The purpose of this investigation was to examine selected biochemical mechanisms known to influence contractility and energy metabolism in the myocardium, with particular emphasis placed on the regulatory role of protein phosphorylation in the ventricular myocardium. The investigation was conducted in three phases; initially the cardiac contraction cycle was examined to determine whether reported fluctuations in myocardial cAMP levels were associated with other biochemical events known to be cAMP-dependent. The second phase involved the determination of specific kinase activities and endogenous substrates in a highly purified cardiac sarcolemmal preparation. In the final phase, ventricular myocytes were utilized to examine the ability of adenosinergic and muscarinic agonists to influence the isoproterenol-induced increases in protein phosphorylation. Studies in the first phase examined cyclic AMP levels and selected kinase activities in hearts frozen at various stages of the cardiac cycle. An automated clamping device, capable of freezing a perfused rat heart in less than 50 msec, was utilized to separate the cardiac cycle into various phases. Three different timing schemes were employed to divide the cycle into 2 to 4 segments. These different timing schemes revealed no significant differences in cAMP during the cardiac cycle. Myocardial cAMP values ranged from 2.5 to 4.1 pmol/min/mg protein in all phases. However, in one scheme there was a tendency for cAMP to be elevated in early systole, with minimal values occurring diastole. There were also no significant differences seen for either glycogen phosphorylase or cAMP-dependent protein kinase (PKA) activity between various phases of the cardiac cycle. Since no significant fluctuations were observed in the levels of cAMP or the activities of PKA or glycogen phosphorylase during a single cardiac contraction cycle, it would appear that these agents do not exert their effects on cardiac function on a beat to beat basis. The second phase of study examined the nature and function of individual protein kinases in the myocardium. Using a highly purified cardiac sarcolemmal preparation, kinase specific, synthetic substrates were employed to quantify the activities of cAMP-dependent (PKA), calcium/calmodulin-dependent (PKCM), calcium/phospholipid-dependent (PKC) and cGMP-dependent (PKG) protein kinases. Additionally, endogenous protein substrates were examined in this preparation to provide possible insight as to the function of these kinases in the heart. The activities of PKA, PKG, PKCM, and PKC in nmol 32P/min/μg protein were as follows: PKA, 1606; PKG, 35.7; PKCM, 353; and PKC, 13.2. Three endogenous protein substrates of apparent molecular weights of 15kD, 28kD and 92kD were phosphorylated. While no endogenous protein phosphorylation was detectable as a result of cG-PK activity, all of the substrates were phosphorylated, to varying degrees, by both PKA and CACM-PK. PKC phosphorylated only the 15kD substrate. Even though several endogenous kinases are evident in the sarcolemmal preparation, cAMP-dependent protein kinase demonstrates the greatest degree of activity. This kinase also appeared to be the most abundant; however, there is some concern as to the source of these kinases in the membrane preparation since endothelial membranes as well as cardiac membranes appeared to be present. Evidence for endothelial contamination was provided by the finding that the membrane preparation contained appreciable amounts of angiotensin converting enzyme (ACE) activity, an enzyme felt to reside in the vascular endothelium. Since studies with this preparation could not exclude contribution of nonmuscle cell membranes a model consisting solely of dispersed ventricular myocytes was developed. The third phase of these studies examined protein phosphorylation in primary cultures of ventricular myocytes. Specifically, these studies examined protein phosphorylation induced by exposure to isoproterenol (ISO), a catecholamine known to effect changes in the phosphorylation state of proteins in the heart by means of a β-adrenergic-mediated/cAMP-dependent mechanism was examined. Additionally, the effects of phenylisopropy-ladenosine (PIA) and carbamyl choline chloride (CARB) were examined with regard to their anti-adrenergic role(s) in this process. Adherent, collagenase-dispersed, radiolabelled (32p) ventricular myocytes exposed to ISO demonstrated a dose and time dependent increase in 32p incorporation into several endogenous protein substrates. When the myocytes were exposed (60 sec) to either PIA or CARB prior to the exposure to ISO, ISO-induced 32p incorporation into protein substrates of apparent molecular weight of 6kD, 31kD and 155kD was reduced up to 67% when compared to the effects of ISO alone. Additionally, both PIA and CARB attenuated the ISO-induced increase in PKA activity in the myocyte, yet only CARB was seen to produce an inhibitory effect on the ISO-induced increase in cAMP levels in the myocytes. The effects of CARB were dose-dependent and inhibited the effects of ISO on 32p incorporation at all doses tested. PIA elicited biphasic effects: lower PIA concentrations were inhibitory in nature, while higher concentrations of PIA appeared to potentiate the increase in 32p incorporation induced by ISO. Based on electrophoretic mobilities (SDS/PAGE) of the 6kD and the 155kD substrates, these substrates have been tentatively identified as the monomeric form of the sarcoplasmic reticulum-associated protein, phospholamban, and the contractile filament-associated protein, C protein, respectively. The 31kD substrate has been identified, by means of immunoblot, as the contractile filament-associated protein, troponin I. The role of protein phosphorylation in the myocardium involves complex, inter-related mechanisms that encompass extracellular, transmembranal and cytoplasmic elements in the heart. It is well understood that certain mechanisms of the contraction cycle known to vary on a beat to beat basis, such as myosin ATPase, involve changes in protein phosphorylation. However, the nature of the various kinases and substrates examined in this study appear to influence longer-term events of myocardial contractility. Mechanisms coupled with hormone action, modulation of second messenger-dependent components, and factors associated with changes in contractility seen with aging and disease are more likely to exhibit changes similar to those described herein. A better understanding of the underlying biochemistry may provide greater insight into the importance of these metabolic changes.

