Figure 4. Stimulation of M receptors enhances UPS proteolytic function and proteasome activities in cultured cardiomyocytes.

A, Pilocarpine (Pilo, 10μM) or saline control (Saline) was administered to neonatal rat ventricular myocytes in culture (NRVMs) 24h after infection with Ad-GFPu and Ad-RFP. The cells were harvested at 48h of pilocarpine treatment. Total protein extracts were used for western blot analyses of the steady state GFPu and RFP. B, Cycloheximide (CHX) chase assay for GFPu. NRVMs were first infected with Ad-GFPu (10MOI) and 24h later treated with pilocarpine (10μM) or saline. After an additional 24 hours, CHX (100μM) was added to the culture media to inhibit protein synthesis. Cells were harvested at baseline (0 min), 5, 10, and 15 min after CHX addition and the cell lysate was used for immunoblot analysis. Representative images (upper panel) and pooled densitometry data from 4 biological repeats (lower panel) are shown. GFPu half-life (t_1/2) in the pilocarpine treated cells was significantly shorter than in the saline control treated cells (8.3±1.3min vs. 18.7±2.5 min; n=4 pairs; p<0.05, paired t-test). C, Proteasome peptidase activity assays. Crude protein extracts from NRVMs that had been treated as in panel A were used for assessing chymotrypsin-like, caspase-like, and trypsin-like activities in the presence (+) and absence (−) of ATP. *p<0.05 vs. Saline, n=12 biological repeats.