Myocardial infarction is a leading cause of morbidity and mortality worldwide. The purpose of this thesis was to develop strategies for the assessment of patients with suspected myocardial infarction using a high-sensitivity cardiac troponin I assay, and to evaluate the relationship between the aetiology of myocardial infarction and long term clinical outcomes to identify opportunities to modify outcomes. In the United Kingdom, approximately 1 million patients present to hospital with chest pain each year and are assessed for suspected myocardial infarction, yet fewer than 20% of patients receive this diagnosis. Prior clinical standards mandated the admission of patients for serial cardiac troponin testing to identify myocardial necrosis and determine if myocardial infarction had occurred. However, new high-sensitivity assays offer a magnitude improvement in diagnostic precision, and as such provide a novel approach to diagnose or exclude myocardial infarction at an earlier stage. In our first study, I evaluate the performance of a high-sensitivity cardiac troponin I assay as a risk stratification tool in patients with suspected acute coronary syndrome. A systematic review and individual patient-level data meta-analysis was performed, including prospective studies measuring high-sensitivity cardiac troponin I in patients with suspected acute coronary syndrome, where the diagnosis was adjudicated according to the universal definition of myocardial infarction. The primary outcome was myocardial infarction or cardiac death during the index hospitalization or at 30 days. Meta-estimates for primary and secondary outcomes were derived using a binomial-normal random effects model. Performance was evaluated in subgroups and across a range of troponin concentrations (2-16 ng/L) using individual patient data. A total of 22,457 patients were included in the meta-analysis (age 62 [15.5] years; n=9,329 (41.5%) women), of whom 2,786 (12.4%) experienced myocardial infarction or cardiac death at 30 days. Cardiac troponin I concentrations were < 5 ng/L at presentation in 11,012 (49%) patients, with a negative predictive value of 99.5% (95% confidence interval [CI] 99.3-99.6) for myocardial infarction or cardiac death at 30 days. Lower thresholds did not improve safety, but did significantly reduce the proportion identified as low risk. This threshold of 5 ng/L formed the basis for the development of a diagnostic pathway for patients with suspected acute coronary syndrome. In a cohort study of 1,218 patients with suspected acute coronary syndrome who underwent high-sensitivity cardiac troponin I measurement at presentation, 3 and 6 or 12 hours, I derived and validated a novel pathway (rule out myocardial infarction if < 5 ng/L at presentation, or change < 3 ng/L and < 99th centile at 3 hours), and compared this with the established European Society of Cardiology 3-hour pathway (rule out myocardial infarction if < 99th centile at presentation, or at 3 hours if symptoms < 6 hours). The primary outcome was a comparison of the negative predictive value (NPV) of both pathways for myocardial infarction or cardiac death at 30 days. The primary outcome was evaluated in pre-specified subgroups stratified by age, gender, time of symptom onset and known ischaemic heart disease. In those < 99th centile at presentation, the ESC pathway ruled out myocardial infarction in 28.1% (342/1,218) and 78.9% (961/1,218) at presentation and 3 hours respectively, missing 18 index and two 30-day events (NPV 97.9%, 95% confidence intervals [CI] 96.9-98.7%). The novel pathway ruled out 40.7% (496/1,218) and 74.2% (904/1,218) at presentation and 3 hours, missing two index and two 30-day events (NPV 99.5%, 95% CI 99.0-99.9%; P < 0.001 for comparison). The NPV of the novel pathway was greater than the ESC pathway overall (P < 0.001), and in all subgroups including those presenting early or known to have ischaemic heart disease. There are a number of additional approaches for the rule out of myocardial infarction. Clinical risk scores apply conventional risk factors to estimate the probability of myocardial infarction. The most widely implemented scores, HEART, EDACS, GRACE and TIMI, have been extensively validated when used alongside contemporary troponin assays, however, their impact on pathways applying high-sensitivity cardiac troponin testing is less clear. In 1,935 patients with suspected acute coronary syndrome, I evaluated the safety and efficacy of our novel pathway or the European Society of Cardiology 3-hour pathway alone, or in conjunction with low-risk TIMI (0 or 1), GRACE (≤108), EDACS (< 16) or HEART (≤3) scores. Myocardial infarction or cardiac death at 30-days occurred in 14.3% (276/1,935). The ESC pathway ruled out 70% with 27 missed events giving a negative predictive value (NPV) of 97.9% (95% confidence interval [CI], 97.1 to 98.6%). Addition of a HEART score ≤3 reduced the proportion ruled out by the ESC pathway to 25%, but improved the NPV to 99.7% (95%CI 