Sergei D. Rybalkin

Cyclic GMP Phosphodiesterases and Regulation of Smooth Muscle Function

Abstract— Cyclic GMP (cGMP) made in response to atrial natriuretic peptide (ANP) or nitric oxide (NO) is an important regulator of short-term changes in smooth muscle tone and longer-term responses to chronic drug treatment or proliferative signals. The ability of smooth muscle cells (SMCs) to utilize different combinations of phosphodiesterase (PDE) isozymes allows cGMP to mediate these multiple processes. For example, PDE5 as a major cGMP-hydrolyzing PDE effectively controls the development of smooth muscle relaxation. In order for contraction to occur, PDE5 is activated and cGMP falls. Conversely, blockade of PDE5 activity allows the relaxation cycle to be prolonged and enhanced. A recently shown direct activation of PDE5 by cGMP binding to the GAF A domain suggests that this regulatory site might be a target for new drug development. The calcium surge associated with vasoconstrictor initiated contraction also activates a calcium/calmodulin-dependent PDE (PDE1A). Together, PDE5 and PDE1A lower cGMP sufficiently to allow contraction. Longer term, both PDE5 and PDE1A mRNA are induced by chronic stimulation of guanylyl cyclase. This induction is a major cause of the tolerance that develops to NO-releasing drugs. Finally, high levels of cGMP or cAMP also act as a brake to attenuate the proliferative response of SMCs to many mitogens. After vessel damage, in order for SMC proliferation to occur, the levels of cGMP and cAMP must be decreased. In humans, this decrease is caused in large part by induction of another Ca2+/calmodulin-dependent PDE (PDE1C) that allows the brake to be released and proliferation to start.

PI3Kγ Modulates the Cardiac Response to Chronic Pressure Overload by Distinct Kinase-Dependent and -Independent Effects

The G protein-coupled, receptor-activated phosphoinositide 3-kinase gamma (PI3Kgamma) mediates inflammatory responses and negatively controls cardiac contractility by reducing cAMP concentration. Here, we report that mice carrying a targeted mutation in the PI3Kgamma gene causing loss of kinase activity (PI3KgammaKD/KD) display reduced inflammatory reactions but no alterations in cardiac contractility. We show that, in PI3KgammaKD/KD hearts, cAMP levels are normal and that PI3Kgamma-deficient mice but not PI3KgammaKD/KD mice develop dramatic myocardial damage after chronic pressure overload induced by transverse aortic constriction (TAC). Finally, our data indicate that PI3Kgamma is an essential component of a complex controlling PDE3B phosphodiesterase-mediated cAMP destruction. Thus, cardiac PI3Kgamma participates in two distinct signaling pathways: a kinase-dependent activity that controls PKB/Akt as well as MAPK phosphorylation and contributes to TAC-induced cardiac remodeling, and a kinase-independent activity that relies on protein interactions to regulate PDE3B activity and negatively modulates cardiac contractility.

PDE5 is converted to an activated state upon cGMP binding to the GAF A domain

cGMP‐specific, cGMP‐binding phosphodiesterase (PDE5) regulates such physiological processes as smooth muscle relaxation and neuronal survival. PDE5 contains two N‐terminal domains (GAF A and GAF B), but the functional roles of these domains have not been determined. Here we show that recombinant PDE5 is activated directly upon cGMP binding to the GAF A domain, and this effect does not require PDE5 phosphorylation. PDE5 exhibited time‐ and concentration‐dependent reversible activation in response to cGMP, with the highest activation (9‐ to 11‐fold) observed at low substrate concentrations (0.1 μM cGMP). A monoclonal antibody directed against GAF A blocked cGMP binding, prevented PDE5 activation and decreased basal activity, revealing that PDE5 in its non‐activated state has low intrinsic catalytic activity. Activated PDE5 showed higher sensitivity towards sildenafil than non‐activated PDE5. The stimulatory effect of cGMP binding on the catalytic activity of PDE5 suggests that this mechanism of enzyme activation may be common among other GAF domain‐containing proteins. The data also suggest that development of agonists and antagonists of PDE5 activity based on binding to this site might be possible.

