Cardiac proteasome functional insufficiency is implicated in a large subset of heart disease and has been experimentally demonstrated to play an essential role in cardiac proteotoxicity, including desmin-related cardiomyopathy and myocardial ischemia-reperfusion (I-R) injury. Pharmacological inhibition of phosphodiesterase 5 (PDE5) via sildenafil for example, which can stabilize cGMP and thereby increase cGMP-dependent protein kinase (PKG) activity, is consistently reported to protect against I-R injury; however, the underlying mechanism is not fully understood. We have recently discovered that PKG activation enhances proteasomal degradation of misfolded proteins (Ranek, et al. Circulation 2013), prompting us to hypothesize that proteasome-priming may contribute to cardioprotection-induced by PDE5 inhibition. Here we used a cardiomyocyte-restricted proteasome inhibition transgenic mouse line (Tg) and non-Tg (Ntg) littermates to interrogate the action of sildenafil on I-R injury created by left anterior descending artery (LAD) ligation (30 min) and release (24 hr). Sildenafil was administered 30 min before LAD ligation. Results showed that (1) the 26S proteasome activity of the Ntg I-R hearts was significantly elevated by sildenafil but this elevation was blocked in the Tg line; (2) the infarct size reduction by sildenafil treatment in Ntg mice was completely abolished in the Tg mice with the same treatment; and (3) systolic and diastolic function impairment after I/R was markedly attenuated in sildenafil-treated Ntg mice, but not in the sildenafil-treated Tg mice. Additionally, immunoprecipitation assays show that PKG interacted with the proteasome in cultured cardiomyocytes, and this interaction appeared to be augmented by sildenafil treatment. Moreover, in vitro incubation of active PKG with purified human 26S proteasomes increased proteasome peptidase activities and the phosphorylation at specific serine residues of a 19S proteasome subunit as revealed by “gel-free” nano-LC-MS/MS. We conclude that active PKG directly interacts with, phosphorylates, and increases the activities of, the proteasome and that proteasome priming mediates to cardioprotection of PDE5 inhibition against I-R injury.
Phosphodiesterase-5 Inhibitor, Tadalafil, Protects Against Myocardial Ischemia/Reperfusion Through Protein-Kinase G-Dependent Generation of Hydrogen Sulfide