Made available in DSpace on 2015-12-06T23:47:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2008 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O Infarto do Miocárdio (IM) é uma das principais causas de morte súbita, insuficiência cardíaca (IC) e perda da qualidade de vida. Como o coração tem capacidade limitada de regeneração, terapias como o implante de células-tronco vem sendo desenvolvidas. O objetivo deste trabalho foi estudar o implante de células mononucleares derivadas da medula-óssea no IM experimental em ratos, avaliando seus efeitos sobre o remodelamento miocárdico, função ventricular global e contratilidade do miocárdico remoto ao infarto. Para tanto, utilizou-se injeção intramiocárdica de células da medula-óssea (BMC) de ratos machos Lewis-inbred, 48 horas após oclusão da artéria coronária interventricular anterior em ratas da mesma linhagem. Após seis semanas, foi analisada função cardíaca global in vivo por ecocardiografia Doppler e avaliação hemodinâmica, seguida do estudo in vitro da mecânica contrátil do miocárdio remanescente representado pelo músculo papilar isolado. Posteriormente, realizaram-se estudos histopatológicos dos processos de fibrose intersticial (coloração com picrossirius red), hipertrofia (volume nuclear do miócito) e densidade capilar (coloração com ácido periódico de Schiff); e avaliação por Western blotting de proteínas envolvidas na contração no tecido remoto ao infarto: Ca2+-ATPase do retículo sarcoplasmático (SERCA2), fosfolamban total (PLB) e fosforilado (PLB-Ser16), trocador Na+ /Ca2+ (NCX), e isoformas α1 e α2 da Na+ /K+ -ATPase (NKA). Os ratos foram distribuídos em infartos moderados (30- 39% do VE = mIM) e grandes (≥ 40% do VE = IM), tratados (grupos mIM+BMC e IM+BMC) ou não tratados (grupos mIM e IM), e comparados ao grupo sem infarto nem terapia (SHAM). P < 0,05 foi considerado significante. A mortalidade durante os procedimentos ou acompanhamento não foi diferente entre os grupos. A análise por PCR identificou cromossomo Y de células de macho implantadas em amostras coletadas de corações de fêmeas desde dez minutos após implante até seis semanas, com intensidade decrescente. Após seis semanas o tamanho do infarto foi discretamente menor em IM+BMC (38 ± 1%) que em IM (44 ± 1%), e também em mIM+BMC (31 ± 1%) comparado aos mIM (34 ± 1% do VE). IM+BMC cursou com menor dilatação ventricular (45 ± 10%) que IM (86 ± 7%) com melhora da fração de encurtamento do VE em 20 ± 9% e menor incidência de hipertensão pulmonar, enquanto mIM e mIM+BMC demonstraram comportamento semelhante. O aumento na pressão diastólica final (PDf) somente detectado em IM (16 ± 2 mm Hg) assim como a depressão da +dP/dtmáx (7.727 ± 367 mm Hg/s) foram prevenidos em IM+BMC (5 ± 2 mm Hg e 9.035 ± 345 mm Hg/s). Os demais parâmetros hemodinâmicos analisados sob condições basais não foram diferentes do grupo SHAM. Entretanto, a sobrecarga pressórica súbita reduziu sensivelmente o volume ejetado em mIM (-46 ± 4%) e IM (-63 ± 4%), mas apenas discretamente em mIM+BMC (-12 ± 6%) e IM+BMC (-20 ± 3%) semelhante aos -9 ± 4% de SHAM. A correlação estabelecida entre aumento na pós-carga e geração de trabalho sistólico mostrou-se positiva nos SHAM (inclinação média de 0,75 ± 0,09) e nos tratados mIM+BMC (0,74 ± 0,12) e IM+BMC (0,52 ± 0,05), mas nitidamente negativa nos grupos infartados sem terapia (mIM: inclinação = -0,38 ± 0,05; e IM: -0,97 ± 0,1). Pelo estudo do músculo papilar, a tensão desenvolvida (g.mm-2) foi deprimida em IM (2,4 ± 0,2) e mIM (3,8 ± 0,4) em relação a mIM+BMC (5,5 ± 0,5), IM+BMC (5,6 ± 0,4) e SHAM (6,1 ± 0,3). Ademais, os coeficientes de inclinação das retas da relação estiramento vs. tensão desenvolvida (g.mm-2 / % do comprimento ótimo) estavam deprimidos nos mIM (0,19 ± 0,01) e IM (0,13 ± 0,02) comparados ao SHAM (0,29 ± 0,02), e normais em mIM+BMC (0,25 ± 0,02) e IM+BMC (0,26 ± 0,03) demonstrando integridade do mecanismo de Frank-Starling no músculo remanescente. Os prejuízos, em mIM e IM, da potenciação pós-pausa de de estímulos, manobra indicadora da integridade da cinética do cálcio no miócito, foram parcialmente prevenidos por BMC. As diminuições, identificadas em mIM e IM, da expressão protéica da SERCA2 (66 ± 7 e 54 ± 6% do SHAM), PLB (72 ± 4 e 52 ± 9%) e PLB-Ser16 (71 ± 6 e 35 ± 10%) foram atenuadas em mIM+BMC e IM+BMC (SERCA2: 84 ± 11 e 94 ± 10%, PLB: 83 ± 7 e 89 ± 7%, e PLB-Ser16: 104 ± 14 e 90 ± 8%). O NCX foi superexpresso em IM (198 ± 29 % do SHAM) e normal nos demais grupos, e enquanto α1-NKA não foi diferente em nenhum grupo, a depressão na quantidade de α2-NKA (mIM: 47 ± 10 e IM: 35 ± 10%) não foi significantemente prevenida com BMC (mIM+BMC: 61 ± 10 e IM+BMC: 63 ± 16% do SHAM). A análise histopatológica mostrou aumento significante do volume nuclear (μm3 ) no miocárdio remodelado de mIM (188 ± 7) e IM (219 ± 9) em comparação ao SHAM (129 ± 8), e prevenção parcial de hipertrofia miocárdica em mIM+BMC (147 ± 8) e IM+BMC (165 ± 8). Já o teor de colágeno intersticial mostrou-se elevado de forma significativa somente nos grandes infartos (2,4 ± 0,1 % da área analisada), mas aparentemente normal em IM+BMC (1,9 ± 0,06 %) comparados ao SHAM (1,5 ± 0,1 %). Na zona de transição entre o infarto e o miocárdio adjacente, além de BMC promover aumento da densidade capilar em mIM+BMC (2.805 ± 109 vs. mIM: 1.933 ± 183 capilares/mm2 ) e IM+BMC (2.702 ± 81 vs. IM: 1.981 ± 115), também houve aumento na relação capilar/cardiomiócito (mIM+BMC: 1,68 ± 0,06 vs. mIM: 1,13 ± 0,02; e IM+BMC: 1,50 ± 0,06 vs. IM: 1,17 ± 0,04). A densidade capilar no miocárdio remoto ao infarto mostrou-se significantemente diminuída nos infartos sem terapia (mIM: 2.924 ± 120; e IM: 2.454 ± 90 capilares/mm2 ) em relação ao SHAM (3.691 ± 248), mas praticamente preservada nos tratados (mIM+BMC: 3.379 ± 103; e IM+BMC: 3.239 ± 129); e também relação entre capilar/cardiomiócito menor em mIM (1,32 ± 0,05 capilar/cardiomiócito) e IM (1,28 ± 0,04) e equivalentes ao SHAM (1,71 ± 0,10) nos grupos mIM+BMC (1,57 ± 0,06) e IM+BMC (1,64 ± 0,02). Finalmente, conclui-se que a terapia com BMC restringe a repercussão pós-infarto observada tanto na função cardíaca global e desempenho ventricular, quanto nos parâmetros de remodelamento estrutural do miocárdio remanescente relacionados à hipertrofia, fibrose e vascularização. Ademais, reduz as alterações moleculares das proteínas relacionadas à cinética intracelular do cálcio preservando, dessa maneira, a função contrátil do miocárdio remoto ao infarto e implante celular. Parece que estes benefícios ocorrem em conjunto com a diminuição da extensão relativa do infarto do miocárdio sem, entretanto, manter dependência a este fenômeno, visto que a maioria dos parâmetros avaliados foi mais eficiente no grupo IM+BMC do que em IM e mesmo em mIM. A reunião destes dados permite sugerir que a terapia com células mononucleares derivadas