Fig. 5.

Neprilysin plays a causative role in PAO accumulation. (A) Representative Western blot showing neprilysin levels in untreated NRCs and CryAB^R120G-infected NRCs in the presence or absence of thiorphan 24 h after infection. (B) PAO accumulation (green) in CryAB^R120G-infected NRCs 24 h after infection was enhanced by thiorphan. Nuclei (blue) were stained with TO-PRO-3. To confirm that the increased PAO levels were not due to NO production, the NO synthase inhibitor N^G-nitro-l-arginine methyl ester (L-NAME) was also added to selected cultures. (C) Overexpression of neprilysin significantly reduces PAO 3 days after infection. (D and E) Quantitation of PAO-positive areas from B and C, respectively, as a percentage of total NRC area. (F) The effect of overexpression or inhibition of neprilysin on the rate of cardiac cell death as measured by the release of adenylate kinase into the medium. Values shown are the average-fold increase relative to control, nontransfected NRCs for three independent series ± SE. ∗, P < 0.005 vs. control; ⋀, P < 0.005 vs. CryAB^R120G-infected NRCs.