Cyclic nucleotide analogs as probes of signaling pathways

To the editor: Cyclic AMP (cAMP) and cyclic GMP (cGMP) are critical second messengers that regulate multiple targets including different cAMPor cGMP-dependent protein kinases (PKAs, PKGs)1,2, exchange proteins directly activated by cAMP (Epacs)3, phosphodiesterases (PDEs)4 and cyclic nucleotide-gated ion channels. Cyclic nucleotide analogs are widely used to study specificity of cellular signaling mediated by these target proteins. However, the selectivities and stabilities of these analogs need to be fully understood to properly interpret results and rigorously assess the mechanisms by which these analogs work in the cell. To better understand the selectivity and cross-reactivity of these analogs, we measured the activation or inhibitory activity of 13 commonly used cyclic nucleotide analogs with isozymes of PKA, PKG and Epac (Table 1), and with 8 different PDEs (Table 2 and Supplementary Tables 1 and 2 online). To measure their stability against hydrolysis, we used isothermal microcalorimetry5, a method that allowed us to evaluate whether or not an analog can function as a substrate or inhibitor for PDEs. We found that indeed some of these analogs were hydrolyzed by multiple PDEs, and other analogs were competitive inhibitors of PDEs. Here we provide half-maximal inhibition constant (Ki) data for all of the non-hydrolyzable analogs, and MichaelisMenten constant (Km) and maximum velocity (Vmax) values for all of the hydrolyzable analogs. Each of these values as well as the analog’s mode of inhibition can be determined in a single experiment (Table 2, Supplementary Methods and Supplementary Figures 1–5 online). The data strongly implied that several of these analogs might, in addition to their primary effects, also cause elevation of cAMP or cGMP indirectly by inhibiting PDEs in the cell. Such an effect could cloud interpretation of the use of these analogs. Similarly, analogs that are PDE substrates also might have their duration of action substantially reduced. To illustrate this point we showed that Sp-8-pCPT-2′O-Me-cAMPS, a highly specific, non-hydrolyzable Epac activator in vitro, can under certain conditions enhance cGMP-PKG and cAMPPKA signaling pathways in intact platelets (Supplementary Fig. 1). Specifically, we observed enhanced phosphorylation of vasodialatorstimulated phosphoprotein (VASP) at both PKA and PKG phosphorylation sites after the addition of Sp-8-pCPT-2′-O-Me-cAMPS. These data indicate that this ‘selective Epac activator’ is able to indirectly activate the cAMP-PKA and cGMP-PKG signaling pathways presumably through inhibition of platelet PDE5 and/or PDE3 (Supplementary Methods and Supplementary Discussion online). We also list in vitro selectivity data for all of the presently available commonly used cyclic nucleotide analogs for different forms of PKA, PKG and Epac I (Table 1). Data for several of these analogs have not

Calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1C) is induced in human arterial smooth muscle cells of the synthetic, proliferative phenotype.

The diversity among cyclic nucleotide phosphodiesterases provides multiple mechanisms for regulation of cAMP and cGMP in the cardiovascular system. Here we report that a calmodulin-stimulated phosphodiesterase (PDE1C) is highly expressed in proliferating human arterial smooth muscle cells (SMCs) in primary culture, but not in the quiescent SMCs of intact human aorta. High levels of PDE1C were found in primary cultures of SMCs derived from explants of human newborn and adult aortas, and in SMCs cultured from severe atherosclerotic lesions. PDE1C was the major cAMP hydrolytic activity in these SMCs. PDE expression patterns in primary SMC cultures from monkey and rat aortas were different from those from human cells. In monkey, high expression of PDE1B was found, whereas PDE1C was not detected. In rat SMCs, PDE1A was the only detectable calmodulin-stimulated PDE. These findings suggest that many of the commonly used animal species may not provide good models for studying the roles of PDEs in proliferation of human SMCs. More importantly, the observation that PDE1C is induced only in proliferating SMCs suggests that it may be both an indicator of proliferation and a possible target for treatment of atherosclerosis or restenosis after angioplasty, conditions in which proliferation of arterial SMCs is negatively modulated by cyclic nucleotides.