de medula-óssea pode agir não só sobre o infarto e zona de transição, mas também na melhora da função do miocárdio remoto, provavelmente por ações parácrinas e/ou endócrinas também mediadoras do processo de remodelamento tecidual e celular. / Myocardium infarction is a major cause of sudden death, heart failure with impaired quality of life. Since the heart exhibits limited regenerative capability, alternative interventions as the stem cell therapy have been developed. Our aim was to study the transplantation of bone marrow-derived mononuclear cells (BMC) in the myocardial infarction in rats, evaluating its effects on the global cardiac function, remodeling and contractility of remnant myocardium. The BMC extracted from male Lewis-inbred rats were intramyocardially injected into the border zone of infarct, 48 hours after coronary occlusion in female rats. After six weeks the in vivo global cardiac function was analyzed by Doppler echocardiography and hemodynamics, and the in vitro mechanics of the remnant myocardium by the isolated papillary muscle study. Thereafter, the following histopathological evaluations were performed: interstitial fibrosis (picrossirius red staining), hypertrophy (myocyte nuclear volume) and capillary density (PAS staining); and myocardium content of calcium handling proteins by Western blotting: sarco-endoplasmic reticulum Ca2+- ATPase (SERCA2), total (PLB) and serine16 phosphorylated phospholamban (PLBSer16), sarcolemmal Na+ /Ca2+ exchanger (NCX), and α1 and α2 isoforms of Na+ /K+ - ATPase (NKA). Rats were distributed in moderate (30-39% of left ventricle = mIM) and large MI (≥ 40% of left ventricle = IM), and treated with BMC (mIM+BMC and IM+BMC groups) or saline injection (mIM and IM groups), compared to shamoperated and placebo-treated group (SHAM). P < 0.05 was considered for statistical significance. Mortality during surgical procedures and follow-up was not different among the groups. The polymerase chain reaction identified Y chromosome of implanted male cells in samples collected from female hearts 10 minutes, 2 days, 1 and 6 weeks following therapy, with decreasing intensity. After six weeks, infarct size was slightly smaller in IM+BMC (38 ± 1% of left ventricle) than in IM (44 ± 1%), and also in mIM+BMC (31 ± 1%) compared to mIM (34 ± 1%). IM+BMC exhibited less ventricular dilatation (45 ± 10%) than IM (86 ± 7%) with improved fractional ventricular shortening by 20 ± 9%, while mIM and mIM+BMC were similar. Increased end-diastolic pressure (PDf) in IM (16 ± 2 mm Hg) and depressed rate of pressure raise (7,727 ± 367 mm Hg/s) were totally prevented by BMC (5 ± 2 mm Hg e 9,035 ± 345 mm Hg/s). Under basal conditions, ejective parameters were not different to SHAM. Notwithstanding, sudden pressure overload expressively reduced stroke volume in mIM (-46 ± 4%) and IM (-63 ± 4%) but just minimally in mIM+BMC (-12 ± 6%) and IM+BMC (-20 ± 3%) similar to SHAM (-9 ± 4%). Correlations between afterload stress and stroke work generation were positive in SHAM (mean slope: 0.75 ± 0.09) and treated mIM+BMC (0.74 ± 0.12) and IM+BMC (0.52 ± 0.05), but were negative in infarcted untreated groups (mIM: -0.38 ± 0.05; and IM: -0.97 ± 0.1). On the papillary muscles study, developed tension was impaired in IM (2.4 ± 0.2 g.mm-2) and mIM (3.8 ± 0.4) and preserved in mIM+BMC (5.5 ± 0.5) and IM+BMC (5.6 ± 0.4) compared to SHAM (6.1 ± 0.3). In addition, slope of the length-tension relation was depressed in mIM (0.19 ± 0.01) and IM (0.13 ± 0.02) in comparison to SHAM (0.29 ± 0.02), and normal in mIM+BMC (0.25 ± 0.02) and IM+BMC (0.26 ± 0.03) showing preservation of Frank-Starling relationship. The impaired post-rest potentiation in mIM and IM (denoting damaged calcium handling) were partially but significantly prevented by BMC. The decrement, identified in mIM and IM, on the protein content of SERCA2 (66 ± 7 and 54 ± 6% of SHAM), PLB (72 ± 4 and 52 ± 9%) and PLB-Ser16 (71 ± 6 and 35 ± 10%), were attenuated in mIM+BMC and IM+BMC (SERCA2: 84 ± 11 and 94 ± 10%, PLB: 83 ± 7 and 89 ± 7%, PLB-Ser16: 104 ± 14 and 90 ± 8%). NCX was over-expressed in IM (198 ± 29 % of SHAM) but normal in BMC groups, and while α1-NKA remained unchanged in all groups, diminished content of α2-NKA (mIM: 47 ± 10 and IM: 35 ± 10%) was not prevented by BMC (mIM+BMC: 61 ± 10 and IM+BMC: 63 ± 16% of SHAM). Histological analysis showed significant increase of nuclear volume (μm3 ) on remodeled myocardium of mIM (188 ± 7) and IM (219 ± 9) in comparison to SHAM (129 ± 8), but partial prevention of the myocyte hypertrophy in mIM+BMC (147 ± 8) and IM+BMC (165 ± 8). Interstitial collagen was significantly increased only in IM (2.4 ± 0.1 % of analyzed area), but apparently normal in IM+BMC (1.9 ± 0.06 %) compared to SHAM (1.5 ± 0.1 %). In the border zone, besides promoting enhanced capillary density in mIM+BMC (2,805 ± 109 vs. mIM: 1,933 ± 183 capillaries/mm2 ) and IM+BMC (2,702 ± 81 vs. IM: 1,981 ± 115), cell therapy also increased capillaries/myocyte ratio (mIM+BMC: 1.68 ± 0.06 vs. mIM: 1.13 ± 0.02; and IM+BMC: 1.50 ± 0.06 vs. IM: 1.17 ± 0.04). In the remodeled myocardium remote to infarct, capillary density was decreased on infarcted untreated groups (mIM: 2,924 ± 120; and IM: 2,454 ± 90 capillaries/mm2 ) in relation to SHAM (3,691 ± 248), but almost totally preserved in mIM+BMC (3,379 ± 103) and IM+BMC (3,239 ± 129); capillaries/myocyte ratio was smaller in mIM (1.32 ± 0.05 capillaries/myocyte) and IM (1.28 ± 0.04), but equivalent to SHAM (1.71 ± 0.10) in mIM+BMC (1.57 ± 0.06) and IM+BMC (1.64 ± 0.02). Thus, the data demonstrates that BMC restrained the postinfarction repercussions concerning global cardiac function and ventricular performance, as well as the structural remodeling in remnant myocardium related to hypertrophy, fibrosis and microvasculature. Furthermore, this therapy reduced the calcium-handling protein changes, thus preserving the contractile behavior of myocardium remote to infarct and cell injection sites. The obtained results suggests that the bone marrow-derived mononuclear cell therapy may act beyond the infarct and border zones, also improving the function of remote tissue, modulating interstitial and cellular remodeling probably throughout paracrine and/or endocrine pathways. / BV UNIFESP: Teses e dissertações