99.0 to 100%, P < 0.001). The novel pathway ruled out 65% with three missed events for a NPV of 99.7% (95%CI 99.4 to 99.9%). No risk score improved the NPV, but all reduced the proportion ruled out (24-47%, P < 0.001 for all). Whilst myocardial infarction due to atherosclerotic plaque rupture and thrombosis (type 1) is well described, the natural disease course of myocardial infarction due to oxygen supply-demand imbalance without atherothrombosis (type 2) is poorly understood. I aimed to define long-term outcomes and explore risk stratification in patients with type 2 myocardial infarction and myocardial injury. Consecutive patients (n=2,122) with elevated cardiac troponin I concentrations (≥0.05 μg/L) were identified at a tertiary cardiac centre. All diagnoses were adjudicated as per the Universal Definition of Myocardial Infarction. The primary outcome was all-cause death. Secondary outcomes included major adverse cardiovascular events (MACE; non-fatal myocardial infarction or cardiovascular death) and non-cardiovascular death. To explore competing risks, cause-specific hazard ratios were obtained using Cox regression models. The adjudicated index diagnosis was type 1 or type 2 myocardial infarction or myocardial injury in 1,171 (55.2%), 429 (20.2%) and 522 (24.6%) patients, respectively. At five years, all-cause death rates were higher in those with type 2 myocardial infarction (62.5%) or myocardial injury (72.4%) compared with type 1 myocardial infarction (36.7%). The majority of excess deaths in those with type 2 myocardial infarction or myocardial injury were due to non-cardiovascular causes (HR 2.32, 95%CI 1.92-2.81, versus type 1 myocardial infarction). Despite this, the observed crude MACE rates were similar between groups (30.6% versus 32.6%), with differences apparent after adjustment for co-variates (HR 0.82, 95%CI 0.69-0.96). Coronary heart disease was an independent predictor of MACE in those with type 2 myocardial infarction or myocardial injury (HR 1.71, 95%CI 1.31-2.24). Patients with type 2 myocardial infarction were less likely to receive secondary prevention therapy, suggesting a treatment gap may exist and there may be potential to modify clinical outcomes. A risk stratification threshold has been defined using high-sensitivity cardiac troponin I which identifies patients at very low risk of myocardial infarction or cardiac death. A diagnostic pathway incorporating this risk stratification threshold appears safer than established guidelines which apply the 99th centile alone. The use of clinical risk scores does not appear to improve the safety of this approach, however, does significantly reduce efficacy. Overall, these findings demonstrate the potential of high-sensitivity cardiac troponin testing to improve the efficiency of the assessment of patients with suspected acute coronary syndrome without compromising patient safety. The observations in those with myocardial injury and infarction have identified a phenotype of patients with type 2 myocardial infarction and coronary artery disease who are at increased cardiovascular risk, and who may benefit from targeted secondary prevention. The studies presented will inform the design of future clinical trials, and may inform international guidelines for the assessment of patients with suspected acute coronary syndrome.

Made available in DSpace on 2014-07-29T15:29:19Z (GMT). No. of bitstreams: 1 dissertacao nancy vieira.pdf: 795865 bytes, checksum: dfeac68319198e6608dcf82885225d60 (MD5) Previous issue date: 2009-11-23 / Bakground: The presence of an increase in troponin I (cTnI) is common in patients who have undergone a percutaneous coronary intervention (PCI), but it is questionable if the myocardial lesion occurring in the intervention allows one to identify patients with a greater risk of presenting adverse cardiac events (ACE) .Objective : The overall objective of the study was to evaluate the immediate evolution of cardiac troponin I (cTnI) in patients undergoing elective coronary angioplasty with stenting, and also compare patients with serum changes after the procedure of coronary angioplasty stenting, with the clinical variables and the occurrence of adverse cardiac events (ACE) following 12 months.. Methods: This is a study of a prospective cut, which evaluates 49 consecutive patients who underwent coronary angioplasty with stent implantation. A cTnI serum dosage was done both before and after the procedure. Patients with a cut off >1.0 ng/mL before implantation were excluded. All the others received follow up for one year, to record the presence of ACE (death, acute myocardial infarction, another angioplasty). Results: cTnI serum elevations occurred in 21(45.7%) of the patients after the PCI. At the end of the follow up period, 11(23.9%) of the patients presented adverse cardiac events. There was no association between the cTnI serum elevation and the presence of ACE in the follow up. Conclusions: The elevation of cTnI found after a coronary angioplasty