Sustained cardiac pressure overload induces hypertrophy and pathological remodeling, frequently leading to heart failure. Genetically engineered hyperstimulation of guanosine 3',5'-cyclic monophosphate (cGMP) synthesis counters this response. Here, we show that blocking the intrinsic catabolism of cGMP with an oral phosphodiesterase-5A (PDE5A) inhibitor (sildenafil) suppresses chamber and myocyte hypertrophy, and improves in vivo heart function in mice exposed to chronic pressure overload induced by transverse aortic constriction. Sildenafil also reverses pre-established hypertrophy induced by pressure load while restoring chamber function to normal. cGMP catabolism by PDE5A increases in pressure-loaded hearts, leading to activation of cGMP-dependent protein kinase with inhibition of PDE5A. PDE5A inhibition deactivates multiple hypertrophy signaling pathways triggered by pressure load (the calcineurin/NFAT, phosphoinositide-3 kinase (PI3K)/Akt, and ERK1/2 signaling pathways). But it does not suppress hypertrophy induced by overexpression of calcineurin in vitro or Akt in vivo, suggesting upstream targeting of these pathways. PDE5A inhibition may provide a new treatment strategy for cardiac hypertrophy and remodeling.

C ardiac adaptation in response to intrinsic or external stress involves a complex process of chamber remodeling and myocyte molecular modifications. A fundamental response to increased biomechanical stress is cardiomyocyte and chamber hypertrophy. Although this may provide initial salutary compensation to the stress, sustained hypertrophic stimulation becomes maladaptive, worsening morbidity and mortality risks because of congestive heart failure and sudden death. 1 Growing evidence highlights oxidative and nitrosative stresses as important mechanisms for this maladaptation. [2][3][4][5][6][7][8][9] Oxidative stress occurs when excess reactive oxygen species (ROS) are generated that cannot be adequately countered by intrinsic antioxidant systems. Superoxide anion (O 2 Ϫ ) can further combine with NO, forming reactive compounds such as peroxynitrite, generating nitroso-redox imbalance. 4 ROS generation is a normal component of oxidative phosphorylation and plays a role in normal redox control of physiological signaling pathways. 5,8,9 However, excessive ROS generation triggers cell dysfunction, lipid peroxidation, and DNA mutagenesis and can lead to irreversible cell damage or death. 5,8,9 In this review, we discuss recent experimental evidence for the role of oxidant stress on cardiac remodeling, focusing on pressureoverload-induced hypertrophy and dilation. ROS, Antioxidant Enzymes, andNitroso-Redox Balance ROS include free radicals such as superoxide (O 2 Ϫ ) and hydroxyl radical and compounds such as hydrogen peroxide (H 2 O 2 ) that can be converted to radicals, and they participate in both normal and pathologic biochemical reactions. 9 O 2 Ϫ is formed intracellularly (Figure 1) by activation of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase or xanthine oxidase (XO), uncoupling of NO synthase (NOS), and electron transport and "leakage" during oxidative phosphorylation in the mitochondria. 5,8,9 H 2 O 2 can generate the highly reactive hydroxyl radical via Fenton chemistry under pathological conditions. 9 Cells also have intrinsic antioxidant systems that counter ROS accumulation. These include enzymes such as catalase, glutathione peroxidases, and superoxide dismutase, and nonenzymatic antioxidants, such as vitamins E, C, beta carotene, ubiquinone, lipotic acid, and urate. 9,10 Superoxide dismutase converts O 2 Ϫ to H 2 O 2 , which is further converted by catalase and glutathione peroxidase to water. The thioredoxin system, including thioredoxin, thioredoxin reductase, and NADPH, forms an additional integrated antioxidant defense system, which operates as a powerful protein-disulfide oxidoreductase. 10 -12 NO is another important reactive molecule controlling cardiovascular homeostasis. NO stimulates the synthesis of intracellular cGMP by activating soluble guanylyl cyclase, and cGMP and its target kinase cGK-1 (protein kinase G 1), in turn, modulate myocyte function, growth, and remodeling. 13 NO also interacts with proteins via S-nitrosylation at specific cysteine residues to alter their fu...

Cardiac pressure load stimulates hypertrophy, often leading to chamber dilation and dysfunction. ROS contribute to this process. Here we show that uncoupling of nitric oxide synthase-3 (NOS3) plays a major role in pressure load-induced myocardial ROS and consequent chamber remodeling/hypertrophy. Chronic transverse aortic constriction (TAC; for 3 and 9 weeks) in control mice induced marked cardiac hypertrophy, dilation, and dysfunction. Mice lacking NOS3 displayed modest and concentric hypertrophy to TAC with preserved function. NOS3 -/-TAC hearts developed less fibrosis, myocyte hypertrophy, and fetal gene re-expression (B-natriuretic peptide and α-skeletal actin). ROS, nitrotyrosine, and gelatinase (MMP-2 and MMP-9) zymogen activity markedly increased in control TAC, but not in NOS3 -/-TAC, hearts. TAC induced NOS3 uncoupling in the heart, reflected by reduced NOS3 dimer and tetrahydrobiopterin (BH4), increased NOS3-dependent generation of ROS, and lowered Ca 2+ -dependent NOS activity. Cotreatment with BH4 prevented NOS3 uncoupling and inhibited ROS, resulting in concentric nondilated hypertrophy. Mice given the antioxidant tetrahydroneopterin as a control did not display changes in TAC response. Thus, pressure overload triggers NOS3 uncoupling as a prominent source of myocardial ROS that contribute to dilatory remodeling and cardiac dysfunction. Reversal of this process by BH4 suggests a potential treatment to ameliorate the pathophysiology of chronic pressure-induced hypertrophy.

The molecular mechanism for priapism is not well characterized. Although the nitric oxide (NO) pathway is known to mediate penile erection under normal conditions, we hypothesized that the mechanism of priapism rests in aberrant downstream signaling of this pathway based on our previous findings that mice lacking the gene for endothelial nitric oxide synthase (eNOS ؊/؊ ) and mice lacking both neuronal NOS (nNOS) and eNOS (nNOS ؊/؊ , eNOS ؊/؊ ) have a tendency for priapic activity. We investigated the role of downstream guanylate cyclase and phosphodiesterase type 5 (PDE5A) expression and function in mediating these responses in eNOS ؊/؊ and nNOS ؊/؊ , eNOS ؊/؊ mice. Erectile responses to both cavernous nerve stimulation and intracavernosal injection of the NO donor diethylamine-NONOate were augmented in eNOS ؊/؊ and nNOS ؊/؊ , eNOS ؊/؊ mice but not in WT or nNOS ؊/؊ mice. PDE5A protein expression and activity and cGMP levels were significantly lower in eNOS ؊/؊ and nNOS ؊/؊ , eNOS ؊/؊ mice, and this effect was reproduced in WT corpus cavernosum exposed to NOS inhibitors. Moreover, cavernous nerve stimulation was associated with a marked augmentation of cavernosal cGMP levels, suggesting that, although lower at baseline, the production of cGMP is unchecked in eNOS ؊/؊ and nNOS ؊/؊ , eNOS ؊/؊ mice upon neurostimulation. Transfection of eNOS ؊/؊ mice with an adenovirus encoding eNOS resulted in a normalization of PDE5A protein and activity as well as a correction of priapic activity. Coupled with the observation that sickle cell disease mice (which show a priapism phenotype) evince dysregulated PDE5A expression͞activity, these data suggest that PDE5A dysregulation is a fundamental mechanism for priapism.endothelial nitric oxide synthase ͉ gene transfer ͉ sickle cell disease

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.