Em pacientes portadores de insuficiência coronariana com prognóstico desfavorável pela presença de disfunção ventricular significativa, a pesquisa de viabilidade miocárdica traz contribuições ao predizer a possibilidade de recuperação contrátil após revascularização. Os segmentos miocárdicos com disfunção contrátil por hipoperfusão podem melhorar o desempenho contrátil restabelecido o aporte sanguíneo local, indicando que a revascularização miocárdica, quando bem indicada, é capaz de melhorar a sobrevida deste grupo de pacientes. Realizou-se pesquisa de tese de doutoramento do Departamento de Radiologia da Universidade de São Paulo na unidade de medicina nuclear da Nuclear Medcenter/Hospital SOCOR em Belo Horizonte, Minas Gerais. O objetivo do estudo foi verificar se a cintilografia de perfusão miocárdica com duplo-isótopo (99mTcsestamibi/ cloreto de tálio-201) como método radioisotópico para identificação do músculo viável, tem sua especificidade aumentada com a inclusão de informações sobre reserva contrátil miocárdica obtidas simultaneamente através do gatedSPECT (imagens tomográficas do coração sincronizadas ao eletrocardiograma) na vigência de baixas doses de dobutamina de forma semelhante ao ecocardiograma. Estudou-se 54 pacientes com infarto do miocárdio prévio, encaminhados ao serviço de medicina nuclear para realização de pesquisa de viabilidade miocárdica. Foram excluídos do estudo os pacientes que no seguimento não foram revascularizados ou que não realizaram controle cintilográfico pós-cirúrgico, uma vez que considerouse como critério de viabilidade a melhora da contratilidade miocárdica após a revascularização. Avaliou-se os parâmetros de viabilidade (integridade celular e reserva contrátil) e o desempenho contrátil pós-cirúrgico de 260 segmentos miocárdicos de treze pacientes revascularizados. Os pacientes estudados foram submetidos a cintilografia de perfusão miocárdica duoisotópica em protocolo de dois dias com imagens tomográficas do coração obtidas em gamma-camera de duas cabeças, modelo Varicam (Elscint) e processadas em estação de trabalho eNTEGRA(GE). As imagens de estresse foram adquiridas em sincronia com o ECG (gated SPECT) em condições basais e na vigência de baixa dose de dobutamina (10 a 15g/Kg/min) 45 minutos após a administração endovenosa do 99mTcsestamibi no pico do exercício isotônico (esforço) ou da ação de agentes farmacológicos (estresse farmacológico) e nas etapas de repouso e redistribuição do cloreto de tálio-201, 20 minutos e quatro a seis horas após a administração endovenosa do radioisótopo em condições basais. Os pacientes operados foram submetidos a um segundo estudo cintilográfico de perfusão miocárdica com gated SPECT, no período mínimo de três meses após o procedimento, para avaliação da performance contrátil pós-cirúrgica. Para análise dos achados cintilográficos, dividiu-se o coração em 20 segmentos que receberam diferentes escores, permitindo a quantificação da perfusão e função miocárdica pelo Cedars Sinai Quantitative Pefusion SPECT QPS/QGS(GE),. Analisou-se o padrão perfusional nas etapas de estresse, repouso e redistribuição e de função (análise do espessamento sistólico, motilidade parietal, valores de fração de ejeção e volumes cardíacos do ventrículo esquerdo) em condições basais e sob estímulo inotrópico. No tratamento estatístico a análise do espessamento sistólico foi o parâmetro considerado significativo para avaliação da reserva contrátil miocárdica pelo método. Houve incremento na especificidade da pesquisa de viabilidade miocárdica pelo método radioisotópico realizado, demonstrando valores de especificidade superiores aos encontrados na literatura. As contribuições do método se mostraram efetivas / In patients with coronariopathy in the setting of ventricular dysfunction having an unpromising prognostic, the myocardial viability must be assessed thus, bringing contribution as it can predict the myocardial dysfunction recovery after revascularization. The myocardial segments with contractile dysfunction as a consequence of hypoperfusion can improve wall motion after perfusion recovery, demonstrating that myocardial revascularization, whenever suggested, can improve survival to this group of patients. This research is a PHD thesis from Radiology Department of São Paulo University and was performed at a nuclear medicine unit - Nuclear Medcenter/SOCOR hospital - in Belo Horizonte, Minas Gerais. The aim of the study was to check if dual isotope perfusion myocardial gated SPECT (99mTc-sestamibi/thallium-201) as a nuclear medicine procedure to the identification of viable myocardium, can improve the method specificity with addition of contractile reserve information obtained simultaneously by gated SPECT with low dose of dobutamine, similar to the echocardiogram. 54 patients with myocardial stroke, referred to the nuclear medicine unit to seek myocardial viability have been studied. Patients that do not have been submitted to revascularization or that did not undergo the post surgery control were excluded, as the parameter considered for viability was the wall motion recovery after revascularization. 260 myocardial segments in 13 patients had their viability parameters (cellular integrity and contractile reserve) as the contractile performance after surgery evaluated. The images were acquired by a Varicam (Elscint) double head gamma camera and processed by eNTEGRA (GE) workstation. The gated SPECT stress images were performed in baseline conditions and with low-dose dobutamine (10 a 15g/Kg/min) 45 minutes after intravenous injection of 99mTc-sestamibi.on the peak of isotonic exercise or pharmacologic stress. The rest and redistribution images were acquired , 20 minutes and 4 or 6 hours after intravenous injection of thallium-201 at rest. The revascularizated patients were also submitted to a second gated SPECT study at least 3 months after surgery for evaluation of the contractile performance. In order to analyze the scintigraphic findings, the heart was divided into 20 segments that received different scores for quantification of myocardial perfusion and function by Cedars Sinai Quantitative Perfusion SPECT QPS/QGS(GE),. The perfusion pattern of stress, rest and redistribution and the parameters of function (wall thickening and motion, ejection fraction and cardiac volumes analysis) at baseline conditions and by inotropic effect. By the statistics analysis wall thickening was considered significant to evaluate the myocardial contractile reserve by this method. There was improvement in the specificity of the radioisotopic research showing specificity values larger than those found in literature. These method contributions were effective