with stent implantation was frequent in our sample, but these elevations in serum did not show any association with clinical variables and the occurrence of adverse cardiac events following 12 months / A presença da elevação da troponina I(cTnI) é comum em pacientes submetidos a uma intervenção coronariana percutânea (ICP), porém é questionado se a lesão miocárdica ocorrida na intervenção permite identificar pacientes com maior risco de apresentar eventos cardíacos adversos. Objetivo: O objetivo geral do estudo foi avaliar o comportamento evolutivo imediato dos níveis séricos de troponina I (cTnI), em pacientes submetidos eletivamente à angioplastia coronária com implante de stent , e também, comparar pacientes com alterações séricas pós-procedimento de angioplastia coronária com implante de stent, com as variáveis clínicas e a ocorrência de eventos cardíacos adversos (ECA) no seguimento de 12 meses. Metodologia: Este é um estudo de coorte prospectivo, que avaliou 49 pacientes consecutivos, submetidos à angioplastia coronariana com implante de stent. Fez-se dosagens séricas de cTnI antes e após o procedimento. Pacientes com cut off > 1,0 ng/mL antes do implante foram excluídos, e os demais foram acompanhados em um ano de seguimento, para registro da presença de ECA (óbito, infarto do miocárdio e nova angioplastia). Resultados: Ocorreram elevações séricas de cTnI em 21(45,7%) dos pacientes após a ICP. Ao término do seguimento 11(23,9%) dos pacientes apresentaram eventos cardíacos adversos. A correlação entre a elevação sérica da cTnI e a presença de ECA no seguimento não mostrou associação. Conclusão: A elevação da cTnI encontrada após uma angioplastia coronariana com o implante de stent foi freqüente em nossa amostra, porém estas elevações séricas não apresentaram nenhuma associação com as variáveis clínicas e a ocorrência de eventos cardíacos adversos no seguimento de 12 meses

Yes / The development of androgenetic alopecia is associated with a risk of developing cardiovascular diseases, but the association of alopecia areata with cardiovascular diseases in humans is largely unexplored. We measured the plasma level of two common cardiovascular disease markers, cardiac troponin I and Creactive protein, in alopecia areata and androgenetic alopecia-affected subjects. Also, we investigated the possible presence of pro-apoptotic factors in the plasma of hair loss subjects. The mean plasma cardiac troponin I level was highest in alopecia areata subjects, moderately higher in androgenetic alopecia subjects, and lowest in subjects without hair loss (p < 0.05). Alopecia areata subjects not receiving treatments had highest levels of cardiac troponin I (p < 0.05). Alopecia areata plasma samples with high cardiac troponin I levels also induced significantly higher rates of cardiomyocyte apoptosis in cell culture assays. The results suggest the potential for increased heart remodelling. Close monitoring of cardiovascular health in alopecia areata subjects, as well as subsets of androgenetic alopecia patients, may be appropriate. / Canadian Institutes of Health Research (CIHR; MOP-82927). EW is the recipient of a Banting Postdoctoral Fellowship (SAC-92845).

Introdução: O tratamento da Síndrome Coronária Aguda (SCA) sem supradesnivelamento do segmento ST (SSST) sofreu grandes avanços nos últimos 20 anos, com a introdução de novos medicamentos e intervenções invasivas, que reduziram significativamente os eventos clínicos graves como morte e re(infarto), em curto, médio e longo prazos, a despeito dessa entidade ainda representar uma alta taxa de mortalidade no mundo ocidental. Entre os medicamentos, os inibidores da enzima conversora da angiotensina (IECA) tiveram um papel fundamental, demonstrando redução desses eventos em pacientes com alto risco cardiovascular. Nesse período, as troponinas cardíacas consolidaram-se como os biomarcadores de necrose miocárdica de escolha para o diagnóstico e avaliação prognóstica nesses pacientes, devido às suas altas sensibilidade e especificidade. Objetivo: Determinar o efeito do uso prévio de IECA na mensuração da troponina I cardíaca em pacientes com SCASSST, e avaliar os desfechos clínicos em até 180 dias. Casuística e métodos: Estudo prospectivo, observacional, em um único centro de cardiologia, realizado entre 8 de setembro de 2009 e 10 de outubro de 2010, com 457 pacientes, consecutivamente internados no Pronto-Socorro com SCASSST. Os pacientes deveriam apresentar sintomas isquêmicos agudos, nas últimas 48 horas. Foram excluídos os que apresentassem elevação do segmento ST, ou qualquer alteração confundidora ao ECG, como ritmo de marcapasso, bloqueio de ramo esquerdo ou fibrilação atrial. Foram selecionados para análise exploratória, dados de história clínica, exame físico, eletrocardiográficos e laboratoriais, com ênfase à troponina I cardíaca. As variáveis com nível de significância menor que 10% nesta análise, foram submetidas a um modelo de regressão logística múltipla. Resultados: Na população