Heart failure

In the 1970s and 1980s, PDE3 inhibitors were discovered to exhibit cardiotonic, inotropic, bronchodilatory and vasodilatory activities in several species, and were initially developed as cardiotonic agents to replace or add to cardiac glycosides in the treatment of HF . However, despite beneficial haemodynamic effects in the short term, chronic use of PDE3 inhibitors was associated with increased cardiac arrhythmias and sudden death . Thus, the use of PDE3 inhibitors is now limited to acute decompensated HF. Nevertheless, PDE3 inhibitors are targeting several functionally distinct isoforms that are co-expressed in the heart, raising the hope that more selective targeting might provide some benefits.

PDE3 is encoded by two genes, PDE3A and PDE3B . Evidence from global PDE3A and PDE3B knockout (KO) mice indicate that PDE3A, but not PDE3B, is responsible for the inotropic and chronotropic effects of PDE3 inhibitors . Three isoforms of PDE3A are expressed in cardiomyocytes, which differ only in their N-terminal domain, giving rise to different intracellular localization . In mice and humans, PDE3A1 controls PLB-SERCA2 activity and Ca ²⁺ reuptake in the sarcoplasmic reticulum ( Fig. 1 ). Because dephosphorylated PLB and depressed SERCA2 activity are a hallmark of HF, PDE3 inhibitors specifically targeting the PDE3A1 associated with PLB-SERCA2 may improve contractile performance and provide therapy for HF . However, currently available PDE3 inhibitors have little selectivity for PDE3A versus PDE3B isoforms, which have similar catalytic domains, and no selectivity for individual PDE3A isoforms, which have identical catalytic domains. Phosphorylation of PDE3A1 was recently shown to regulate its interaction with SERCA2 . Targeting this mechanism may offer an alternative to selectively enhance contractility without the harmful effects of global inhibition of PDE3 activity.

The second major PDE involved in cAMP hydrolysis in the heart is the cAMP-specific PDE4. The PDE4 family is encoded by four genes ( PDE4A–D ). Most of our knowledge of the roles of individual PDE4 subtypes in the heart is limited to PDE4D. KO of the PDE4D gene in mice leads to PKA hyperphosphorylation of RyR2, increased sensitivity to exercise-induced arrhythmias and late-onset dilated cardiomyopathy . PDE4D isoforms are localized in multiple compartments of the cardiomyocyte. For instance, PDE4D3 is localized at the perinuclear region, where it is part of a macromolecular complex organized by the scaffold protein muscle AKAP and comprising Epac1 and the kinase ERK5 to regulate cardiomyocyte hypertrophy . This isoform is also present at the sarcolemma, where it associates through another AKAP with slowly activating delayed rectifier potassium channels controlling cardiac repolarization , and at myofilaments, in association with another scaffold protein, myomegalin . In addition, distinct PDE4D isoforms have been shown to interact with β ₁ -ARs and β ₂ -ARs, either directly or indirectly through β-arrestin, and to shape specific physiological or pathophysiological responses . Finally, similar to PDE3A, PDE4D also associates with the PLB/SERCA2 complex and regulates SERCA pump activity in the mouse heart ( Fig. 1 ).

A role for PDE4B in the heart emerged recently when it was identified as an integral component of the LTCC complex, and the main PDE regulating the LTCC current during β-AR stimulation ( Fig. 1 ). PDE4B KO mice, like PDE4D KO mice, have an increased susceptibility to ventricular arrhythmias during catecholamine stimulation, which may be the result of enhanced Ca ²⁺ influx through the LTCC . Although RyR2 phosphorylation by PKA did not seem to be affected in adult hearts from PDE4B KO mice , a recent study indicates that it was increased in neonatal myocytes lacking PDE4B suggesting that altered RyR2 regulation may also contribute to this arrhythmic phenotype. In a recent study in rat ventricular myocytes, we showed that under β-AR stimulation, inhibition of PDE4 (as well as inhibition of PDE3) exerted inotropic effects via PKA but led to spontaneous diastolic Ca ²⁺ waves via both PKA and CaMKII, suggesting the potential use of CaMKII inhibitors as adjuncts to PDE inhibition to limit their proarrhythmic effects .