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The geraniol (C10H18O) is an acyclic monoterpene alcohol, present in the essential oil of some medicinal plants, herbs and citrus fruits, especially species of the genus Cymbopogon. Were described biochemical and pharmacological properties such as anticonvulsant action, analgesic, antinflammatory, antioxidant, anticancer and antimicrobial activities. In this study we sought to characterize the effects produced by geraniol on contractility, electrical activity and its possible antiarrhythmic potential in mammalian heart. For this, we used guinea-pig (Cavia porcellus) and mice (Mus musculus) of C57Bl/6J strain. The contractile studies were performed in the left atria drawn 1GF and stimulated with pulses of suprathreshold current, maintained in Cuba for isolated organ submerged in modified Tyrode solution (8 mL) and aerated with carbogenic mixture (95% O2 and 5% CO2). The force of atrial contraction was recorded by an isometric transducer. Electrocardiographic recordings were performed on isolated heart under constant aortic perfusion flow (8 mL/min) in a Langendorff system. To study the effects of geraniol on current membrane, experiments were performed using the technique of patch-clamp in rat ventricular cardiomyocytes setup whole-cell. In the atrium, geraniol reduced the force of contraction (~ 98%, EC50 = 1510 ± 160 M) whereas nifedipine, used as positive control, showed a EC50 of 0.90 ± 0.66 M. Geraniol, at 3 mM, decreased the positive inotropism of both CaCl2 and BAY K8644. In ventricular cardiomyocytes, the ICa,L was reduced by 50.7% (n = 5, p < 0.0001) after perfusion with 300 M of geraniol. Furthermore, geraniol prolonged the action potential duration (APD) measured at 50% of repolarization (49.7%, n = 5, p < 0.05), without changing the resting potential. The increase in APD can be attributed to blockade of K+ channel transient outward (Ito) (59.7%, n = 4, p < 0.001), the K+ current non-inactivated (Iss) (39.2 %, n = 4, p < 0.05) and K+ current to inward rectifier (Ik1) (33.7%, n = 4, p < 0.0001). In isolated heart, geraniol increased PRi and QTi without affecting the QRS (n = 6) complex, and reduced both left ventricular pressure (83%) and heart rate (16.5%). Furthermore, geraniol delayed time for the start of ouabain-induced arrhythmias in 128%, preventing in 30% the increase of diastolic tension, however, without affect the positive inotropic effect induced by ouabain (n = 6). Geraniol exerts negative inotropic and chronotropic responses in the mammalian heart by decreasing the L-type Ca2+ current and prolongs the duration of ventricular action potential by reducing potassium currents voltage-dependent. Such effects may be responsible for the antiarrhythmic effect of geraniol front the arrhythmias induced by ouabain in vitro. / O geraniol (C10H18O) é um monoterpeno alcoólico acíclico, presente no óleo essencial de algumas plantas medicinais, frutas cítricas e ervas aromáticas, principalmente espécies do gênero Cymbopogon. São descritas propriedades bioquímicas e farmacológicas, tais como ação anticonvulsivante, analgésica, anti-inflamatória, antioxidante, anticancerígena e antimicrobiana. Neste trabalho buscou-se caracterizar os efeitos produzidos pelo geraniol sobre a contratilidade, atividade elétrica e seu possível potencial antiarrítmico em coração de mamífero. Para tanto, foram utilizados cobaia (Cavia porcellus) e camundongos (Mus musculus) da linhagem C57Bl/6J. Os estudos contráteis foram realizados em átrio esquerdo estirado a 1gf e estimulados com pulsos de corrente supralimiares, mantido em cuba para órgão isolado, submerso em solução de Tyrode modificada (8 mL) e aerado com mistura carbogênica (95 % O2 e 5 % CO2). A força de contração atrial foi captada por um transdutor isométrico. Os registros eletrocardiográficos foram realizados em coração isolado, sob perfusão aórtica de fluxo constante (8 mL/min), em sistema de Langendorff. Para estudar os efeitos do geraniol sobre as correntes de membrana, foram executados experimentos através da técnica de patch-clamp , na configuração whole-cell , em cardiomiócitos ventriculares de camundongo. No átrio, o geraniol reduziu a força de contração (~ 98%, EC50 = 1510 ± 160 M) enquanto que a nifedipina, usada como controle positivo, apresentou uma EC50 de 0,90 ± 0,66 M. O geraniol, na concentração de 3 mM, diminuiu o inotropismo positivo de ambos CaCl2 e BAY K8644. Em cardiomiócito ventricular, a ICa,L foi reduzida em 50,7% (n = 5, p < 0,0001), após a perfusão com 300 μM de geraniol. Além disso, o geraniol prolongou a duração do potencial de ação (DPA), medida a 50 % da repolarização (49,7%, n = 5, p < 0,05), sem alterar o potencial de repouso. O aumento da DPA pode ser atribuído ao bloqueio dos canais para K+ transient-outward (Ito) (59,7 %, n = 4, p < 0,001), canal de K+ não-inativado (Iss) (39,2 %, n = 4, p < 0,05) e do canal para K+ inward rectifier (IK1) (33,7% , n = 4, p < 0,0001). Em coração isolado (n = 6), o geraniol aumentou o PRi e QTi sem afetar a duração do complexo QRS, reduzindo a pressão ventricular esquerda (83%) e a frequência cardíaca (16,5%). Além disso, o geraniol retardou o tempo para o início das arritmias induzidas por ouabaína em 128%, evitando em 30% o aumento da tensão diastólica sem, contudo, afetar o efeito inotrópico positivo da ouabaína (n = 6). O geraniol exerce respostas inotrópicas e cronotrópicas negativas no coração de mamífero, por meio da diminuição das correntes para Ca2+ tipo-L e, prolonga a duração do potencial de ação ventricular por reduzir as correntes para K+ dependentes de voltagem. Tais efeitos podem ser responsáveis pelo efeito antiarrítmico do geraniol frente às arritmias induzidas por ouabaína in vitro .

Orientador: Orlando Petrucci Junior / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T04:53:27Z (GMT). No. of bitstreams: 1 Vilarinho_KarlosAlexandredeSousa_D.pdf: 8722911 bytes, checksum: 025ab56f3339aaf3caf0958e795ae882 (MD5) Previous issue date: 2012 / Resumo: As lesões de isquemia e reperfusão miocárdica continuam sendo um desafio ao cirurgião cardíaco. A eritropoietina tem demonstrado efeito protetor contra lesões por isquemia e/ou reperfusão em corações adultos. Seu papel em corações neonatais ainda não foi esclarecido. Objetivo: avaliar o uso da eritropoietina em corações neonatais submetidos a isquemia e reperfusão. Material e métodos: suínos neonatos foram divididos em grupos de acordo com o momento da administração da eritropoietina (EPO- administrada três minutos antes da isquemia; EPO24- administrada 24 horas antes da isquemia; Controlenão recebeu eritropoietina) e submetidos a 45 minutos de isquemia miocárdica por oclusão da art. interventricular anterior e 90 minutos de reperfusão e avaliados índices de contratilidade derivados de curvas de volume vs. pressão obtidas por meio de cristais sonomicrométricos e pressão intraventricular. As vias da Akt e ERK ½ foram avaliados por western blot. Resultados: os grupos foram semelhantes na avaliação antes da isquemia. Não observamos diferenças entre os grupos em relação a frequência cardíaca, débito cardíaco e volume sistólico do ventrículo esquerdo. Observamos melhora da elastância máxima no grupo EPO aos 60 e 90 minutos de reperfusão, e melhora do trabalho sistólico prérecrutável e da dP/dt máxima nos dois grupos que receberam eritropoietina ao final da isquemia e durante toda a reperfusão. Não houve diferença entre os grupos nos índices de função diastólica. A eritropoietina promoveu fosforilação da Akt, mas não da ERK, e menor expressão de proteínas