Role of Ca2+/Calmodulin-Stimulated Cyclic Nucleotide Phosphodiesterase 1 in Mediating Cardiomyocyte Hypertrophy

Rationale: Cyclic nucleotide phosphodiesterases (PDEs) through the degradation of cGMP play critical roles in maintaining cardiomyocyte homeostasis. Ca2+/calmodulin (CaM)–activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca2+/CaM and cGMP signaling; however, its function in cardiomyocytes is unknown. Objective: Herein, we investigate the role of Ca2+/CaM–stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes and in the heart in vivo. Methods and Results: Inhibition of PDE1 activity using a PDE1-selective inhibitor, IC86340, or downregulation of PDE1A using siRNA prevented phenylephrine induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal and adult rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic isoproterenol infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts; however, PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated neonatal and adult rat ventricular myocytes treated with neurohumoral stimuli such as angiotensin II (Ang II) and isoproterenol. Furthermore, PDE1A plays a critical role in phenylephrine-induced reduction of intracellular cGMP- and cGMP-dependent protein kinase (PKG) activity and thereby cardiomyocyte hypertrophy in vitro. Conclusions: These results elucidate a novel role for Ca2+/CaM–stimulated PDE1, particularly PDE1A, in regulating pathological cardiomyocyte hypertrophy via a cGMP/PKG–dependent mechanism, thereby demonstrating Ca2+ and cGMP signaling cross-talk during cardiac hypertrophy.

Chronic pulmonary hypertension increases fetal lung cGMP phosphodiesterase activity

An experimental ovine fetal model for perinatal pulmonary hypertension of the neonate (PPHN) was characterized by altered pulmonary vasoreactivity and structure. Because past studies had suggested impaired nitric oxide-cGMP cascade in this experimental model, we hypothesized that elevated phosphodiesterase (PDE) activity may contribute to altered vascular reactivity and structure in experimental PPHN. Therefore, we studied the effects of the PDE inhibitors zaprinast and dipyridamole on fetal pulmonary vascular resistance and PDE5 activity, protein, mRNA, and localization in normal and pulmonary hypertensive fetal lambs. Infusion of dipyridamole and zaprinast lowered pulmonary vascular resistance by 55 and 35%, respectively, in hypertensive animals. In comparison with control animals, lung cGMP PDE activity was elevated in hypertensive fetal lambs (150%). Increased PDE5 activity was not associated with either an increased PDE5 protein or mRNA level. Immunocytochemistry demonstrated that PDE5 was localized to vascular smooth muscle. We concluded that PDE5 activity was increased in experimental PPHN, possibly by posttranslational phosphorylation. We speculated that these increases in cGMP PDE activity contributed to altered pulmonary vasoreactivity in experimental perinatal pulmonary hypertension.An experimental ovine fetal model for perinatal pulmonary hypertension of the neonate (PPHN) was characterized by altered pulmonary vasoreactivity and structure. Because past studies had suggested impaired nitric oxide-cGMP cascade in this experimental model, we hypothesized that elevated phosphodiesterase (PDE) activity may contribute to altered vascular reactivity and structure in experimental PPHN. Therefore, we studied the effects of the PDE inhibitors zaprinast and dipyridamole on fetal pulmonary vascular resistance and PDE5 activity, protein, mRNA, and localization in normal and pulmonary hypertensive fetal lambs. Infusion of dipyridamole and zaprinast lowered pulmonary vascular resistance by 55 and 35%, respectively, in hypertensive animals. In comparison with control animals, lung cGMP PDE activity was elevated in hypertensive fetal lambs (150%). Increased PDE5 activity was not associated with either an increased PDE5 protein or mRNA level. Immunocytochemistry demonstrated that PDE5 was localized to vascular smooth muscle. We concluded that PDE5 activity was increased in experimental PPHN, possibly by posttranslational phosphorylation. We speculated that these increases in cGMP PDE activity contributed to altered pulmonary vasoreactivity in experimental perinatal pulmonary hypertension.

Cardiac hypertrophy is not amplified by deletion of cGMP-dependent protein kinase I in cardiomyocytes.

It has been suggested that cGMP kinase I (cGKI) dampens cardiac hypertrophy. We have compared the effect of isoproterenol (ISO) and transverse aortic constriction (TAC) on hypertrophy in WT [control (CTR)] mice, total cGKI-KO mice, and cGKIβ rescue mice (βRM) lacking cGKI specifically in cardiomyocytes (CMs). Infusion of ISO did not change the expression of cGKI in the hearts of CTR mice or βRM but raised the heart weight by ∼20% in both. An identical hypertrophic growth response was measured in CMs from CTR mice and βRM and in isolated adult CMs cultured with or without 1 μM ISO. In both genotypes, ISO infusion induced similar changes in the expression of hypertrophy-associated cardiac genes and significant elevation of serum atrial natriuretic peptide and total cardiac cGMP. No differences in cardiac hypertrophy were obtained by 7-day ISO infusion in 4- to 6-week-old conventional cGKI-KO and CTR mice. Furthermore, TAC-induced hypertrophy of CTR mice and βRM was not different and did not result in changes of the cGMP-hydrolyzing phosphodiesterase activities in hypertropic hearts or CMs. These results strongly suggest that cardiac myocyte cGKI does not affect the development of heart hypertrophy induced by pressure overload or chronic ISO infusion.

