Abstract 17771: p27 Positively Regulates Autophagy in Resting ind Nutrient-deprived Cardiomyocytes
Background: The role of p27 (a regulator of cell growth and division) in autophagy, the catabolic process that removes unwanted proteins and organelles, has not been examined in terminally differentiated cells such as cardiomyocytes (CM).
Results: Treatment (0.1 µM for 4 h) with a p27 fusion protein (TAT.p27) induced microtubule-associated protein LC3-II, an established marker of autophagy, as determined by Western blot (WB) in neonatal rat CM in vitro, under basal conditions (2.2±0.1 fold, P<0.05;TAT.βgal: 1.0±0.1, P=NS; vs. untreated controls) and after glucose deprivation (4.5±0.6 fold, P<0.05;TAT.βgal: 2.7±0.2, P=NS; vs. starvation control: 2.6±0.1). Immunofluorescence (IF) for LC3-positive cytosolic dots revealed similar results. These effects were preserved in presence of the lysozyme inhibitor bafilomycin-A1, supporting a direct effect of p27 on LC3 synthesis. Compared to untreated controls, TAT.p27-treated mice also manifest greater markers of autophagy in the heart at rest (1.6±0.1 fold, P<0.05; saline: 1.0±0.1, P=NS; TAT.βgal: 1.0±0.1, P=NS) and following 48 h food deprivation (3.0±0.1 fold, P<0.05; saline: 2.2±0.1, p=NS; TAT.βgal: 2.3±0.2, P=NS), as compared to fasted controls (2.3±0.12). Similar results were found with IF for LC3-positive dots. Conversely, lentivirus-delivered shRNA against p27 successfully reduced p27 levels (67.5±0.1% vs. mock shRNA, P<0.05) and suppressed basal (0.7±0.1 fold, P<0.05) and glucose-deprived levels of autophagy in CM in vitro (0.4±0.1 fold, P<0.05) vs mock shRNA controls. Of note, only after glucose deprivation did CM prevented from activation of autophagy by p27 knockdown undergo apoptosis as determined by WB for cleaved caspase 3 (2.0±0.1 vs. 1.3±0.1 mock, P<0.05). Note all above results represent N=3 experiments. Using p27 knockout mice, we found that p27 is not required for basal levels of cardiac autophagy, but needed to increase autophagy in response to food deprivation.
Conclusion: p27 positively regulates autophagy in CM in vitro and in vivo at rest and after metabolic stress. Only after metabolic stress did reduced levels of p27 result in apoptosis. These data support the idea of using TAT.p27 to enhance autophagy flux and prevent apoptosis in models of heart disease.
- © 2012 by American Heart Association, Inc.