pró-apoptóticas. Conclusão: A eritropoietina protegeu a função sistólica do ventrículo esquerdo de corações neonatais submetidos a isquemia e reperfusão. Este resultado foi provavelmente mediado por ativação da via Akt / Abstract: Background: The effect of erythropoietin (EPOT) on neonatal hearts is not well understood. The current hypothesis is that erythropoietin has protective effects against ischemia-reperfusion when administered prior to ischemia induction. Methods: Systolic and diastolic indices, as well as the Akt and extracellular regulated kinase (ERK) signaling pathways, were studied in vivo using a neonatal pig heart model. Regional ischemia was induced for 45 min by ligation of the left anterior descending artery, followed by 90 min of reperfusion. The treatment groups consisted of: 1) untreated controls, 2) treatment with erythropoietin 3 min prior to ischemia, and 3) treatment with erythropoietin 24 h before ischemia. Sophisticated myocardial contractility indices were assessed by pressure/volume loops of the left ventricle. The Akt and ERK pathways were evaluated via western blot. Results: Elastance was found to be higher in the group receiving erythropoietin 3 min prior to ischemia. In addition, preload recruitable stroke work was higher for both groups receiving erythropoietin prior to ischemia when compared to controls. The time constant of the isovolumic relaxation and end diastolic pressure volume relationship did not differ between the three groups after 90 min of reperfusion. Furthermore, erythropoietin treatment enhanced phosphorylation of Akt, but not ERK, and erythropoietin treated animals showed lower levels of apoptosis-related proteins. Conclusions: Erythropoietin had a protective effect on neonatal systolic function after ischemia/reperfusion injury, but no effect on diastolic function. This cardioprotective effect might be mediated by activation of the Akt pathway / Doutorado / Fisiopatologia Cirúrgica / Doutor em Ciências

Abstract The heart adapts to hemodynamic overload with cardiac hypertrophy. Initially the increase in heart mass normalizes wall stress and permits normal cardiac function. In the long term pathological growth is associated with increased heart size, loss of functional myocytes and fibrotic replacement, heart dilatation and cardiac dysfunction, which can ultimately lead to heart failure. Vasoactive peptides participate in the regulation of cardiac contractility in an auto/paracrine way, but the peptidergic signaling pathways are largely unknown. The present study aimed to characterize the signaling mechanisms mediating the positive inotropic effect of endothelin-1 (ET-1) and the effects of prolactin releasing peptide (PrRP) on cardiac contractility in the isolated perfused rat heart preparation. The study demonstrated that mitogen-activated protein kinases (MAPK) have opposing roles in the regulation of cardiac contractility stimulated by ET-1. Extracellular signal-regulated kinase 1/2 (ERK1/2) mediated positive inotropic response to ET-1 was found to be counterbalanced by p38-MAPK. In addition, the effect of ET-1 was partly dependent on enhanced NADPH oxidase reactive oxygen species (ROS) generation, which activated the ERK1/2 pathway. In contrast, β-adrenergic inotropic effect was limited by stimulation of ROS production via negating phospholamban phosphorylation. The positive inotropic effect of ET-1 was counterbalanced by guanylyl cyclase (GC)-cGMP-protein kinase G (PKG) pathway and neuronal nitric oxide synthase (nNOS). PrRP was found to exert a direct positive inotropic effect which was independent of cAMP and was suppressed by concurrent activation of protein kinase Cα (PKCα) and protein phosphatase 1 (PP1), and dephosphorylation of phospholamban. In conclusion, in the present study signaling pathways in the acute regulation of cardiac contractility stimulated by ET-1 were characterized. The results suggest that the positive inotropic effect of ET-1 is mediated by ROS and ERK1/2 while p38-MAPK counterbalances the effect of ET-1. In addition, GC-cGMP-PKG pathway and nNOS modulate the response to ET-1. The study also established the previously unknown cardiac effects of PrRP. The findings provide a better understanding of molecular mechanisms involved in the regulation of cardiac contractility, and may indicate potential targets for novel therapeutic interventions. / Tiivistelmä Sydänlihaksen lisääntynyt mekaaninen kuormitus esimerkiksi verenpainetaudin tai sydäninfarktin yhteydessä voi johtaa sydämen kammion seinämän paksuuntumiseen eli hypertrofiaan. Hypertrofia auttaa sydäntä sopeutumaan lisääntyneeseen työmäärään, mutta se on myös sydän- ja verisuonitautitapahtumien riskitekijä. Pitkään jatkuva kuormitus johtaa usein sydämen pumppausvoiman heikkenemiseen ja sydämen vajaatoimintaan. Sydän tuottaa useita paikallisesti vaikuttavia vasoaktiivisia tekijöitä, jotka osallistuvat sydämen supistuvuuden säätelyyn. Väitöskirjatutkimuksessa tutkittiin signaalinvälitysjärjestelmiä, jotka osallistuvat endoteliini-1:n (ET-1) sydämen supistusvoimaa lisäävän eli positiivisen inotrooppisen vaikutuksen muodostumiseen sekä selvitettiin prolaktiinia vapauttavan peptidin (PrRP) vaikutuksia sydämen supistusvoimaan käyttämällä koemallina eristettyä, perfusoitua rotan sydäntä. Väitöskirjatyön tutkimustulokset osoittivat, että mitogeeni-aktivoituvat proteiinikinaasit (MAPK) ERK1/2 ja p38-MAPK osallistuvat ET-1:n inotrooppisen vaikutuksen säätelyyn. ERK1/2 välitti, ja p38-MAPK rajoitti ET-1:n lisäämää supistusvoiman kasvua. NADPH-oksidaasin tuottamat reaktiiviset happiradikaalit välittivät sydämessä ET-1:n inotrooppista vaikutusta ja ERK1/2 fosforylaatiota. Toisaalta NADPH-oksidaasin tuottamat happiradikaalit rajoittivat β-adrenergisen agonistin inotrooppista vaikutusta. Guanylaattisyklaasi (GC)-cGMP-proteiinikinaasi G (PKG) -järjestelmä ja neuronaalinen typpioksidisyntaasi (nNOS) vaimensivat ET-1:n lisäämää sydämen supistusvoiman kasvua. PrRP lisäsi sydämen inotropiaa syklisestä AMP:stä riippumattomalla tavalla, mutta vasteen säätelyyn havaittiin osallistuvan proteiinikinaasi C ja proteiinifosfataasi 1. Väitöskirjatutkimuksessa saatiin uutta tietoa solunsisäisistä viestinvälitysjärjestelmistä, jotka osallistuvat sydämen supistusvoimaan säätelyyn. Havaintojen perusteella ET-1:n positiivisen inotrooppisen vaikutuksen muodostumista välittävät reaktiiviset happiradikaalit ja ERK1/2, kun taas p38-MAPK rajoittaa vastetta. Lisäksi GC-cGMP-PKG -järjestelmä ja nNOS osallistuvat ET-1:n vaikutusten säätelyyn. Tutkimuksessa havaittiin myös, että PrRP vaikuttaa sydämen supistuvuuteen. Solutason mekanismien yksityiskohtien tunteminen voi mahdollistaa tulevaisuudessa sydän- ja verisuonisairauksien, kuten sydämen hypertrofian ja vajaatoiminnan, hoitomenetelmien tehostumisen ja mahdollisesti uudentyyppisten hoitomenetelmien kehittämisen.

