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.

The cardiac pathological response to sustained pressure overload involves myocyte hypertrophy and dysfunction along with interstitial changes such as fibrosis and reduced capillary density. These changes are orchestrated by mechanical forces and factors secreted between cells. One such secreted factor is TGF-β, which is generated by and interacts with multiple cell types. Here we have shown that TGF-β suppression in cardiomyocytes was required to protect against maladaptive remodeling and involved noncanonical (non-Smad-related) signaling. Mouse hearts subjected to pressure overload and treated with a TGF-β-neutralizing Ab had suppressed Smad activation in the interstitium but not in myocytes, and noncanonical (TGF-β-activated kinase 1 [TAK1]) activation remained. Although fibrosis was greatly reduced, chamber dysfunction and dilation persisted. Induced myocyte knockdown of TGF-β type 2 receptor (TβR2) blocked all maladaptive responses, inhibiting myocyte and interstitial Smad and TAK1. Myocyte knockdown of TβR1 suppressed myocyte but not interstitial Smad, nor TAK1, modestly reducing fibrosis without improving chamber function or hypertrophy. Only TβR2 knockdown preserved capillary density after pressure overload, enhancing BMP7, a regulator of the endothelial-mesenchymal transition. BMP7 enhancement also was coupled to TAK1 suppression. Thus, myocyte targeting is required to modulate TGF-β in hearts subjected to pressure overload, with noncanonical pathways predominantly affecting the maladaptive hypertrophy/dysfunction.

Objectives We tested the hypothesis that bi-directional, gene-targeted regulation of cardiomyocyte cGMP-selective phosphodiesterase type 5 (PDE5) influences maladaptive remodeling in hearts subjected to sustained pressure-overload. Background PDE5 expression is up-regulated in human hypertrophied and failing hearts, and its inhibition (e.g. by sildenafil) stimulates protein kinase G activity, suppressing and reversing maladaptive hypertrophy, fibrosis, and contractile dysfunction. Sildenafil is currently being clinically tested for the treatment of heart failure. However, new studies have questioned the role of myocyte PDE5 and protein kinase G (PKG) to this process, proposing alternative targets and mechanisms. Methods Mice with doxycycline controllable myocyte-specific PDE5 gene-expression were generated (medium (me-TG) and high (hi-TG) expression lines), and subjected to sustained pressure-overload. Results Rest myocyte and heart function, histology, and molecular profiling were normal in both TG-lines versus controls at 2 months of age. However, upon exposure to pressure-overload (aortic banding), TG hearts developed more eccentric remodeling, maladaptive molecular signaling, depressed function, and amplified fibrosis with up-regulation of tissue growth factor signaling pathway. PKG activation was inhibited in TG myocytes versus control. After establishing a severe cardiomyopathic state, hi-TG mice received doxycycline to suppress PDE5 expression/activity only in myocytes. This in turn enhanced PKG activity, and reversed all of the previously amplified maladaptive responses despite sustained pressure-overload. Sildenafil was also effective in this regard. Conclusions These data strongly support a primary role of myocyte PDE5 regulation to myocardial pathobiology and PDE5 targeting therapy in vivo, and reveal a novel mechanism of myocyte-orchestrated extracellular matrix remodeling via PDE5/cGMP-PKG regulatory pathways

Rationale Soluble guanylyl cyclase (sGC) generates cyclic guanosine monophophate (cGMP) upon activation by nitric oxide (NO). Cardiac NO-sGC-cGMP signaling blunts cardiac stress responses, including pressure-overload induced hypertrophy. The latter itself depresses signaling via this pathway by reducing NO generation and enhancing cGMP hydrolysis. Objective We tested the hypothesis that the sGC response to NO also declines with pressure-overload stress, and assessed the role of heme-oxidation and altered intracellular compartmentation of sGC as potential mechanisms. Methods and Results C57BL/6 mice subjected to transverse aortic constriction developed cardiac hypertrophy and dysfunction. NO-stimulated sGC activity was markedly depressed, while NO- and heme-independent sGC activation by BAY 60-2770 was preserved. Total sGCα1 and β1 expression were unchanged by TAC, however sGCβ1 subunits shifted out of caveolin-enriched microdomains. NO-stimulated sGC activity was 2–3-fold greater in Cav3-containing lipid raft versus non-lipid raft domains in control, and 6-fold greater after TAC. In contrast, BAY 60-2770 responses were >10 fold higher in non-Cav3 domains with and without TAC, declining about 60% after TAC within each compartment. Mice genetically lacking Cav3 had reduced NO and BAY-stimulated sGC activity in microdomains containing Cav3 for controls, but no change within non-Cav3-enriched domains. Conclusions Pressure-overload depresses NO/heme-dependent sGC activation in the heart, consistent with enhanced oxidation. The data reveal a novel additional mechanism for reduced NO-coupled sGC activity related to dynamic shifts in membrane microdomain localization, with Cav3-microdomains protecting sGC from heme-oxidation and facilitating NO-responsiveness. Translocation of sGC out of this domain favors sGC oxidation and contributes to depressed NO-stimulated sGC activity.