Role of cGMP Signaling and Phosphodiesterase-5 inhibitors in Cardioprotection

Abstract

cGMP is an important intracellular second messenger that mediates multiple tissue and cellular responses. The cGMP pathway is a key element in the pathophysiology of the heart and its modulation by drugs such as phosphodiesterase-5 (PDE-5) inhibitors and guanylate cyclase activators may represent a promising therapeutic approach for acute myocardial infarction, cardiac hypertrophy, heart failure and doxorubicin cardiotoxicity in patients. In addition, PDE-5 inhibitors may prove as innovative therapeutic agents for enhancing the chemosensitivity of doxorubicin while providing concurrent cardiac benefit.

Introduction

Cyclic guanosine 3’,5’-monophosphate (cGMP) is a critical intracellular second messenger regulating fundamental physiological processes in the myocardium, from acute contraction/relaxation to chronic gene expression, cell growth and apoptosis. Several studies have shown that cGMP inhibits hypertrophy, reduces ischemia-reperfusion (I/R) injury, regulates contractile function and cardiac remodeling (1–3). cGMP is generated from the cytosolic purine nucleotide GTP by guanylyl cylases (GCs) using Mg²⁺ or Mn²⁺ as cofactors. Two isoforms of GCs exist in vertebrate cells and tissues: a nitric oxide (NO)-sensitive cytosolic or soluble guanylyl cyclase (sGC) and natriuretic peptide (NP)-activated plasma membrane bound, particulate guanylyl cyclase (pGC). Once produced, the effects of cGMP occur through three main groups of cellular target molecules: cGMP-dependent protein kinases (PKGs), cGMP-gated cation channels and phosphodiesterases (PDEs). cGMP positively regulates PKG, but inhibits/activates PDEs, which are predominant in the cardiovascular system (4;5).

Regulation of cGMP by Phosphodiesterases

The cGMP pool in the cell is tightly controlled by PDEs which specifically cleave the 3', 5’-cyclic phosphate moiety of cAMP and/or cGMP to produce the corresponding 5' nucleotide. Currently 21 PDE genes have been cloned and are classified into 11 families according to their sequence of homology, biochemical and pharmacological properties (6). The PDEs vary in their substrate specificity for cAMP and cGMP: PDE-5, PDE-6 and PDE-9 are specific for cGMP; PDE-4, PDE-7 and PDE-8 are specific for cAMP; and PDE-1, PDE-2, PDE-3, PDE-10 and PDE-11 have mixed specificity for cAMP/cGMP (7). PDE-5 selectively hydrolyzes cGMP, and its inhibition increases cGMP bioavailability. The abundance of PDE-5 in smooth muscles and its role in regulating their contractile tone has made PDE-5 an important drug target for the treatment of erectile dysfunction (6), leading to the development of potent PDE-5 inhibitors, such as sildenafil (Viagra™ and Revatio™), vardenafil (Levitra™) and tadalafil (Cialis™ and Adcirca™). Revatio™ and Adcirca™ have also been approved for treatment of pulmonary hypertension. Earlier studies showed that PDE-5 is not present in normal cardiomyocytes (8;9) although later investigations revealed its expression in canine (10), mouse cardiomyocytes (1,11,12), and human heart (13,14). PDE5 expression is increased in hypertrophic human right ventricle, as well as failing left ventricular tissue (13–15). A gene silencing model also confirmed PDE-5 protein expression (16), while a recent report still questioned its presence in adult mouse cardiac myocytes (17). Since cGMP-hydrolytic activity is also attributable to PDE-1 and PDE-3, Vandeput et al. suggested that the effects of sildenafil on cGMP hydrolysis were due to inhibition of both PDE-5 and PDE-1 in the left ventricles of normal and failing mouse hearts (14).

cGMP in Pre and Post-Conditioning

Nitric oxide (NO) triggers various physiological responses by binding and activating sGC to produce cGMP from GTP (18). NO-cGMP-PKG signaling pathway is involved in the cardioprotective action in I/R injury as a survival signal (19;20). In cardiomyocytes, cGMP reduced the effects of myocyte stunning following simulated I/R (21). NO donor, S-nitroso-N-acetyl-L,L-penicillamine mimicked the preconditioning (PC)-like effect by inducing cGMP (22). Moreover, the activation of NO/cGMP/PKG pathway inhibited the elevation of intracellular Ca²⁺ concentrations by phosphorylating target proteins responsible for intracellular Ca²⁺ homeostasis during I/R injury in Chinese hamster ovary (CHO) cells (23).

Ischemic PC rapidly increased cGMP levels via sGC during ischemia leading to delayed protective effect (24 hrs later) against myocardial stunning and infarction in conscious rabbits (24). Bradykinin, one of the triggers of PC caused receptor-mediated production of NO resulting in cGMP production, activation of PKG, and opening of mitochondrial K_ATP (mitoK_ATP) channel in rabbit heart and cardiomyocytes (25). Opening of mitoK_ATP channels causes partial compensation of the membrane potential, which enables additional protons to be pumped out to form a H⁺ electrochemical gradient for both ATP synthesis and Ca²⁺ transport (26). The cGMP/PKG pathway also confers ischemic post conditioning protection in part by delaying normalization of pHi during reperfusion, probably via PKG-dependent inhibition of Na⁺/H⁺-exchanger in rat heart (27).

cGMP Modulatory Drugs for Cardioprotection

cGMP modulatory drugs induce cardioprotective effect through PKG as outlined in Figure 1. Natriuretic peptides (NP) exert biological effects by binding to membrane associated pGC. Atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are three structurally related, but genetically distinct, signaling molecules that regulate the cardiovascular, skeletal, nervous, reproductive and other systems by activating pGC and elevating intracellular cGMP concentrations. ANP is primarily stored in atrial granules and secreted in response to atrial stretch. BNP is also in atrial granules but is found in highest level in ventricles of stressed hearts (28). CNP is found at lower concentration in vascular endothelium and is present in higher concentration in chondrocytes where it stimulates long bone growth (29).