L'analyse quantitative de la fonction contractile myocardique constitue un enjeu majeur pour le dépistage, le traitement et le suivi des maladies cardio-vasculaires, première cause de mortalité dans les pays développés. Dans ce contexte, l'Imagerie par Résonance Magnétique (IRM) s'impose comme une modalité privilégiée pour l'exploration dynamique du coeur, renseignant, d'une part, sur l'évolution des parois (ciné IRM), et permettant, d'autre part, d'accéder à des informations cinématiques au sein du myocarde (IRM de marquage). L'exploitation quantitative de ces données est néanmoins actuellement limitée par la quasi-absence de méthodologies fiables, robustes et reproductibles d'estimation de mouvement non rigide à partir de séquences d'images acquises dans cette modalité.<br /><br />Cette thèse se propose de démontrer que les techniques de recalage non rigide statistique constituent un cadre approprié pour l'estimation des déformations myocardiques en IRM, leur quantification à des fins diagnostiques, et leur modélisation en vue d'établir une référence numérique de normalité. Ses contributions concernent :<br /><br /> 1. l'élaboration d'une méthode robuste non supervisée d'estimation des déplacements myocardiques à partir de séquences d'IRM de marquage. Elle permet l'obtention de mesures de mouvement fiables en tout point du myocarde, à tout instant du cycle cardiaque et sous incidence de coupe arbitraire.<br /><br /> 2. le développement d'un outil de quantification dynamique des déformations à l'échelle du pixel et du segment myocardique, intégrant un étape de segmentation automatique du coeur par recalage d'images ciné IRM acquises conjointement aux données de marquage. Pour le coeur sain, la comparaison des mesures obtenues à des valeurs de référence issues d'une synthèse approfondie de la littérature médicale démontre une excellente corrélation. Pour des coeurs pathologiques, les expériences menées ont montré la pertinence d'une analyse quantitative multiparamétrique pour localiser et caractériser les zones atteintes.<br /><br /> 3. la construction d'un modèle statistique (atlas) de contraction d'un coeur sain. Cet atlas fournit des modèles quantitatifs de référence locaux et segmentaires pour les paramètres de déformation. Son intégration, en tant que modèle de mouvement, au processus de recalage des données d'IRM de marquage conduit en outre à une description très compacte des déplacements myocardiques sans perte de précision notable.

Introdução: Nos pacientes coronarianos, portadores de disfunção ventricular esquerda (DVE), a mortalidade cirúrgica da revascularização miocárdica é 3 a 4 vezes maior do que a encontrada em pacientes com função ventricular normal, sendo fundamental a seleção daqueles que efetivamente poderão ser beneficiados pela cirurgia. As metanálises indicam que a pesquisa da viabilidade miocárdica é útil nesta seleção, impactando em melhora da contratilidade ventricular e redução de mortalidade quando a revascularização é realizada em pacientes com ventrículo esquerdo viável; entretanto, os estudos clínicos randomizados não encontraram os mesmos resultados. Isso porque, apesar de 50% desses pacientes apresentarem quantidades substanciais de viabilidade miocárdica, nem todos conseguem melhorar a contratilidade ventricular esquerda após a revascularização, devido à existência de outros fatores que interferem nessa melhora. Objetivos: Determinar os fatores preditores de melhora da contratilidade ventricular esquerda em pacientes com fração de ejeção < 50% submetidos à cirurgia de revascularização miocárdica (CRM) isolada e o tempo necessário para a ocorrência dessa melhora contrátil. Métodos: Estudo prospectivo observacional que avaliou pacientes coronarianos com DVE submetidos a eletrocardiograma e ecocardiograma no pré-operatório e 1, 3, 6, 9 e 12 meses após a CRM, e à ressonância magnética cardíaca (RMC) com estresse farmacológico com dipiridamol e realce tardio com gadolínio no pré-operatório e após 3 e 12 meses da revascularização, buscando associações entre a melhora da contratilidade ventricular esquerda e as diversas variáveis dos pacientes. Resultados: Foram estudados 306 segmentos miocárdicos de 18 pacientes, com idade de 59,5 + 7,4 anos. Ocorreu melhora contrátil em 47 (29%) segmentos do ventrículo esquerdo que apresentavam alterações contráteis pré-operatórias (p < 0,0001). A análise multivariada identificou três fatores preditores de melhora da contratilidade ventricular esquerda: a ausência de onda Q patológica, que aumenta em 172% a chance de melhora, a presença de viabilidade miocárdica, que aumenta em 282% a chance de melhora e a ausência de isquemia miocárdica, que aumenta em 392% a chance de melhora (razão de chances 2,72, IC 95%, 1,24 a 5,92, p = 0,012; razão de chances 3,82, IC 95%, 1,79 a 8,16, p = 0,0005; e razão de chances 4,92, IC 95%, 2,13 a 11,36, p = 0,0002, respectivamente). Em 9 (75%) pacientes a melhora da contratilidade ventricular ocorreu nos 3 primeiros meses após a CRM e em 3 (25%) pacientes ocorreu nos 9 meses seguintes Conclusões: Os três fatores preditores de melhora da contratilidade ventricular esquerda encontrados foram a ausência de onda Q patológica no eletrocardiograma, e a presença de viabilidade e ausência de isquemia miocárdicas na RMC. A recuperação da contratilidade ventricular esquerda ocorreu predominantemente nos 3 primeiros meses após a CRM, no entanto foi verificada uma melhora progressiva até o final dos 12 meses de seguimento. / Introduction: In patients with coronary artery disease (CAD) and left ventricular dysfunction, the surgical mortality from coronary artery bypass graft (CABG) is 3 to 4 times higher than that reported for patients with normal ventricular function, and selecting those who can effectively benefit from the surgery is essential. Meta-analyses have indicate that myocardial viability assessment is useful in this selection, impacting on left ventricular contractility improvement and mortality reduction when revascularization is performed in patients with viable left ventricles; However, randomized clinical trials have not found the same results. Although 50% of these patients have substantial myocardial viability, not all of them can improve left ventricular contractility after revascularization, due to other factors that interfere with this improvement. Objectives: This study aims to determine the predictors of improvement in left ventricular contractility in patients with an ejection fraction of <50% who underwent isolated CABG, as well as the time required for this improvement in contractility. Methods: This prospective observational study assessed patients with CAD and left ventricular dysfunction who underwent electrocardiography and echocardiography during the preoperative period and 1, 3, 6, 9, and 12 months after CABG and cardiac magnetic resonance with pharmacological stress with dipyridamole and late gadolinium enhancement in the preoperative period and 3 and 12 months after revascularization, to determine the associations between the evolution of left ventricular contractility and several patient-related variables. Results: A total of 306 myocardial segments of the 18 patients, aged 59.5 ± 7.4 years, were studied. There was a contractile improvement in 47 (29%) segments of the left ventricle that presented preoperative contractile abnormalities (p < 0.0001). The multivariate analysis identified three predictors of left ventricular contractility improvement: the absence of pathological Q waves, which increases the chance of improvement by 172% (odds ratio (OR) 2.72, 95% confidence interval (CI), 1.24-5.92, p = 0.012), the presence of myocardial viability, which increases the chance of improvement by 282% (OR 3.82, 95% CI, 1.79-8.16, p = 0.0005), and the absence of myocardial ischemia, which increases the chances of improvement by 392%, (OR 4.92, 95% CI, 2.13-11.36, p = 0.0002). In 9 (75%) patients the improvement in ventricular contractility occurred in the first 3 months after CABG, and in 3 (25%) patients, it occurred in the following 9 months. Conclusions: The three predictors of left ventricular contractility improvement were the absence of pathological Q waves on an electrocardiogram, the presence of myocardial viability and the absence of signs of ischemia on cardiac MRI. The improvement in left ventricular contractility occurred predominantly in the first three months after CABG, but a progressive recovery was observed until the end of the 12-month follow-up period.

