Abstract 187: Autophagy in the Myocardium of Mice Subjected to Ischemia Followed by Reperfusion
Introduction: Autophagy has been thought by many to be cardioprotective, but its extent early after myocardial ischemic insults remains unclear. We characterized the magnitude of early autophagy in myocardium of murine hearts subjected to ischemia with or without reperfusion.
Methods: 10 week old transgenic GFP-LC3 (a reporter transgene for autophagy) mice on a C57Bl6 genetic background and normal C57Bl6 mice were subjected to left anterior descending coronary ligation for 1 or 4 hours followed by 24 hours of reperfusion (1HTL, 4HTL) or to 24 hours of persistent ligation (24HPL). Sections of hearts from the GFP-LC3 mice were analyzed by fluorescence microscopy. Left ventricular (LV) homogenates of hearts from C57Bl6 mice were analyzed by Western blotting for LC3-I to LC3-II conversion (a marker of autophagic flux), for phospho-S6 ribosomal protein (Ser235/236) (a marker of mTOR activation and presumably consequent down regulation of autophagy), and by electron microscopy (EM).
Results: Fluorescent GFP-LC3 dots were absent in the center of infarct zones and reduced markedly in the peri-infarct zones compared with the number in normal zones and in sham controls (normal LV 77 ± 17 dots per high power field, n = 6; sham 79 ± 4, 1HTL 31 ± 10; 4HTL 9 ± 5; 24HPL 32 ± 9, n =3 and p ≤ 0.05 compared with sham and normal hearts for all other groups). The ratio of LC3-II to LC3-I did not increase in LV homogenates from hearts subjected to ischemia (sham 1.0 ± 0.04; 1HTL 0.92 ± 0.17, 4HTL 1.03 ± 0.07, 24HPL 1.11 ± 0.15, n = 3 for all groups). Phosphorylation of S6 ribosomal protein increased in LV homogenates in hearts from all groups subjected to ischemia (sham 1.0 ± 0.1; 1HTL 1.46 ± 0.3, 4HTL 1.77 ± 0.18, 24HPL 1.55 ± 0.28, p ≤ 0.05 for all groups compared to sham, n = 3 for all groups) consistent with down regulation of autophagy. Virtually no EM recognizable autophagic cells were evident in infarct or peri-infarct zones.
Conclusions: Autophagy is virtually absent within 24 hours in the center of zones of mouse hearts subjected to ischemia reperfusion and is decreased significantly in the peri-infarct zones. mTOR is activated consistent with suppression of autophagy. Thus, pharmacologic augmentation of autophagy is an attractive target for limiting infarct size early after the onset of ischemia.
- © 2010 by American Heart Association, Inc.