Abstract P069: Interplay Between the Ubiquitin-Proteasome System and Autophagy in the Heart
The ubiquitin-proteasome system (UPS) and autophagy are responsible for the degradation of most cellular proteins. Dysfunctions in the UPS and autophagy are both implicated in cardiac pathogenesis. The UPS and autophagy, thought to be two parallel pathways, seem to interact. Pharmacologically induced proteasome inhibition (PSMI) was shown to increase autophagosomes in cultured cardiomyocytes (CMs) but this observation remains to be demonstrated in intact animals and it is unclear whether the increased autophagosomes are caused by an increased autophagic flux. A recent report shows that chronic autophagic inhibition (AI) impairs the degradation of proteasomal substrates in a p62-dependent manner in cultured cells but this has not been tested in intact animals or CMs. Here we sought to address these important issues. Using the GFP-LC3 reporter, we demonstrate in both cell cultures and intact mice that PSMI upregulates p62 and increases autophagic flux in CMs. This is consistent with the increased autophagic flux in proteinopathic mouse hearts in which proteasome function is inadequate. We then tested the effect of AI on the degradation of a surrogate proteasome substrate (GFPu) in cultured CMs. GFPu was expressed in cultured neonatal rat ventricular myocytes (NRVMs) via adenoviral delivery. Compared with the vehicle treatment, a 24-hr treatment but not 6-hr treatment of autophagy inhibitor 3-methyladenine (3-MA) or lysosome inhibitor bafilomycin A1 (BFA) significantly increased GFPu and p62 proteins in cultured NRVMs. Next, we tested the effect of AI on UPS-mediated protein degradation in intact mice. Transgenic mice overexpressing GFPdgn, a previously validated surrogate proteasome substrate, were treated with BFA (1.6 mg/kg/12hr, i. p.). BFA induced lysosome inhibition was verified by a significant increase in LC3-II and p62. Myocardial GFPdgn protein levels were significantly increased 24hrs but not 3hrs after the first BFA injection. The protein level of representative proteasome subunits was not affected by AI. Our data demonstrate that PSMI activates autophagy while chronic AI impairs the degradation of proteasome substrates in the heart. It will be interesting and important to test the role of p62 in the interplay.
- © 2011 by American Heart Association, Inc.