Adenosine is a nucleoside that plays an important role in the regulation of contractility in the heart. Adenosine receptors are G-protein coupled and those implicated in regulation of contractility are presumed to act via modulating the activity of adenylyl cyclase and cAMP content of cardiomyocytes. Adenosine A1 receptors (A1R) reduce the contractile response of the myocardium to β-adrenergic stimulation. This is known as anti adrenergic action. The A2A adenosine receptor (A2AR) has the opposite effect of increasing contractile responsiveness of the myocardium. The A2AR also appears to attenuate the effects of A1R. The effects of these receptors have been primarily studied in the rat heart and with the utilization of cardiomyocyte preparations. With the increasing use of receptor knockout murine models and murine models of various pathological states, it is of importance to comprehensively study the effects of adenosine receptors on regulation of contractility in the murine heart. The following studies examine the adenosinergic regulation of myocardial contractility in isolated murine hearts. In addition, adenosinergic control of contractility is examined in hearts isolated from A2AR knockout animals. Responses to adenosinergic stimulation in murine isolated hearts are found to be comparable to those observed in the rat, with A1R exhibiting an anti adrenergic action and A2AR conversely enhancing contractility. A significant part of the A2AR effect was found to occur via inhibition of the A1R antiadrenergic action. A part of the anti adrenergic action of A1R has previously been shown to be the result of protein phosphatase 2A activation and localization to membranes. Additional experiments in the present study examine the effect of adenosinergic signaling on PP2A in myocardial extracts from wild type and A2AR knockout hearts. A2AR activation was found to decrease the activity of PP2A and enhance localization of the active enzyme to the cytosol; away from its presumed sites of action. In the A2AR knockout the response to A1R activation was enhanced compared with the wild type and basal PP2A activity was reduced. It is concluded that A2AR modulation of PP2A activity may account for the attenuation of the A1R effect by A2AR observed in the contractile studies.

Introdução: Nos pacientes coronarianos, portadores de disfunção ventricular esquerda (DVE), a mortalidade cirúrgica da revascularização miocárdica é 3 a 4 vezes maior do que a encontrada em pacientes com função ventricular normal, sendo fundamental a seleção daqueles que efetivamente poderão ser beneficiados pela cirurgia. As metanálises indicam que a pesquisa da viabilidade miocárdica é útil nesta seleção, impactando em melhora da contratilidade ventricular e redução de mortalidade quando a revascularização é realizada em pacientes com ventrículo esquerdo viável; entretanto, os estudos clínicos randomizados não encontraram os mesmos resultados. Isso porque, apesar de 50% desses pacientes apresentarem quantidades substanciais de viabilidade miocárdica, nem todos conseguem melhorar a contratilidade ventricular esquerda após a revascularização, devido à existência de outros fatores que interferem nessa melhora. Objetivos: Determinar os fatores preditores de melhora da contratilidade ventricular esquerda em pacientes com fração de ejeção < 50% submetidos à cirurgia de revascularização miocárdica (CRM) isolada e o tempo necessário para a ocorrência dessa melhora contrátil. Métodos: Estudo prospectivo observacional que avaliou pacientes coronarianos com DVE submetidos a eletrocardiograma e ecocardiograma no pré-operatório e 1, 3, 6, 9 e 12 meses após a CRM, e à ressonância magnética cardíaca (RMC) com estresse farmacológico com dipiridamol e realce tardio com gadolínio no pré-operatório e após 3 e 12 meses da revascularização, buscando associações entre a melhora da contratilidade ventricular esquerda e as diversas variáveis dos pacientes. Resultados: Foram estudados 306 segmentos miocárdicos de 18 pacientes, com idade de 59,5 + 7,4 anos. Ocorreu melhora contrátil em 47 (29%) segmentos do ventrículo esquerdo que apresentavam alterações contráteis pré-operatórias (p < 0,0001). A análise multivariada identificou três fatores preditores de melhora da contratilidade ventricular esquerda: a ausência de onda Q patológica, que aumenta em 172% a chance de melhora, a presença de viabilidade miocárdica, que aumenta em 282% a chance de melhora e a ausência de isquemia miocárdica, que aumenta em 392% a chance de melhora (razão de chances 2,72, IC 95%, 1,24 a 5,92, p = 0,012; razão de chances 3,82, IC 95%, 1,79 a 8,16, p = 0,0005; e razão de chances 4,92, IC 95%, 2,13 a 11,36, p = 0,0002, respectivamente). Em 9 (75%) pacientes a melhora da contratilidade ventricular ocorreu nos 3 primeiros meses após a CRM e em 3 (25%) pacientes ocorreu nos 9 meses seguintes Conclusões: Os três fatores preditores de melhora da contratilidade ventricular esquerda encontrados foram a ausência de onda Q patológica no eletrocardiograma, e a presença de viabilidade e ausência de isquemia miocárdicas na RMC. A recuperação da contratilidade ventricular esquerda ocorreu predominantemente nos 3 primeiros meses após a CRM, no entanto foi verificada uma melhora progressiva até o final dos 12 meses de seguimento. / Introduction: In patients with coronary artery disease (CAD) and left ventricular dysfunction, the surgical mortality from coronary artery bypass graft (CABG) is 3 to 4 times higher than that reported for patients with normal ventricular function, and selecting those who can effectively benefit from the surgery is essential. Meta-analyses have indicate that myocardial viability assessment is useful in this selection, impacting on left ventricular contractility improvement and mortality reduction when revascularization is performed in patients with viable left ventricles; However, randomized clinical trials have not found the same results. Although 50% of these patients have substantial myocardial viability, not all of them can improve left ventricular contractility after revascularization, due to other factors that interfere with this improvement. Objectives: This study aims to determine the predictors of improvement in left ventricular contractility in patients with an ejection fraction of <50% who underwent isolated CABG, as well as the time required for this improvement in contractility. Methods: This prospective observational study assessed patients with CAD and left ventricular dysfunction who underwent electrocardiography and echocardiography during the preoperative period and 1, 3, 6, 9, and 12 months after CABG and cardiac magnetic resonance with pharmacological stress with dipyridamole and late gadolinium enhancement in the preoperative period and 3 and 12 months after revascularization, to determine the associations between the evolution of left ventricular contractility and several patient-related variables. Results: A total of 306 myocardial segments of the 18 patients, aged 59.5 ± 7.4 years, were studied. There was a contractile improvement in 47 (29%) segments of the left ventricle that presented preoperative contractile abnormalities (p < 0.0001). The multivariate analysis identified three predictors of left ventricular contractility improvement: the absence of pathological Q waves, which increases the chance of improvement by 172% (odds ratio (OR) 2.72, 95% confidence interval (CI), 1.24-5.92, p = 0.012), the presence of myocardial viability, which increases the chance of improvement by 282% (OR 3.82, 95% CI, 1.79-8.16, p = 0.0005), and the absence of myocardial ischemia, which increases the chances of improvement by 392%, (OR 4.92, 95% CI, 2.13-11.36, p = 0.0002). In 9 (75%) patients the improvement in ventricular contractility occurred in the first 3 months after CABG, and in 3 (25%) patients, it occurred in the following 9 months. Conclusions: The three predictors of left ventricular contractility improvement were the absence of pathological Q waves on an electrocardiogram, the presence of myocardial viability and the absence of signs of ischemia on cardiac MRI. The improvement in left ventricular contractility occurred predominantly in the first three months after CABG, but a progressive recovery was observed until the end of the 12-month follow-up period.