Effects of Autophagy Inhibitor, 3-methyladenine, on Isolated Atria Subjected to Simulated Ischemia-Reperfusion

HERMANN, ROMINA; MESTRE CORDERO, VICTORIA E.; RUSIECKI, TATIANA M.; FERNÁNDEZ PAZOS, M.MERCEDES; VÉLEZ, DÉBORA E.; REZNIK, FEDERICO; SAVINO, ENRIQUE A.; MARINA PRENDES, M.GABRIELA; VARELA, ALICIA

Buenos Aires

Congreso; 20th Annual Scientific Meeting of the International Society of Cardiovascular Pharmacotherapy (ISCP); 2015

Autophagy is a cellular catabolic process that occurs constitutively at a low rate in all eukaryotic cells, mainly for house-keeping purposes. Although autophagy is a prominent feature of myocardial ischemia and reperfusion, the functional significance is unclear and controversial. In order to gain a deeper insight into the role of autophagy in myocardial ischemia-reperfusion, we explored the effects of the pharmacological inhibitor of autophagy 3-methyladenine (3-MA, 5mM) on isolated rat left atria subjected to simulated ischemia (75min)-reperfusion (75min) (Is-Rs).The results showed a significant enhance in the LC3-II/LC3-I ratio, which is an established indicator of autophagy, during Rs. This was prevented by 3-MA which also increased p62 protein, one of the specific substrates that are degraded through the autophagy-lysosomal pathway. Electron micrographs showed double membrane autophagosome like structures during Rs, which were absent in the presence of 3-MA. These findings suggest that autophagy is upregulated during Rs and that 3-MA inhibits the autophagic flux, under the present experimental conditions. Moreover, ultrastructural analysis showed a deterioration of mitochondrial morphology, which was more severe in the presence of 3-MA, without affecting cell viability. To assess whether these deleterious effects of 3-MA on mitochondrial morphology were accompanied by changes in mitochondrial ATP synthesis, the rate of ATP synthesis by isolated mitochondria at the end of Is-Rs was determined. Results showed that, 3-MA attenuated the rate of ATP synthesis in isolated mitochondria. Changes in ATP content of isolated atria subjected to Is-Rs showed a markedly decreased at the end of Is, reaching similar values in 3-MA-treated and non-treated atria, while at the end of Rs, ATP of 3-MA-treated and non-treated atria recovered to 32% and 51%, respectively. When analyzing contractile parameters, the results showed a gradual recovery during Rs, which were notaffected by 3-MA. However, when maximal inotropic response was assessed by adding isoproterenol 2 μM to the incubation medium at the end of the Is-Rs period, an attenuation in cardiac contractility was observed in the presence of 3-MA. Furthermore, when Is-Rs experiments were performed in the presence of 3-MA, tachyarrhythmias were observed throughout the reperfusion period, with frequencies ranging between 270 and 300 contractions per minute. This phenomenon was not observed in absence of the autophagy inhibitor. When the experiments were performed in the presence of 3-MA and the inner membrane anion channel blocker (PK11195), a significant attenuation of incidence of tachyarrhythmias was observed, suggesting involvement of mitochondria in the 3-MA-induced arrhythmias.Present results suggest an association between the inhibition of autophagy and functional alterations of the cells that have undergone sublethal stress and been able to recover in this experimental model of moderate ischemia.