estudada, observou-se que a idade média era de 62,1 anos (DP=11,04) e 291 pacientes (63,7%) do gênero masculino. Fatores de risco como hipertensão arterial sistêmica (85,3%) e dislipidemia (75,9%) foram os mais prevalentes, além da presença de SCA prévia em 275 (60,2%) pacientes; com 49,5% dos pacientes já submetidos a alguma revascularização prévia (Intervenção Coronária Percutânea(ICP) ou Revascularização do Miocárdio (RM), além de 35,0% de diabéticos. Na avaliação de eventos em 180 dias, ocorreram 28 óbitos (6,1%): 11 por choque cardiogênico, 8 por infarto agudo do miocárdio, 3 por choque séptico, além de outras causas. Foi elaborado um modelo de análise estatística, onde foram analisadas as variáveis que interferiam com a liberação de troponina. Por esse modelo, observou-se que cada 1mg/dL a mais na glicemia de admissão, aumentava a chance da troponina ser maior que 0,5 ng/mL em 0,8% (p=0,0034);o uso de IECA previamente à internação reduzia a chance da troponina ser maior que 0,5 ng/mL em 40,6% (p=0,0482) e a presença de infradesnivelamento do segmento ST igual ou maior a 0,5 mm, em uma ou mais derivações, aumentava a chance da troponina ser maior que 0,5 ng/mL em 2,6 vezes (p=0,0016). A estatística C para este modelo foi de 0,77. Conclusão: Os dados apresentados nesta pesquisa em um centro terciário de cardiologia, mostraram uma correlação inequívoca entre o uso de IECA e a redução do marcador de necrose miocárdica troponina I cardíaca, utilizado como medida qualitativa.Porém, ainda não há dados disponíveis para se afirmar que esta redução poderia levar a um número menor de eventos clínicos graves como morte e re(infarto), no período de 180 dias. / Introduction: The last 20 years have seen great advances in the management of non-ST elevation acute coronary syndrome (NSTE-ACS). The introduction of novel drugs and invasive interventions significantly reduced major clinical events such as death and (re)infarction in the short, medium and long term. Yet, mortality rates remain high in the Western world. Among these drugs, angiotensin-converting enzyme (ACE) inhibitors have played a critical role in reducing these events in patients at high cardiovascular risk. In the meantime, cardiac troponins became firmly established as the myocardial necrosis biomarkers of choice to make the diagnosis and prognosis of these patients, due to their high sensitivity and specificity. Objective: To determine the effect of prior use of ECA inhibitors in serum levels of cardiac troponin I in patients with NSTE-ACS and to assess the clinical outcomes up to 180 days. Patients and methods: This was a prospective, observational study conducted at a single tertiary cardiology center from September 8, 2009 to October 10, 2010 with 457 consecutive patients admitted to the emergency department for NSTE-ACS. Only patients with acute ischemic symptoms within the past 48 hours were included in the study. Those with ST-segment elevation or any confounding ECG factor, such as pacemaker rhythm, left bundle branch block, or atrial fibrillation, were excluded. Study population underwent exploratory analysis, clinical history, physical examination, ECG, and laboratory tests, particularly for cardiac troponin I. Variables with a significance level less then 10% were entered into a multiple logistic regression model. Results: The mean age of the study population was 62.1 years (SD = 11.04), and 291 patients (63.7%) were male. Risk factors such as hypertension (85.3%) and dyslipidemia (75.9%) were the most prevalent, followed by previous ACS in 275 (60.2%) patients; 49.5% of the patients had already undergone previous revascularization procedures (either percutaneous coronary intervention [PCI] or coronary artery bypass grafting [CABG]). Diabetes was present in 35% of the patients. At the 180-day assessment, 28 patients (6.1%) had died: 11 as a result of cardiogenic shock, 8 of acute myocardial infarction, and 3 of septic shock, among other causes. In this study, a statistical model was developed to determine which variables affected troponin release. This model showed that each 1mg/dL increase in admission blood glucose increased the likelihood of troponin being higher than 0.5 ng/mL by 0.8% (p=0.0034); the use of ACE inhibitors prior to admission reduced the likelihood of troponin being higher than 0.5 ng/mL by 40.6% (p=0.0482), and the presence of ST-segment depression >= 0.5 mm in one or more ECG leads increased 2.6 times the likelihood of troponin being higher than 0.5 ng/mL (p=0.0016). The C-statistic for this model was 0.77. Conclusion: The data from this study conducted at a tertiary cardiology center show an unequivocal relationship between the use of ACE inhibitors and decreased levels of cardiac troponin I, a biomarker of myocardial necrosis used as a qualitative measure. However, there is no available data to determine whether or not this decrease would result in a lower number of major clinical events, such as death and re(infarction) within 180 days.

