Abstract 16677: Lipid-overload Impairs Cardiomyocyte Autophagosome Turnover by Activating a Protein kinase C (PKC)-NADPH oxidase 2 (NOX2) Pathway
Obesity regulates autophagy in a tissue-dependent manner to influence tissue homeostasis in beneficial or deleterious ways. The regulation of cardiomyocyte autophagy in obesity is incompletely understood. High-fat feeding in mice increased autophagosome number in the heart and treatment of these animals with chloroquine (CQ) did not further increase their numbers, suggesting impaired turnover. To elucidate the underlying mechanisms, H9C2 cardiomyocytes (CM) were treated with vehicle (veh) or 500 μM palmitate (PAL) for 4h. PAL treatment significantly induced autophagosome abundance as revealed by an increase in LC3-II levels (~2.5 fold) and autophagosome number (~4-fold) compared with vehicle treatment. Inhibition of autophagosome turnover with CQ led to the accumulation of LC3-II and autophagosomes to a greater extent in veh-treated CMs than in PAL-treated CMs relative to their respective CQ-untreated controls. PAL significantly induced superoxide (O2-) generation (~3.8 fold) and impaired lysosomal acidification and lysosomal Cathepsin L (CatL) activity. Treatment with the superoxide scavenger tiron, significantly reduced LC3-II levels (~35%) in PAL-treated CMs and restored lysosomal acidification and Cat L activity. Moreover, inhibition of NOX2 activity by siRNA-mediated silencing of its regulatory subunit p47phox or by apocynin significantly reduced LC3-II levels (~31%), normalized autophagosome accumulation and CatL activity, and suppressed O2- generation to basal levels. PAL induced NOX2 activity by facilitating the translocation of p47phox to the plasma membrane which was partially blunted by the inhibition of PKC-α/βII with Gö6976. Inhibition of PKC-α/βII prevented the accumulation of LC3-II (~37%), restored Cat L activity and suppressed O2- production to basal levels in PAL-treated CMs. Thus, activation of a PKC-NOX2 pathway represents a novel mechanism that impairs lysosomal acidification and enzyme activity, leading to inhibition of autophagosome turnover in lipid-overloaded CMs.
- © 2013 by American Heart Association, Inc.