Soluble guanylate cyclase activation increases proteasome activities and protects against proteotoxicity in cardiomyocytes

Abstract

Background The ubiquitin-proteasome system (UPS) mediates the selective degradation of misfolded proteins and most cellular proteins that are normal but no longer needed, thereby playing important roles in diverse cellular processes. Proteasome malfunction and proteotoxicity are implicated in the genesis of a large subset of cardiovascular diseases. We previously discovered that activation of the cGMP-dependent protein kinase (PKG) primes the proteasome. Here we sought to test whether activation of the soluble guanylate cyclase (sGC) could activate the proteasome and improve protein quality control in cardiomyocytes. Methods and Results In cultured neonatal rat ventricular myocytes (NRVMs), treatment with an sGC activator (cinaciguat) led to increases in the levels of Ser239-phosphorylated vasodilator-stimulated phosphoprotein (VASP) in a dose-dependent fashion and increased the activities of 26S proteasomes as assessed with in-gel proteasome activity assays, revealing that sGC activation increases PKG activity and stimulates proteasome activities in cardiomyocytes. Adenoviral vectors expressing degron CL1-fused green fluorescent proteins (GFPu, a proven UPS substrate) or red fluorescent proteins (RFP, a stable protein control) were used as a reporter system for UPS performance. Cinaciguat treatment enhanced the degradation of GFPu in NRVMs. In NRVMs with adenovirus-mediated overexpression of a human-disease-linked misfolded protein HA-tagged CryABR120G, the proteasome-mediated flux of CryABR120G was significantly enhanced by cinaciguat treatment. Consistently, cinaciguat treatment significantly reduced the levels of CryABR120G oligomers, indicating that the cinaciguat treatment promotes proteasomal degradation of the misfolded species of CryABR120G. Furthermore, the LDH leakage and PKCδ cleavage in NRVMs overexpressing CryABR120G were significantly attenuated by cinaciguat treatment. Conclusions Activation of sGC with cinaciguat increases PKG activity, activates the 26S proteasome, facilitates the degradation of misfolded proteins, and thereby protects against proteotoxicity in cardiomyocytes.