Cyclic guanosine monophosphate (cGMP) is a second messenger molecule that transduces nitric oxide (NO) and natriuretic peptide (NP) coupled signaling, stimulating phosphorylation changes by protein kinase G (PKG). Enhancing cGMP synthesis or blocking its degradation by phosphodiesterase type 5A (PDE5A) protects against cardiovascular disease1,2. However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation3. Furthermore, though PDE5A regulates NO-generated cGMP4,5, NO-signaling is often depressed by heart disease6. PDEs controlling NP-coupled cGMP remain uncertain. Here we show that cGMP-selective PDE9A7,8 is expressed in mammalian heart including humans, and is upregulated by hypertrophy and cardiac failure. PDE9A regulates NP rather than NO-stimulated cGMP in heart myocytes and muscle, and its genetic or selective pharmacological inhibition protects against pathological responses to neuro-hormones, and sustained pressure-overload stress. PDE9A inhibition reverses pre-established heart disease independent of NO-synthase (NOS) activity, whereas PDE5A inhibition requires active NOS. Transcription factor activation and phospho-proteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation. Thus, unlike PDE5A, PDE9A can regulate cGMP signaling independent of the NO-pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target.

Rationale Recent studies suggest an important role of autophagy in protection against αB-crystallin-based (CryABR120G) desmin-related cardiomyopathies (DRC) but this has not been demonstrated in a different model of cardiac proteinopathy. Mechanisms underlying the response of cardiomyocytes to proteotoxic stress remain incompletely understood. Objective First, to determine whether and how the autophagic activity is changed in a mouse model of desminopathy; second, to investigate the role of p62 in the protein quality control of cardiomyocytes. Methods and Results Using an autophagosome reporter and determining changes in LC3-II protein levels in response to lysosomal inhibition, we found significantly increased autophagic flux in mouse hearts with transgenic overexpression of a DRC-linked mutant desmin. Similarly, autophagic flux was increased in cultured neonatal rat ventricular myocytes (NRVMs) expressing a mutant desmin. Suppression of autophagy by 3-methyladenine increased, whereas enhancement of autophagy by rapamycin reduced, the ability of a comparable level of mutant desmin overexpression to accumulate ubiquitinated proteins in NRVMs. Furthermore, p62 mRNA and protein expression was significantly upregulated in cardiomyocytes by transgenic overexpression of the mutant desmin or CryABR120G both in intact mice and in vitro. p62 depletion impaired aggresome and autophagosome formation, exacerbated cell injury, and decreased cell viability in cultured NRVMs expressing the misfolded proteins. Conclusions Autophagic flux is increased in desminopathic hearts and, as previously suggested in CryABR120G-based DRC, this increased autophagic flux serves as an adaptive response to overexpression of misfolded proteins. p62 is upregulated in mouse proteinopathic hearts. p62 promotes aggresome formation and autophagy activation and protects cardiomyocytes against proteotoxic stress.

Background Proteasome functional insufficiency is implicated in a large subset of cardiovascular diseases and may play an important role in their pathogenesis. The regulation of proteasome function is poorly understood, hindering the development of effective strategies to improve proteasome function. Methods and Results Protein kinase G (PKG) was manipulated genetically and pharmacologically in cultured cardiomyocytes. Activation of PKG increased proteasome peptidase activities, facilitated proteasome-mediated degradation of surrogate (GFPu) and bona fide misfolded proteins (CryABR120G), and attenuated CryABR120G overexpression-induced accumulation of ubiquitinated proteins and cellular injury. PKG inhibition elicited the opposite responses. Differences in the abundance of the key 26S proteasome subunits Rpt6 and □5 between PKG manipulated and the control groups were not statistically significant, but the isoelectric points of were shifted by PKG activation. In transgenic mice expressing a surrogate substrate (GFPdgn), PKG activation by sildenafil increased myocardial proteasome activities and significantly decreased myocardial GFPdgn protein levels. Sildenafil treatment significantly increased myocardial PKG activity and significantly reduced myocardial accumulation of CryABR120G, ubiquitin conjugates, and aberrant protein aggregates in mice with CryABR120G-based desmin-related cardiomyopathy. No discernible effect on bona fide native substrates of the ubiquitin-proteasome system was observed from PKG manipulation in vitro or in vivo. Conclusions PKG positively regulates proteasome activities and proteasome-mediated degradation of misfolded proteins likely through posttranslational modifications to proteasome subunits. This may be a new mechanism underlying the benefit of PKG stimulation in treating cardiac diseases. Stimulation of PKG by measures such as sildenafil administration is potentially a new therapeutic strategy to treat cardiac proteinopathies.

Background Autophagy is essential to intracellular homeostasis and involved in the pathophysiology of a variety of diseases. Mechanisms regulating selective autophagy remain poorly understood. The COP9 signalosome (CSN) is a conserved protein complex consisting of 8 subunits (CSN1 through CSN8) and known to regulate the ubiquitin-proteasome system (UPS). However, it is unknown whether CSN plays a role in autophagy. Methods and Results Marked increases in LC3-II and p62 proteins were observed upon Csn8 depletion in the cardiomyocytes of mouse hearts with cardiomyocyte-restricted knockout of the gene encoding CSN subunit 8 (CR-Csn8KO). The increases in autophagosomes were confirmed by probing with GFP-LC3 and electron microscopy. Autophagic flux assessments revealed that defective autophagosome removal was the cause of autophagosome accumulation and occurred prior to a global UPS impairment in Csn8-deficient hearts. Analyzing the prevalence of different stages of autophagic vacuoles revealed defective autophagosome maturation. Down-regulation of Rab7 was found to strikingly co-localize with the autophagosome accumulation at the individual cardiomyocyte level. A significantly higher percent of cardiomyocytes with autophagosome accumulation underwent necrosis in CR-Csn8KO hearts. Chronic lysosomal inhibition with Chloroquine induced cardiomyocyte necrosis in mice. Rab7 knockdown impaired autophagosome maturation of non-selective and selective autophagy and exacerbated cell death induced by proteasome inhibition in cultured cardiomyocytes. Conclusions: (1) Csn8/CSN is a central regulator in not only the proteasomal proteolytic pathway but also selective autophagy; (2) likely through regulating the expression of Rab7, Csn8/CSN plays a critical role in autophagosome maturation; and (3) impaired autophagosome maturation causes cardiomyocytes to undergo necrosis.