As stated above, phosphorylation of certain PDE3 and PDE4 isoforms by PKA activates these enzymes, and this constitutes powerful negative feedback for cAMP signals in cardiomyocytes. This regulation has been shown to be facilitated by spatial proximity of PKA and PDEs assembled by the perinuclear muscle AKAP or by phosphoinositide 3-kinase γ isoform, which, in addition to its lipid kinase function, also acts as an AKAP, facilitating the phosphorylation of PDE3A, PDE3B, PDE4A and PDE4B by PKA .

Although these studies underline the critical role of PDE4 in controlling β-AR stimulation in rodents, this family contributes less to the regulation of cardiac contractility in humans, where PDE3 predominates . However, in human atrial strips, inhibition of PDE3 and also PDE4 potentiates the arrhythmogenic effect of β-AR stimulation, and PDE4 activity tends to decrease in the atria of patients with atrial fibrillation . Further understanding of the role of PDE4 in humans may also be important for the proarrhythmic effect of PDE3 inhibitors, because PDE3 inhibitors, such as milrinone and enoximone, may also inhibit PDE4 in cardiac preparations . In cardiac hypertrophy and HF, there are profound alterations of the expression and activity of PDE3 and PDE4. In a model of pathological hypertrophy induced by pressure overload in rats, we found that the expression and activity of PDE3A, PDE4A and PDE4B were decreased, and that this was associated with a blunted regulation of subsarcolemmal cAMP generated by β-ARs by PDE3 and PDE4 . In contrast, in a model of cardiac hypertrophy induced by angiotensin II, increased PDE4 activity was observed, accompanied by an increase in the 69-kDa-PDE4A isoform and a decrease in the expression of the 52- and 76-kDa PDE4D isoforms. These results suggest that the level of expression of the isoforms of PDE3 and PDE4 is specifically regulated by the type of stimulus used to induce cardiac hypertrophy and the stage of the disease. While an increase in cAMP-PDE can participate in desensitization of the β-AR pathway, a decrease could represent a compensatory mechanism to restore cAMP concentrations and inotropism. However, lower PDE also alters the degree of cAMP confinement, which could lead to illegitimate or excessive activation of certain pools of PKA or Epac, hence promoting maladaptive remodelling and rhythmic disturbances. This is supported by the results of a recent study showing that the specific PDE4D5 isoform regulates activation of hypertrophic programme by Epac1 upon stimulation of β ₂ -AR receptors . In a very recent study, the local regulation of cAMP by PDEs in the vicinity of SERCA2 was compared using transgenic mice with cardiac expression of a PLB-targeted cAMP biosensor subjected to transaortic constriction . In agreement with their known localization within the SERCA2 complex , both PDE3 and PDE4 were found to regulate cAMP in this microdomain. Interestingly, during hypertrophy and early HF, there was a specific rearrangement of PDEs regulating this specific cAMP pool, with a decreased contribution from PDE4 and an increased contribution from PDE2 . These results indicate that PDE alterations in cardiac disease include redistribution of PDE variants in discrete microcompartments of cardiomyocytes.

PDE3 and PDE4 also provide the major cAMP-degrading activities in VSMCs ( Fig. 2 ), and their expression is also modified in the aorta of rats with HF . Importantly, endothelial dysfunction in HF leads to activation of PDE3 in VSMCs because of the loss of endothelial NO production and, consequently, of PDE3 inhibition by cGMP. This leads to a loss of relaxation induced by β-AR and PDE4 inhibition, thus suggesting that inhibition of vascular PDE3 may constitute an attractive approach to restoring normal vasorelaxation in HF .

The dual specific PDE2 represents a minor part of cAMP hydrolytic activity in the normal heart, but the cAMP hydrolytic activity of this PDE is stimulated 5- to 30-fold by cGMP, which was shown to inhibit cardiac LTCC in various species, including humans . Subsequently, measurements with Förster resonance energy transfer-based sensors in neonatal rat cardiomyocytes showed that by decreasing the concentration of cAMP, PDE2 counteracts the effects of β-AR stimulation downstream of β ₃ -ARs . In contrast to PDE3 and PDE4, the expression and activity of which are generally decreased in pathological hypertrophy and HF , we found recently that PDE2 is increased in animal models as well as in human HF . PDE2 inhibition partially restores β-AR responsiveness in diseased cardiomyocytes, suggesting that PDE2 enhancement in HF constitutes a protective mechanism against excessive β-AR stimulation. However, according to another recent study, PDE2 could exert a prohypertrophic effect by blunting PKA-mediated phosphorylation of nuclear factor of activated T cells (NFAT) . Further studies are needed to fully understand the role of PDE2 in HF.