CONTEXTO: Estudos observacionais têm demonstrado alterações na variabilidade da freqüência cardíaca em pacientes em sepse, embora não exista publicação correlacionando lesão cardíaca causada pela inflamação sistêmica e redução da variabilidade da freqüência cardíaca. OBJETIVOS: Determinar, em pacientes em sepse, se a lesão cardíaca é causa das alterações da variabilidade da freqüência cardíaca e sua correlação com a mortalidade. Analisar também a associação entre evolução clínica e variabilidade da freqüência cardíaca, troponina, lesão celular à microscopia eletrônica e óptica e variáveis hemodinâmicas. MÉTODO: Estudo observacional, prospectivo, entre pacientes que desenvolveram sepse grave ou choque séptico, analisando sobrevida. Foram analisados: variabilidade da freqüência cardíca (alta e baixa freqüência, através da monitorização cardíaca contínua), dosagem dos níveis séricos de troponina, creatinofosfoquinase e creatinofosfoquinase fração MB, proteína C-reativa, alterações morfológicas e funcionais das células cardíacas por microscopia óptica e eletrônica e imunoistoquímica. Os dados hemodinâmicos foram obtidos por ecocardiograma e medida direta por cateter de artéria pulmonar. RESULTADOS: Dos 31 pacientes incluídos, 12 (38,7%) morreram durante o acompanhamento de 6 dias e 13 sobreviveram até o 28o dia (41,9%); 6 pacientes (19,4%) morreram nas primeiras 6 horas do estudo. O índice APACHE, utilizado como marcador prognóstico, foi igual nos dois grupos (27 +/- 2,6 nos sobreviventes e 26 +/- 2 nos não sobreviventes). A troponina plasmática se mostrou marcador de disfunção miocárdica na sepse (sobreviventes: 0,53 +/- 0,13 mg/ml, e não-sobreviventes: 2,31 +/- 1,01 mg/ml, p < 0,05), com informação prognostica, e se correlacionou com os piores dados hemodinâmicos do grupo de não-sobreviventes (índice de trabalho sistólico do ventrículo esquerdo no grupo de sobreviventes de 48,1 +/- 6,7 g.m/m2 e no de não-sobreviventes de 34,0 +/- 6,7 g.m/m2, p < 0,05). A variabilidade da freqüência cardíaca diminuída se correlacionou com pior prognóstico e a análise multivariada apontou a baixa freqüência cardíaca como uma variável independente para predizer alta da unidade de terapia intensiva (280 +/- 25 ms2 nos sobreviventes e 84 +/- 7,2 ms2 nos pacientes que evoluiram a óbito, freqüência mínima 129 +/- 19 ms2 e 65 +/- 9 ms2 na freqüência máxima, p < 0,05). A microscopia do coração evidenciou infiltrado inflamatório difuso e aumento do número de mitocôndrias e destruição destas organelas. CONCLUSÃO: Nossos resultados sugerem que a alteração da variabilidade da freqüência cardíaca está relacionada com a evolução para óbito, e correlaciona-se com a lesão cardíaca diagnosticada pela dosagem de troponina sérica e alteração do índice do trabalho sistólico do ventrículo esquerdo por sístole. A histologia do coração demonstrou importante lesão celular do coração, com destruição e aumento do número de mitocôndrias. / CONTEXT: Observational studies have shown alterations in the variability of heart rate in patients with sepsis, although there is not a published study showing correlation to cardiac lesion caused by systemic inflammation and reduction on the heart rate variability. OBJECTIVE: To determine, in patients in sepsis, if the cardiac lesion is caused by the alteration on the heart rate variability and access its correlation with mortality; and also, to analyze the association between clinical evolution and cardiac rate variability, plasmatic troponin, cellular lesion on electronic microscopy and optic and the patients\' hemodynamic data. METHOD: Observational study among patients that developed sepsis or septic shock, analyzing the survivors\' rate. The heart rate variability was analyzed (high and low rate through continuous cardiac monitorization), as well as plasma levels of troponin, CK, CK-MB, C-reactive protein, the morphologic alterations and of cardiac cells function, through electronic and optic microscopy and through immunohistochemistry. The hemodynamics\' data were analyzed though echocardiogram and pulmonary artery catheter. RESULTS: Among 31 patients included, 12 (38.7%) of them died during the course of 6 days and 13 (41.9%) survived until the 28th day, 6 (19.4%) died within the first 6 hours of the study. The APACHE index, used as a prognostic marker, was the same in both groups (27 +/- 2.6 in the survivor group and 26 +/- 2.0 in the non-survivors group). The plasmatic troponin became a marker in the myocardial dysfunction in sepsis (survivors 0.53 +/- 0.13 mg/ml and non-survivors 2.31 +/- 1.01 mg/ml, p < 0.05), with prognostic information, and it correlated with the worst hemodynamic parameters among non-survivors group (left ventricle systolic index was 48.1 +/-6.7 g.m/m2 in survivors and 34.0 +/- 6.7 g.m/m2 in non survivors group, p < 0.05). The decrease on the heart rate variability was correlated with the worst prognoses and analyses pointed to a low rate as an independent variable to predict the patients discharge from the intensive care unit (280 +/- 25 ms2 among survivors and 84 +/-7.2ms2 among non-survivors). The heart microscopy showed diffused inflammatory infiltration and increase in the number of mitochondrias and lesions in these organelles. CONCLUSION: Our results suggest that the alteration of the variability on the heart rate is related with the evolution to death, and correlated to cardiac lesion, evidenced through the dosage of troponin, and to alteration of the systolic workload of the left ventricle by systole. The heart histology demonstrated important cellular lesion of the heart, with a raise in number of and damage to mitochondrias.

