whether the increases in MMP levels after APT reflect the severity of this condition. We examined whether the circulating levels of these biomarkers increase in proportion to the severity of experimental APT induced in anesthetized dogs. APT was induced with autologous blood clots (saline, 1, 3, or 5 mL/kg) injected into the right atrium. Hemodynamic evaluations were carried out for 120 min. Gelatin zymography of MMP-2 and MMP-9 from plasma samples were performed and serum CTI levels were determined at baseline and 120 min after APT. Our results sugest that while no significant increases in pro-MMP-2 levels were found after APT, pro-MMP-9 levels increased by 80% only after 5 mL/kg of clot embolization. Serum CTI and plasma pro-MMP-9 levels correlated positively with pulmonary vascular resistance (p=0.007 and rs=0.833 for troponin I, and p=0.034 and rs=0.684 for pro-MMP-9) and with pulmonary artery pressure (p=0.005 and rs=0.610 for troponin I, and p=0.022 and rs=0.720 for pro-MMP-9). We conclude that circulating CTI and pro-MMP-9 increase in proportion to the severity of APT, although the increases in plasma pro-MMP-9 are less clear with less severe APT. These findings may be relevant for clinical APT.

Abstract The first aim of this study was to investigate the relationship between Doppler ultrasonographic parameters and biochemical markers of human fetal cardiac dysfunction and myocardial cell damage in pregnancies complicated by placental insufficiency and/or fetal growth restriction. Our second aim was to examine fetal central and peripheral hemodynamic characteristics associated with retrograde aortic isthmus net blood flow. Fetuses with significant myocardial cell damage (cTnT > 0.10 ng/ml) had increased pulsatility in the blood velocity waveforms of ductus venosus, left hepatic vein and inferior vena cava, and had more often atrial pulsations in the umbilical vein. Their umbilical artery NT-proANP concentrations were higher than in fetuses without myocardial cell damage. The proportion of left ventricular cardiac output of the combined cardiac output was greater and the corresponding proportion of the right ventricle was less than in fetuses with only increased NT-proANP levels ( > 1145 pmol/l). Tricuspid regurgitation was present more often and the right ventricular fractional shortening was less in fetuses with myocardial cell damage than in fetuses with normal umbilical artery cTnT levels. In fetuses with placental insufficiency and/or growth restriction (n = 48), umbilical artery NT-proANP concentrations showed a significant positive correlation with ductus venosus, left hepatic vein and inferior vena cava pulsatility index values for veins. Fetuses with placental insufficiency and antegrade aortic isthmus net blood flow demonstrated a shift in their right ventricular cardiac output from the pulmonary to the systemic circulation, and foramen ovale volume blood flow made up the majority of the left ventricular cardiac output. Fetuses with retrograde aortic isthmus net blood flow failed to demonstrate these changes, and they had signs of increased left atrial pressure. In addition, right ventricular fractional shortening was decreased and the pulsatility in the ductus venosus blood velocity waveforms was increased. In conclusion, human fetal myocardial cell damage was associated with a rise in systemic venous pressure, a change in the distribution of cardiac output towards the left ventricle and a rise in right ventricular afterload. Fetuses with retrograde aortic isthmus net blood flow failed to rearrange the distribution of the cardiac output and they had signs of increased left atrial pressure. In addition, right ventricular afterload and pulsatility in the ductus venosus blood velocity waveforms were increased.

Numerous troponin T (TnT) isoforms are generated by alternative RNA splicing primarily in its NH2-terminal hypervariable region, but the functions of these isoforms are not completely understood. In this dissertation work, calcium and terbium binding behavior of several forms of TnT were investigated by spectroscopic and radioactive techniques. Chicken breast muscle TnT binds calcium and terbium through its NH2-terminal Tx motif (HEEAH)n with high affinity (10-6 mM) and fast on-rate (106 - 107 M-1 s-1). Chicken leg muscle TnT and a human cardiac TnT NH2-terminal fragment, which both lack the Tx motif on their NH2-terminal regions, do not have affinities for calcium in the physiological range. Computational predictions on TnT N47 suggest that the TnT NH2-terminal region might fold into an elongated structure with at least one high affinity metal ion binding pocket comprised primarily of the Tx motif sequence and several lower affinity binding sites. In addition, calcium binding to TnT N47 might alter its conformation and flexibility. Luminescence resonance energy transfer measurements and other experimental observations are consistent with the computational predictions suggesting the computational simulated atomic model is reasonable. TnT mutations are responsible for 15% of familiar hypertrophic cardiomyopathy (FHC) cases with a phenotype of relatively mild hypertrophy, but a high incidence of sudden death. Detection of those genetic mutations would facilitate the clinical diagnosis and initiation of treatment at an early stage. This dissertation also investigated a novel hybridization proximity assay (HYPA) combining molecular beacon and luminescence resonance energy transfer (LRET) technologies. Experimental results suggest that a shared stem probe design produces a more consistent response upon hybridization, whereas the internally labeled probe was less consistent, but can yield the highest responses. Using the optimally designed molecular probes, the HYPA provides a detection of alterations in nucleic acid structure of as little as a single nucleotide. This novel HYPA is expected to expand its applications in the analysis and screening of genetic diseases.