Similar to PDE2, PDE1 and PDE5 have been reported to be overexpressed in pathological hypertrophy and HF . Because PDE1 and PDE5 preferentially (PDE1A) or specifically (PDE5) degrade cGMP, their increase in HF can clearly be seen as maladaptive. Accordingly, transgenic mice with cardiac-specific overexpression of PDE5 are predisposed to adverse remodelling after myocardial infarction , whereas pharmacological inhibition of PDE1 or PDE5 reduces hypertrophy and improves cardiac pressure and volume overload. Numerous animal studies have shown that PDE5 inhibitors protect against ischaemia/reperfusion injury, doxorubicin cardiotoxicity, ischaemic and diabetic cardiomyopathy and Duchenne muscular dystrophy . However, it remains controversial whether significant concentrations of PDE5 are expressed in the myocardium, raising the possibility that the beneficial effects of PDE5 inhibitors involve other mechanisms, including inhibition of PDE1 . In patients with systolic HF, the PDE5 inhibitor sildenafil decreased pulmonary vascular pressure and increased peak oxygen consumption and cardiac index . Sildenafil also improved left ventricular diastolic function, cardiac geometry and clinical status in patients with systolic HF and improved diabetic cardiomyopathy . However, despite encouraging results in an initial single-centre study , chronic therapy with sildenafil was not associated with clinical benefit in patients with diastolic HF in a larger multicentre study . Ongoing trials with PDE5 inhibitors include testing for gender response to tadalafil in left ventricular hypertrophy associated with diabetic cardiomyopathy ( NCT01803828 ).

Two other PDEs were recently proposed to participate in cGMP degradation in the heart. Experiments performed in isolated cardiomyocytes from transgenic mice expressing a Förster resonance energy transfer-based cGMP biosensor have suggested that PDE3, which is classically known to degrade cAMP preferentially, may also be involved in the control of cGMP concentrations . In addition, the cGMP-specific PDE9 was found to be expressed in rodent and human hearts, and to be upregulated in hypertrophy and HF . PDE9 genetic ablation or pharmacological inhibition appears to protect the heart against pathological remodelling during pressure overload. Moreover, PDE9 inhibition reverses pre-established heart disease in a NO synthase activity-independent manner, whereas PDE5 inhibition requires active NO synthase, which is decreased in HF; this is because PDE9 seems to hydrolyze specifically cGMP generated by natriuretic peptides, whereas PDE5 controls cGMP generated by NO . We have shown previously that PDE2 is critical for the regulation of subsarcolemmal cGMP concentrations in response to particulate GC activation in adult cardiomyocytes , thus raising the question of whether PDE2 and PDE9 exert redundant or distinct regulation of natriuretic peptide signalling.