Introdução: Existe ampla variação no prognóstico de pacientes na fase subaguda após um episódio de síndrome coronariana aguda. O uso da troponina cardíaca de alta sensibilidade pode auxiliar na identificação de pacientes com maior risco de complicações em médio e longo prazo. Objetivos: Identificar, nos pacientes com SCA, a frequência de níveis persistentemente elevados de troponina; avaliar a relação entre a elevação persistente dos níveis de troponina e a incidência de desfechos adversos, como: morte por todas as causas, morte cardiovascular e morte por infarto agudo do miocárdio, pelo período de seguimento de até cinco anos após o evento índice; e avaliar se a incidência de desfechos adversos está relacionada a elevação dos níveis de troponina mesmo abaixo do valor de corte para o 99º percentil do método. Métodos: Foram avaliados todos os pacientes recrutados no estudo ERICO (Estratégia de Registro da Insuficiência Coronariana) com diagnóstico de angina instável (AI), infarto agudo do miocárdio (IAM) sem supradesnivelamento de segmento ST e IAM com supradesnivelamento de segmento ST. Níveis de troponina I de alta sensibilidade foram medidos em 525 pacientes no período de 25 a 90 dias após um episódio de síndrome coronariana aguda (SCA). Os participantes foram divididos em tercis de acordo com os níveis de troponina e seguidos entre fevereiro de 2009 e dezembro de 2015. Os desfechos analisados foram: mortalidade por todas as causas, mortalidade cardiovascular e incidência de infarto agudo do miocárdio. Resultados: Pacientes no tercil superior de troponina colhida entre 25 e 90 dias tiveram maior taxa de risco (TR) de mortalidade por todas as causas quando comparados com o tercil inferior, na análise não ajustada (TR: 5,17, intervalo de confiança de 95% [IC 95%]: 2,41-11,10) e ajustada por idade e sexo (TR: 4,93, IC95%: 2,29-10,64). Estes achados persistiram mesmo após o ajuste para fatores de risco cardiovascular conhecidos no modelo multivariado número 1 (TR: 5,24, IC95%: 2,08-13,20), e análise posterior com ajuste pela taxa de filtração glomerular abaixo de 60 ml/min/1.73m2 e fração de ejeção do ventrículo esquerdo abaixo de 0,40 (TR: 6,47, IC95% 1,77-23,66). A mortalidade cardiovascular foi significativamente maior no tercil superior após ajuste para idade e sexo (TR: 6,51, IC 95% 1,92-22,10) e no primeiro modelo de ajuste multivariado (TR: 7,47, IC 95%: 1,62-34,41); houve tendência não estatisticamente significativa de maior mortalidade cardiovascular no segundo modelo (TR: 4,52, IC 95%: 0,71-28,62). Não houve diferenças significativas entre os tercis em relação à incidência de infarto agudo do miocárdio. Conclusão: Nosso estudo demonstrou que níveis de troponina de alta sensibilidade medidos na fase subaguda após um episódio de SCA são preditores independentes de mortalidade por todas as causas / Introduction: Prognosis of patients in the stable phase after an acute coronary syndrome (ACS) event is widely variable. The use of high-sensitivity cardiac troponin I can aid in the identification of patients at higher risk for long-term adverse outcomes. Objectives: To identify, among ACS patients, the frequency of persistently elevated troponin levels 25 to 90 days after the event; to evaluate the relation between persistently elevated troponin levels and incidence of adverse outcomes, such as: allcause mortality, cardiovascular mortality, and myocardial infarction mortality; and to evaluate if the incidence of adverse outcomes is related to elevations in troponin levels even below the 99th percentile of the essay. Methods: All 525 patients recruited in the ERICO study (Acute Coronary Syndrome Registry Strategy) with a diagnosis of unstable angina (UA), non-ST elevation myocardial infarction (NSTEMI) or ST elevation myocardial infarction (STEMI) who had blood samples available 25 to 90 days after the ACS event had high sensitivity cardiac troponin I levels measured; these patients were then divided into tertiles and followed from February 2009 to December 2015. We evaluated all-cause mortality, cardiovascular mortality and myocardial infarction as endpoints during follow-up. Results: Patients in the highest tertile had a greater hazard ratio (HR) for all-cause mortality compared to the lowest tertile, on crude analysis (HR: 5.17, 95% Confidence Interval [95% CI]: 2.41-11.10) and after adjustment for age and sex (HR: 4.93, 95% CI: 2.29-10.64). These findings persisted even after adjustment for known cardiovascular risk factors on multivariate model 1 (HR: 5.24, 95% CI: 2.08-13.20), and further adjustment for estimated glomerular filtration rate < 60 ml/min/1.73m2 and left ventricular ejection fraction < 0.40 (HR: 6.47, 95% CI: 1.77-23.66). Cardiovascular mortality was significantly higher in the highest tertile after adjustment for age and sex (HR: 6.51, 95% CI: 1.92-22.10) and in the first model of multivariate adjustment (HR: 7.47, 95% CI: 1.62-34.41); there was a nonsignificant trend towards higher cardiovascular mortality in the second model of multivariate adjustment (HR: 4.52, 95% CI: 0.71-28.62). Conclusion: In conclusion, our study showed that elevated high sensitivity cardiac troponin I levels measured in the stabilized phase after an ACS event are independent predictors of long-term mortality

