Effects of autophagy inhibitor, 3-methyladenine on isolated atria subjected to simulated ischemia reperfusion.

HERMANN R; V MESTRE CORDERO; TATIANA RUSIECKI; M FERNANDEZ PAZOS; D VELEZ; F REZNIK; ENRIQUE A. SAVINO; MARÍA G. MARINA PRENDES; A VARELA

Congreso; Annual Cientific Meeting of the International Society of Cardiovascular Pharmacoterapy; 2015

Effects of autophagy inhibitor, 3-methyladenine on isolated atria subjected to simulated ischemia reperfusion.Background and aims : Autophagy is a cellular catabolic process that occurs constitutively at a low rate in all eukaryoticcells, mainly for house-keeping purposes. Although autophagy is a prominent feature of myocardial ischemia andreperfusion, the functional significance is unclear and controversial. In order to gain a deeper insight into the role ofautophagy in myocardial ischemia-reperfusion, we explored the effects of the pharmacological inhibitor of autophagy 3-methyladenine (3-MA).Methods: Isolated rat left atria subjected to simulated 75-min ischemia/75-min reperfusion (Is-Rs) in the presence orabsence of 3-MA (5mM) were used.Results: The results showed a significant enhance in the LC3-II/LC3-I ratio, an established indicator of autophagosomeformation, during Rs. This was prevented by 3-MA which also increased p62 protein, one of the specific substrates thatare degraded through the autophagy-lysosomal pathway. Electron micrographs showed double membraneautophagosome like structures during Rs, which were absent in the presence of 3-MA. These findings suggest thatautophagy is upregulated during Rs and that 3-MA inhibits the autophagic flux, under the present experimentalconditions. Moreover, ultrastructural analysis showed a deterioration of mitochondrial morphology, which was moresevere in the presence of 3-MA, without affecting cell viability. To assess whether these deleterious effects of 3-MA onmitochondrial morphology were accompanied by changes in mitochondrial ATP synthesis, the rate of ATP synthesis byisolated mitochondria at the end of Is-Rs was determined. Results showed that 3-MA attenuated the rate of ATPsynthesis in isolated mitochondria. Changes in ATP content of isolated atria subjected to Is-Rs showed a markedlydecreased 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. Contractile parameters gradually recoveredduring Rs and were not affected by 3-MA. However, when maximal inotropic response was assessed by addingisoproterenol 2 μM  to the incubation medium at the end of the Is-Rs period, an attenuation in cardiac contractility wasobserved 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 300contractions per minute. This phenomenon was not observed in absence of the autophagy inhibitor. When theexperiments were performed in the presence of 3-MA and the inner membrane anion channel blocker (PK11195), asignificant attenuation of incidence of tachyarrhythmias was observed, suggesting involvement of mitochondria in the 3-MA-induced arrhythmias. Conclusion: Present results suggest an association between the inhibition of autophagy and functional alterations of thecells that have undergone sublethal stress and been able to recover in this experimental model of moderate ischemia.