Ischaemia/reperfusion injury

Manipulation of PDE activity may also prove protective in the context of ischaemia-reperfusion injury. Indeed, PDE5 inhibitors were shown to reduce infarct size in rabbits and mice; they also decreased cell death in isolated cardiomyocytes, suggesting that part of this effect is independent of vasodilation. Several mechanisms appear to be involved in these effects, including increased NO synthase expression, cGMP elevation, PKG activation and the opening of mitochondrial adenosine triphosphate-sensitive potassium channels and Ca ²⁺ -activated potassium channels . PDE3 inhibitors have also been reported to reduce infarct size when applied before sustained ischaemia, thus mimicking the cardioprotection conferred by ischaemic preconditioning . A recent study using KO mice for either PDE3A or PDE3B strongly suggested that PDE3B is the isoform mediating the cardioprotective effect of PDE3 inhibitors in this context. Indeed, PDE3B KO mice, but not PDE3A KO mice, were protected against ischaemia-reperfusion injury. This protective effect appears to involve cAMP/PKA-mediated opening of mitochondrial Ca ²⁺ -activated potassium channels and assembly of ischaemia-induced caveolin 3-enriched fractions . Somehow at odds with the above-mentioned cardioprotective effect of PDE3 inhibitors, mice with cardiac-specific overexpression of PDE3A1 were protected during ischaemia-reperfusion injury. In addition to regulating SERCA2, PDE3A1 also acts as a negative regulator of cardiomyocyte apoptosis, by controlling the expression of the transcriptional repressor and proapoptotic factor, inducible cAMP early repressor (ICER) . Inhibition of this mechanism in mice with cardiac-specific overexpression of PDE3A1 was associated with protection during ischaemia-reperfusion . Collectively, these studies suggest that PDE3A and PDE3B may play an opposite role during ischaemia-reperfusion, which may be linked to their differential localization and the control of discrete cAMP pools in cardiomyocytes .

Erectile dysfunction

PDE5 is highly abundant in vascular smooth muscle, and by limiting the breakdown of cGMP, PDE5 inhibition potentiates the vasorelaxant effect of the NO/cGMP pathway initiated by endothelial NO production or exogenous NO produced by NO donors ( Fig. 2 ). Thus, PDE5 inhibitors were initially proposed as potent vasodilators to treat coronary heart disease. In the mid-1980s, the Pfizer group developed sildenafil (UK-92480, a derivative of zaprinast) as an orally available PDE5 inhibitor (half maximal inhibitory concentration [IC ₅₀ ] for PDE5 ∼5 nM and 10× selectivity over other PDEs). Sildenafil turned out to be ineffective against angina, but was reported to induce enhanced penile erections in a number of volunteers participating in these trials. Thereafter, extensive research focused on this unexpected side effect.

Penile erection is dependent on the NO/cGMP pathway. Upon sexual stimulation, NO released in the corpora cavernosa by non-cholinergic/non-adrenergic neurons and endothelial cells promotes relaxation of surrounding smooth muscle cells (SMCs) by increasing intracellular cGMP concentrations. Relaxation of intracavernosal smooth muscle and dilatation of penile arteries promote the expanding of sinusoidal spaces, resulting in blood filling and penile erection. PDE5 is the predominant PDE in penile SMCs . Thus, PDE5 inhibitors enhance the erectile response by potentiating the effects of cGMP triggered by NO release .

In 1998, sildenafil (Viagra ^® ; Pfizer, New York City, NY, USA) received approval as the first oral treatment for erectile dysfunction in men. Three other PDE5 inhibitors (tadalafil, vardenafil and, more recently, avanafil) are now commercialized. No current evidence suggests a difference in their efficacy. However, sildenafil and vardenafil exhibit lower selectivity for PDE5 over PDE6, the retinal PDE, which might explain the visual disturbances experienced by some patients. According to their action mechanism, all PDE5 inhibitors require sexual stimulation to be effective, and their association with organic nitrates is contraindicated. Nevertheless, an increase in vascular PDE expression, especially PDE1A and PDE5A, has been observed in rat models of nitrate tolerance; PDE inhibition, with zaprinast and vinpocetine , respectively, was effective in reversing this tolerance, suggesting that PDE upregulation is involved in the development of nitrate tolerance. The potential application of PDE inhibitors, especially PDE1 and PDE5 inhibitors, in limiting the development of nitrate tolerance remains to be evaluated in humans.

Pulmonary hypertension

Pulmonary arterial hypertension (PAH) is characterized by an increase in mean pulmonary arterial pressure, leading to a progressive functional decline with right HF and, eventually, death . The pulmonary artery remodelling underlying this disease includes pulmonary vasoconstriction, in situ thrombosis, medial hypertrophy and intimal proliferation, leading to occlusion of the small- to mid-sized pulmonary arterioles and the formation of plexiform lesions. Endothelial dysfunction, leading to an imbalance in the production of vasodilator/antiproliferating factors in favour of vasoconstrictor/proliferating factors, appears to be one of the main pathobiological mechanisms of the disease, and is the rationale for current therapeutics. Impairment of the arterial pulmonary NO/cGMP/PDE5 pathway is supported by a decrease in NO synthase expression in endothelial cells from PAH patients , and upregulation of PDE5 in their VSMCs. Thus, PDE5 inhibition counteracts this deleterious process, promoting cGMP accumulation, resulting in inhibition of pulmonary vasoconstriction and VSMC growth and remodelling.