A troponina I (TnI) é a sub-unidade inibitória do complexo troponina, responsável pela regulação da contração do músculo esquelético. Foi demonstrado que sua ação inibitória sobre a Mg2+ATPase da actomiosina, deve-se principalmente à região entre os resíduos 96 e 116 (região do peptídeo inibitório). Para estudar o mecanismo de inibição a nível molecular, produzimos três mutantes na região do peptídeo inibitório através de mutações sítio-dirigidas. Substituímos os resíduos lisina 105 por ácido glutâmico (K105E), fenilalanina 106 por tirosina (F106Y) e arginina 113 por ácido glutâmico (R113E). As troponinas I mutantes foram expressas em E.coli, purificadas e ensaiadas em sua atividade inibitória, interações com os outros componentes do complexo regulatório e sua capacidade regulatória. Os resultados obtidos indicam que a mutação na posição 105 alterou a interação da proteína com a tropomiosina, diminuindo sua atividade inibitória e afinidade pela actina-tropomiosina. A substituição na posição 113 alterou a interação da proteína com a actina e com a actina-tropomiosina, também diminuindo a atividade inibitória na presença de tropomiosina e inviabilizando a inibição na ausência de tropomiosina. Já a substituição na posição 106 não produziu alteração detectável. Concluímos que o resíduo 105 faz parte do sítio de ligação da troponina I ao complexo actina-tropomiosina e que o resíduo 113 participa diretamente do mecanismo de inibição. Desta forma, definimos duas interfaces de interação da troponina I com o filamento de actina-tropomiosina, necessárias a ligação da troponina I ao filamento e inibição da ATPase. / Troponin I (TnI) is the inhibitory subunit of the troponin complex, responsible for the regulation of skeletal muscle contraction. It has been demonstrated that TnI\'s inhibitory action on Mg2+ATPase of actomyosin is due principally to the region between residues 96 and 116 (the inhibitory region). To study the inhibitory mechanism at the molecular level, we produced three mutants of the inhibitory region by site-directed mutagenesis. We substituted lysine 105 for glutamic acid (K105E), phenylalanine 106 for tyrosine (F106Y) and arginine 113 for glutamic acid (R113E). The TnI mutants were expressed in E. coli, purified and analyzed for their inhibitory activity, interaction with other components of the regulatory complex and regulatory capacity. The results indicate that the mutation in K105E modified the interaction of TnI with tropomyosin, reduced its inhibitory activity and actin-tropomyosin affinity. The mutant R113E displayed modified interaction with actin and actin-tropomyosin, reduced inhibitory activity in the presence of tropomyosin and essentially no inhibitory activity in the absence of tropomyosin. The mutant F106Y behaved essentially like wild-type TnI. We conclude that residue 105 is part of the site by which troponin I binds to the actin-tropomyosin and that residue 113 participates directly in the inhibitory mechanism. In this way, we have defined two interfaces between troponin I and the actin-tropomyosin which are necessary for binding TnI to the filament and to inhibit the actomyosin ATPase.

Da interação da actina com a miosina resulta a contração muscular. Esta interação é controlada pela concentração de íons cálcio, que atuam sobre um sistema regulatório associado ao filamento de actina. O sistema regulatório é composto de uma molécula de tropomiosina e um complexo protéico composto de três polipeptídeos chamado troponina. Uma das subunidades, troponina C, e o receptor de ions cálcio; outra, troponina T, liga-se à tropomiosina; e a terceira, troponina I, é a responsável pela inibição da atividade ATPásica da actomiosina. Esta dissertação descreve o desenvolvimento do processo de expressão da troponina I em bactéria, sua purificação e caracterização. Partindo de um cDNA de troponina I músculo esquelético de galinha, procedemos à construção de um vetor de expressão da proteína em E.coli com o sistema pET. Inicialmente construímos um vetor de expressão capaz de produzir a troponina I fundida a 18 aminoácidos. A proteína foi purificada e caracterizada, comportando-se de forma semelhante à troponina I selvagem. Numa segunda etapa, foram feitas mutações sítio-dirigidas para a construção de um novo sítio de restrição que possibilitou construir um vetor de expressão da troponina I a partir de sua metionina inicial. Entretanto não foi obtida expressão com esta construção. Para solucionar o problema, foram feitas novas mutações que alteraram os codons AGG presentes nos 25 primeiros codons do gene da proteína por codons CGT, sem alterar o aminoácido codificado. Nesta construção final foi obtida a expressão da proteína sem fusão, que purificada e caracterizada, comporta-se de modo idêntico à troponina I selvagem. / Muscle contraction results from the interaction of myosin with actin and this interaction is controlled by the calcium ion concentration which acts on the regulatory system associated with the actin filaments. This regulatory system comprises one tropomyosin molecule and a complex composed of three polypeptide subunits. One of this subunits, troponin C binds calcium ions; another, troponin T, binds to tropomyosin while the third, troponin I, inhibits the ATPase activity of actomyosin. This dissertation describes the development of the methodology to express troponin I in bacteria and its subsequent purification and characterization. Starting with a troponin I cDNA from chicken skeletal muscle, a pET expression vector was constructed. The initial vector resulted the expression of a troponin I fusion protein having an additional 18 amino acids. This protein was purified and characterized and found to behave like wild type troponin I. Subsequently, employing site-directed mutagenesis, a new vector was made which allowed the expression of the protein starting at its initial methionine codon and lacking the extra amino acids. However, no expression was achieved using this vector and, to circumvent this, further mutations were introduced in the first 25 codons which substituted AGG for CGT without altering the amino acid sequence. This final construct permitted the expression of a non fusion troponin I which, after purification and characterization, was found to behave identically to the wild type protein.

