Abstract 320: Activation of Protein Kinase G Enhances Proteasome-Mediated Degradation of Misfolded Proteins

Abstract

The Ubiquitin Proteasome System (UPS) plays a pivotal role in the intracellular protein homeostasis, thereby regulating a variety of cellular processes including protein quality control (PQC). Inadequate PQC and proteasome functional insufficiency are associated with and may play an important role in adverse cardiac remodeling and cardiac failure (CHF). This is best reflected by the increased presence of misfolded proteins and protein aggregates in failing human hearts. While the regulatory mechanisms of these processes remain poorly understood they carry significant clinical implications. Reduced protein kinase G (PKG) activity is well documented in CHF. Increasing the activity PKG, during various cardiovascular diseases improves patient prognosis. We hypothesize that PKG plays a role in regulating UPS function. Using both genetic and/or pharmacological manipulation of PKG activity in cultured cardiomyocytes and intact mice and taking advantage of a previously validated surrogate misfolded protein (GFPu or GFPdgn) as well as a bona fide misfolded protein (R120G-CryAB) that is known to cause proteinopathy in human and mice, our studies have revealed a significant role for PKG in UPS regulation. In cultured cardiomyocytes, PKG inhibition was achieved by PKG-specific siRNA transfection or administration of PKG pharmacological inhibitor while PKG activity was induced by adenoviral delivery of a constitutively active PKG or by the treatment of phosphodiesterase 5A inhibitor Sildenafil. PKG activation significantly stimulated UPS activity in cultured cardiomyocytes as indicated by a reduced protein level of GFPu and increased proteasome activities; while PKG inhibition reduced UPS proteolytic activity. Furthermore PKG activation resulted in enhanced removal of R120G-CryAB. These findings from cell culture studies were further corroborated by in vivo findings from manipulating the muscarinic (M) receptor in GFPdgn transgenic mice. The M receptor signals through PKG. Activating the M receptor deceased, while antagonizing the M receptor increased, the steady protein level of GFPdgn in the heart. Hence, we conclude that PKG positively regulates the UPS thereby supporting cardiomyocyte PQC.