In the SUPER study, a 12-week treatment with the PDE5 inhibitor sildenafil showed improvements in exercise capacity, New York Heart Association functional class and pulmonary haemodynamics in patients with symptomatic PAH . The improvements were largely sustained after 3 years of treatment (SUPER-2 study) . Sildenafil (Revatio ^® ; Pfizer) was approved in 2005 for the long-term treatment of patients with class II and III PAH. Tadalafil (Adcirca ^® ; Eli Lilly and Company, Indianapolis, IN, USA) was also commercialized based on similar clinical benefits .

Other PDE families may be critical in the pathogenesis of PAH. It has been shown that not only cGMP-PDE activity, but also cAMP-PDE activity is increased in rat pulmonary arteries isolated from a model of chronic hypoxia-induced PAH .

Expression of PDE1A, PDE1C and PDE3B is enhanced in pulmonary artery SMCs from both idiopathic and secondary PAH patients compared with control pulmonary artery SMCs . Methylxanthine derivatives, which exhibit low PDE1 selectivity, were shown to be protective in different preclinical models of PAH . More recently, the selective PDE2 inhibitor BAY 60-7550 was shown to have beneficial effects on pulmonary vasoconstriction, remodelling and right ventricular hypertrophy in both hypoxia- and bleomycin-induced pulmonary hypertension in mice. The authors also reported that BAY 60-7550 reduced the proliferation of isolated pulmonary artery SMCs from PAH patients . Additional benefits were observed when BAY 60-7550 was given in conjunction with cAMP- or cGMP-elevating agents (the prostacyclin analogue treprostinil, sildenafil, atrial natriuretic peptide or NO donor). The dual promotion of cGMP and cAMP signalling upon PDE2 inhibition might be an attractive perspective in the treatment of PAH.

Post-angioplasty restenosis/atherosclerosis

Intimal hyperplasia and luminal stenosis are the key characteristics of several different vascular disorders, such as atherosclerosis and post-angioplasty restenosis .

To prevent restenosis after percutaneous coronary intervention, the most effective therapy is local delivery of antiproliferative reagents via drug-eluting stents, containing drugs, such as sirolimus and paclitaxel . However, first-generation drug-eluting stents also attenuate re-endothelialization and can lead to increased in-stent thrombosis ; they also remain ineffective in treating vascular disorders with diffuse neointimal lesions. Thus, the development of novel therapeutic strategies is currently in high demand.

Under normal conditions, SMCs residing in the media of vessels are quiescent, with a low turnover rate and insignificant secretory activity. These SMCs are highly differentiated cells that exhibit a contractile phenotype by expressing large amounts of contractile proteins, and principally function to maintain vascular tone. However, SMCs also retain a degree of plasticity to allow phenotypic modulation. Under vascular injury, SMCs undergo profound metamorphosis, changing from a quiescent/contractile phenotype to an active/synthetic phenotype with proliferative and migratory properties . Interestingly, this phenotype switch is associated with modification of PDE expression profile. Given the antiproliferative and antimigratory properties of cyclic nucleotides, PDEs appear of great interest in these vascular proliferative diseases.

The properties of PDE3 inhibition, leading to vasodilatation and inhibition of platelet aggregation and VSMC proliferation, appeared to be favourable in this context. Several clinical trials were designed to evaluate the benefits of the already approved and orally available PDE3 inhibitor, cilostazol. On a background of usual antiplatelet therapy with aspirin and clopidogrel, cilostazol was shown to be effective in attenuating post-angioplasty restenosis, especially in patients at high risk of restenosis 6 months after stent implantation (CREST trial) , and in patients with diabetes mellitus implanted with a drug-eluting stent (DECLARE-DIABETES trial) . Cilostazol compared with aspirin also reduced the progression of carotid atherosclerosis at 2 years in patients with type 2 diabetes (DAPC trial) . Despite the possible benefit of cilostazol in vascular diseases, its use is limited by tolerability, as some patients often report drug discontinuation because of headache, diarrhoea, dizziness or increased heart rate .

