Rapid restoration of blood flow to ischemic myocardium is essential, however it causes further injury called reperfusion injury. Apoptosis contributes significantly to cardiomyocyte cell death during ischemia reperfusion injury, in which caspase family proteases play an essential role as they are the executioners of apoptosis. Caspase inhibitors showed promising cardioprotective results when administered before ischemia or at the start of reperfusion. However, before applying them in pre clinical studies of myocardial ischemia, several investigations needed to be taken to determine their therapeutic window post reperfusion, their effect on functional recovery of myocardium post ischemia, their mechanism of action. Methods Isolated perfused rat hearts were subjected to 35 min ischemia followed by 2 hr reperfusion where caspase inhibitors [broad spectrum caspase inhibitor (ZVAD, 0.1µM), specific caspase 3 inhibitor (DEVD, 0.07µM)] were added at the start of reperfusion, 15, 30 and 60 min after starting reperfusion at the presence or absence of Wortmannin (WORT, 100nM, PI3-kinase inhibitor). Hearts underwent triphenyl tetrazolium staining for infarct size assessment, or were frozen for Western blot analysis. Freshly isolated adult rat ventricular myocytes were subjected to 6 hr hypoxia followed by either 18 hr, where caspase inhibitors (ZVAD, 25µM and DEVD, 25µM) were added at the start of reoxygenation, 15, 30 and 60 min after starting reoxygenation at the presence or absence of Wortmannin (WORT, 100nM). Cardiomyocytes were analysed for viability, apoptosis, necrosis and intracellular caspase-3 activity using flow cytometry analysis. Isolated adult rat ventricular papillary muscles were subjected to 35 min hypoxia followed by 100 min reperfusion where caspase inhibitors [ZVAD (0.1 µM, 2.5µM) and DEVD (2.5µM)] were added at the start of reperfusion throughout. Power output was measured using work loop technique.

O objetivo desta tese é avaliar a expressão do miRNA-423-5p, em diferentes sítios, em modelo experimental de remodelamento cardíaco após insulto isquêmico em ratos. Os animais foram randomizados em grupos SHAM (cirurgia sem oclusão da artéria coronária descendente anterior esquerda) ou IAM (cirurgia com ligadura da artéria coronária descendente anterior esquerda) e acompanhados por 1, 7, 28 e 90 dias. Após o tempo de seguimento, os animais foram submetidos ao ecocardiograma e eutanasiados. Foi retirado sangue do plexo retroorbital, sangue venoso e arterial e coletado tecido do músculo gastrocnêmio e do ventrículo esquerdo separando as áreas remota (REM), infartada (INF) e peri-infartada (PERI). A partir da homogeneização dos tecidos, foi realizada a extração de miRNA e sua expressão quantificada pelo método de PCR em tempo real. Também foi mensurado os níveis plasmáticos do peptídeo natriurético cerebral (BNP). Os dados foram analisados pela teste de ANOVA de uma e duas vias e correlações através do programa estatístico SPSS 21.0. Quanto a caracterização do modelo utilizado, podemos verificar que a fração de ejeção do ventrículo esquerdo dos ratos IAM foram menores que os do grupo SHAM e os percentuais de área acinética foram iguais em todos os grupos IAM, como esperado. Também observamos que o miRNA-423-5p é expresso no coração, nos diferentes segmentos analisados, apresentando variação significativa nos tempos avaliados, correlacionado-se positivamente com o tamanho do infarto e negativamente com a fração de ejeção do ventrículo esquerdo. Diante deste cenário, nossos achados solidificam o conceito de que a expressão do miRNA-423-p se altera ao longo do tempo após insulto isquêmico e pode ter papel relevante no remodelamento cardíaco de origem isquêmica. / The objective of this project was to evaluate the expression of miRNA-423-5p in an experimental model of cardiac remodeling after ischemic injury in rats. Animals were randomized to SHAM group (surgery without occlusion of the left anterior descending coronary artery) or acute myocardial infaction (AMI) group (surgery with ligation of the left anterior descending coronary artery) and followed for 1, 7, 28 and 90 days. After the follow-up period, the animals were submitted to echocardiography and euthanized. Blood from the retroorbital plexus, venous and arterial blood was collected; and gastrocnemius and left ventricle tissue was collected, separating the remote (REM), infarcted (INF) and peri-infarcted (PERI) areas. From the homogenization of tissues, miRNA was extracted and its expression quantified by real-time PCR. Plasma levels of brain natriuretic peptide (BNP) were also measured by ELISA. Data were analyzed by one-way and two-way ANOVA and coefficient correlations were calculated using the statistical package SPSS 21.0. Regarding the experimental model, we could verify that the left ventricular ejection fraction of the AMI rats were reduced compared to the SHAM group and the percentages of akinetic area were the same in all AMI groups, as expected. We also observed that miRNA-423-5p is expressed in the heart in the different segments analyzed, showing significant variation in the different periodos that were evaluated, is positively correlated with infarct size and negatively with left ventricular ejection fraction. In this scenario, our findings solidify the concept that miRNA-423-p expression changes over time after an ischemic insult and may play a relevant role in the cardiac remodeling of ischemic origin.

Heart failure is an important medical disease and impacts millions of people throughout the world. In order to treat this problem, biomaterial injectable treatment injected into the myocardium of the failing LV are currently being developed. Through this treatment, the biomaterial material injections can reduce wall stresses during the cardiac remodeling process. By using computational techniques to analyze the effects of a treatment involving the injection of biomaterial material into the LV after MI, the material parameters of the hydrogel injections can be optimized. The results shows that the hydrogel injections could reduce the global average fiber stress and the transmural average stress seen from optimization. These results indicated that the hydrogel injections could influence the stiffness in passive LV tissue, but there is still need for more research on the active part of ventricular contraction. Conclusion: hydrogel injection is a viable way to alter ventricular mechanical properties.