The fast-skeletal-muscle-fiber-specific expression of the troponin I(fast) (TnIfast) gene is driven by an Intronic Regulatory Element (IRE) located within the first intron of the gene. The IRE is a 148 bp transcriptional enhancer that contains several known and suspected cis-regulatory elements. These include the E-box, the closely-spaced MEF2 site and CACT box, the CACC site, and the CAGG element. Previous loss-of-function studies performed using the quail TnIfast IRE suggest that its activity depended on the MEF2 and CACT elements. The goal of my thesis research was to determine whether the MEF2 and CACT sites were not only necessary, but also sufficient, to support IRE activity. I prepared head-to-tail multimers of a 27-bp IRE segment that consisted largely of the near-adjacent MEF2 and CACT elements and did not contain any other known/suspected elements. These multimers were cloned upstream of a reporter gene consisting of the minimal promoter of the quail TnIfast gene linked to sequences encoding human placental alkaline phosphatase. The transcriptional capabilities of the constructs were assessed by gene transfer into the mouse soleus muscle in vivo by intramuscular injection/electroporation, and histochemical analysis of reporter enzyme plap expression including quantitative microdensitometry. I found that expression of these constructs was readily detectable and that it was markedly reduced by prior mutation of the CACT and, especially, of the MEF2 sites. These data indicate that the short DNA segment containing MEF2 and CACT elements is sufficient to drive expression in skeletal muscle and confirms the functional importance of these specific elements. / Although constructs containing the wild-type IRE 27-bp region were expressed, there was little preferential expression in fast fibers, in contrast to expression driven by the complete 148-bp IRE. Thus my results indicate that the MEF2 and CACT elements are not sufficient to drive fast fiber-type-specific expression, and suggest that additional elements outside of the 27-bp region tested are also necessary for fiber-type-specificity.

Da interação da actina com a miosina resulta a contração muscular. Esta interação é controlada pela concentração de íons cálcio, que atuam sobre um sistema regulatório associado ao filamento de actina. O sistema regulatório é composto de uma molécula de tropomiosina e um complexo protéico composto de três polipeptídeos chamado troponina. Uma das subunidades, troponina C, e o receptor de ions cálcio; outra, troponina T, liga-se à tropomiosina; e a terceira, troponina I, é a responsável pela inibição da atividade ATPásica da actomiosina. Esta dissertação descreve o desenvolvimento do processo de expressão da troponina I em bactéria, sua purificação e caracterização. Partindo de um cDNA de troponina I músculo esquelético de galinha, procedemos à construção de um vetor de expressão da proteína em E.coli com o sistema pET. Inicialmente construímos um vetor de expressão capaz de produzir a troponina I fundida a 18 aminoácidos. A proteína foi purificada e caracterizada, comportando-se de forma semelhante à troponina I selvagem. Numa segunda etapa, foram feitas mutações sítio-dirigidas para a construção de um novo sítio de restrição que possibilitou construir um vetor de expressão da troponina I a partir de sua metionina inicial. Entretanto não foi obtida expressão com esta construção. Para solucionar o problema, foram feitas novas mutações que alteraram os codons AGG presentes nos 25 primeiros codons do gene da proteína por codons CGT, sem alterar o aminoácido codificado. Nesta construção final foi obtida a expressão da proteína sem fusão, que purificada e caracterizada, comporta-se de modo idêntico à troponina I selvagem. / Muscle contraction results from the interaction of myosin with actin and this interaction is controlled by the calcium ion concentration which acts on the regulatory system associated with the actin filaments. This regulatory system comprises one tropomyosin molecule and a complex composed of three polypeptide subunits. One of this subunits, troponin C binds calcium ions; another, troponin T, binds to tropomyosin while the third, troponin I, inhibits the ATPase activity of actomyosin. This dissertation describes the development of the methodology to express troponin I in bacteria and its subsequent purification and characterization. Starting with a troponin I cDNA from chicken skeletal muscle, a pET expression vector was constructed. The initial vector resulted the expression of a troponin I fusion protein having an additional 18 amino acids. This protein was purified and characterized and found to behave like wild type troponin I. Subsequently, employing site-directed mutagenesis, a new vector was made which allowed the expression of the protein starting at its initial methionine codon and lacking the extra amino acids. However, no expression was achieved using this vector and, to circumvent this, further mutations were introduced in the first 25 codons which substituted AGG for CGT without altering the amino acid sequence. This final construct permitted the expression of a non fusion troponin I which, after purification and characterization, was found to behave identically to the wild type protein.