PDE1C is considered as a marker of VSMC proliferation in rodent as well as in human vessels ( Fig. 2 ). Indeed, PDE1C expression was reported to be minor in the normal human aorta or saphenous vein, but readily upregulated ex vivo in SMCs cultured from these vessels or isolated from aortic atherosclerosis lesions , as well as in vivo in neointimal human coronary artery lesions . Ex vivo inhibition of PDE1C using antisense oligonucleotides or a PDE1 inhibitor resulted in suppression of proliferation of SMCs isolated from the normal aorta or from lesions of atherosclerosis . More importantly, in vivo deficiency of PDE1C ( PDE1C KO) or inhibition of PDE1 through perivascular application of IC86340 was shown to attenuate injury-induced neointimal formation in mouse carotid artery . In this study, the authors identified a novel mechanism involved in the beneficial effects of PDE1C inhibition, a decrease in platelet-derived growth factor receptor β expression, via a cAMP/PKA-dependent mechanism contributing to lysosomal-dependent receptor degradation in a low-density lipoprotein receptor-related protein 1-dependent manner. Thus PDE1C inhibition appears to offer novel therapeutic strategies in vascular hyperplasic disorders.

Angiogenesis

Cyclic nucleotide signalling pathways are considered to modulate several components of tumourigenesis, among them angiogenesis, which is a fundamental process in tumour growth and metastasis. The vascular endothelial growth factor (VEGF) pathway is well established as one of the key regulators of this process , and current antiangiogenic therapies rely on blocking VEGF activity. VEGF-induced angiogenesis is partly mediated by NO through endothelial NO synthase activation and the subsequent increase in endothelial cGMP concentration. In human umbilical vein endothelial cells, VEGF has been shown to increase PDE2 and PDE4 activities and decrease PDE5 activity. Treatment of these cells with a combination of PDE2 and PDE4 inhibitors increased cAMP concentrations and decreased VEGF-induced human umbilical vein endothelial cell migration, proliferation and cell cycle progression. This treatment also reduced the total capillary surface of the chicken embryo chorioallantoic membrane, used as an in vivo preclinical model of angiogenesis . More recently, it was reported that a PDE4 inhibitor (intraperitoneal administration for 36 days) reduced in vivo the growth of A549 lung tumour xenografts in nude mice by attenuating proliferation and angiogenesis, evaluated by Ki67 and CD31 staining, respectively . Overall, these preclinical data assessing the role of PDEs in the angiogenesis process strengthen the interest in PDE inhibitors in cancer therapy.

Systemic hypertension

PDE inhibitors are currently not indicated as antihypertensive drugs, despite their vasodilatory properties. Indeed, milrinone was reported to decrease arterial pressure after intravenous infusion , but as its chronic oral administration in HF patients was shown to increase mortality, most probably because of arrhythmias and cardiac arrest , its clinical use was restricted to acute and end-stage treatment of HF. Recently, Maass et al. identified six missense PDE3A mutations causing an autosomal dominant hypertension in brachydactyly type E patients. The mutated PDE3A exhibited a gain-of-function, with increased cAMP hydrolytic activity, responsible for VSMC hyperplasia and increased vascular resistance. This elegant study should boost the interest in selective PDE3 inhibitors in the treatment of hypertension, especially in association with brachydactyly.

Intermittent claudication

Cilostazol, a selective PDE3 inhibitor, was approved for intermittent claudication, a relatively common lower-extremity peripheral arterial disease, characterized by ischaemia-induced leg pain or cramping, and for which pharmacological therapy is limited. Cilostazol exerts dual inhibitory properties on PDE3 and adenosine uptake, which might explain the minimal cardiac effects compared with other PDE3 inhibitors . By inhibiting platelet aggregation and promoting arterial vasodilation, cilostazol was shown to increase walking distance and to reduce the clinical symptoms of intermittent claudication . However, its use is controversial because of the modest benefit-risk balance (in particular, because of the risk of side effects affecting the heart or serious bleeding), and it has been withdrawn in some countries (e.g. France).