Roles of cGMP-dependent protein kinase I (cGKI) and PDE5 in the regulation of Ang II-induced cardiac hypertrophy and fibrosis

Significance It has been reported that elevation of cGMP and activation of cGMP-dependent protein kinase I (cGKI) by inhibition of PDE5 activity with sildenafil prevents cardiac hypertrophy. We studied the roles of cGKI and PDE5 inhibition on angiotensin (AII)-induced hypertrophy and fibrosis using control (Ctr) mice and mice expressing cGKI only in cells where the smooth muscle SM22α promotor is active (βRM). In Ctr mice, sildenafil did not reduce the AII-induced increase of cardiomyocyte (CM) size or myocyte hypertrophic markers but did reduce fibrosis. In βRM mice, sildenafil had little or no effect on markers of fibrosis. These results show that the sildenafil/cGMP/cGKI cascade can have an inhibitory effect on cardiac fibrosis but not on CM hypertrophy. Conflicting results have been reported for the roles of cGMP and cGMP-dependent protein kinase I (cGKI) in various pathological conditions leading to cardiac hypertrophy and fibrosis. A cardioprotective effect of cGMP/cGKI has been reported in whole animals and isolated cardiomyocytes, but recent evidence from a mouse model expressing cGKIβ only in smooth muscle (βRM) but not in cardiomyocytes, endothelial cells, or fibroblasts has forced a reevaluation of the requirement for cGKI activity in the cardiomyocyte antihypertrophic effects of cGMP. In particular, βRM mice developed the same hypertrophy as WT controls when subjected to thoracic aortic constriction or isoproterenol infusion. Here, we challenged βRM and WT (Ctr) littermate control mice with angiotensin II (AII) infusion (7 d; 2 mg⋅kg−1⋅d−1) to induce hypertrophy. Both genotypes developed cardiac hypertrophy, which was more pronounced in Ctr animals. Cardiomyocyte size and interstitial fibrosis were increased equally in both genotypes. Addition of sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, in the drinking water had a small effect in reducing myocyte hypertrophy in WT mice and no effect in βRM mice. However, sildenafil substantially blocked the increase in collagen I, fibronectin 1, TGFβ, and CTGF mRNA in Ctr but not in βRM hearts. These data indicate that, for the initial phase of AII-induced cardiac hypertrophy, lack of cardiomyocyte cGKI activity does not worsen hypertrophic growth. However, expression of cGKI in one or more cell types other than smooth muscle is necessary to allow the antifibrotic effect of sildenafil.

Turning on cGMP-dependent pathways to treat cardiac dysfunctions: boom, bust, and beyond

cGMP inhibits hypertrophy, decreases fibrosis, and protects against cardiac ischemia-reperfusion (I/R) injury. Gene-targeting studies have not defined a clear role for its major downstream effector, cGMP-dependent protein kinase I (cGKI), in cardiac hypertrophy, but do implicate cGMP-cGKI signaling in fibrosis and I/R injury. No direct cGKI activators have advanced to clinical trials, whereas cardiac trials of agents that modulate cGMP via particulate or soluble guanylyl cyclases (GCs) and phosphodiesterase 5 (PDE5) are ongoing. Here we review concerns arising from preclinical and clinical studies that question whether targeting the cGMP pathway remains an encouraging concept for management of heart dysfunction. So far, trial results for GC modulators are inconclusive, and sildenafil, a PDE5 inhibitor, although cardioprotective in mouse models, has not shown positive clinical results. Preclinical cardioprotection observed for sildenafil may result from inhibition of PDE5 in non-cardiomyocytes or off-target effects, possibly on PDE1C. On the basis of such mechanistic considerations, re-evaluation of the cellular localization of drug target(s) and intervention protocols for cGMP-elevating agents may be needed.