Figure 1. Myocardial protection by upregulation of cGMP.

Cardioprotective modalities including preconditioning, post conditioning, ANP/BNP, NO donors and Cinaciguat generate cGMP through activation of sGC/pGC. cGMP exerts cardioprotective effect against ischemia/reperfusion injury through activation of PKG.

In the cardiovascular system, NPs have growth suppressive, antiproliferative and antihypertrophic actions on vascular smooth muscle cells, cardiomyocytes and fibroblasts (30). ANP/BNP exert myocardial protective effects against I/R injury through a cGMP-PKG-dependent modulation of mitoK^ATP channels (31). ANP also protects against reoxygenation-induced hypercontracture in cardiomyocytes by stimulating cGMP synthesis (32). Administration of ANP at reperfusion protected against I/R injury (33,34) exerted antiapoptotic effects in rat cardiomyocytes through cGMP-PKG and by inducing phosphatidylinositol 3-kinase-protein kinase B (PI3K/AKT) signaling (35). cGMP analog, 8-Br-cGMP, or elevation of intracellular cGMP concentration via the sGC activator NO or BNP exerted cardioprotective effects through PKG activation (36).

Cinaciguat (BAY 58-2667) activates sGC independent of NO (37). This drug preferentially activates sGC when the heme iron is oxidized (Fe³⁺) or the heme moiety is missing, which makes this enzyme insensitive to both its endogenous ligand NO and exogenous nitrovasodilators (37). These unique attributes make Cinaciguat an attractive molecule for protection against reperfusion injury. Indeed, Cinaciguat induces pre- or post-conditioning like effect against I/R in rabbit, rat (38), mouse (39) and dog heart (40) by activating PKG and opening of the mitoK_ATP channels (39). Intriguingly, this drug caused PKG-dependent generation of hydrogen sulfide in mouse cardiomyocytes and protected against simulated ischemia/reoxygenation (SI-RO) injury, similar to tadalafil in the heart (3). Hydrogen sulfide causes cardioprotection through opening of sarcolemmal K_ATP channels in rat heart and cardiomyocytes (41).

PDE-5 inhibitors are promising drugs for cardiovascular protection. Our laboratory first demonstrated the cardioprotective effect of sildenafil against I/R injury (42). Rabbits treated with sildenafil before ischemia showed significant reduction of infarct size, which was mediated by opening of mitoK_ATP channels (42). Sildenafil also reduced cell death due to necrosis and apoptosis in isolated cardiomyocytes suggesting that the cytoprotective effect of this drug was independent of its vascular/hypotensive effect (11). Vardenafil is 20-fold more potent than sildenafil for inhibiting purified PDE-5 (43) and it also had similar protective effect against I/R injury in rabbits (44). Moreover, both drugs reduced infarct size when infused at reperfusion through opening of mitoK_ATP channel (45). Tadalafil is a long-acting PDE-5A inhibitor, which has a half-life of 17.5 h (46) and is effective for up to 36 hours for improving erectile function. Tadalafil also reduced infarct size and improved cardiac function following I/R in mice (3).

Mechanistically, the cardioprotective effect of sildenafil was dependent on enhanced NO generation through eNOS/iNOS (47), activation of protein kinase C (PKC) (48), and opening of mitoK_ATP channels (42). Sildenafil also increased cGMP accumulation and PKG activation in mouse cardiomyocytes and heart (11,47). The PKG-dependent cytoprotective mechanism of sildenafil involves phosphorylation of ERK and GSK3β, induction of Bcl-2 and opening of mitoK_ATP channels (49) (Figure 2). Interestingly, sildenafil increased Bcl-2 expression which was absent in iNOS-deficient cardiomyocytes thereby suggesting a link of NO signaling with the expression of antiapoptotic protein (49). Overexpression of PKG1α also reduced in adult rat cardiomyocyte injury after ischemia which involved inhibition of active caspase-3, phosphorylation of Akt, ERK, and JNK, and increased expression of NOS and Bcl-2 as well as decreased Bax expression (50).

Figure 2. Mechanism of cardioprotection by PDE-5 inhibitor, sildenafil.

Sildenafil treatment triggers signaling cascade resulting in increased expression of eNOS/iNOS and activation of cGMP dependent PKG. PKG subsequently causes phosphorylation of ERK1/2 and GSK3β in conjunction with increase in the Bcl-2, which inhibits apoptosis through attenuation in cytochrome C release and inhibition of the mitochondrial permeability transition pore (MPTP). PKG also opens mitoK_ATP channels which limits against ischemia/reperfusion injury through preservation of ATP and decrease in Ca2+ influx in the mitochondria.

cGMP Signaling in Hypertrophy and Heart Failure

Sildenafil exerts anti-hypertrophic effects in mice with pressure-overload in the absence of vascular unloading (1). The antihypertrophic effects coexisted with PKG activation, and its targets included RGS2 (regulator of G protein-coupled signaling 2) (51), as well as calcineurin-NFAT-TRPC6 (52). In the hypertrophied right ventricular myocardium, PDE-5 is upregulated, PKG activity is inhibited and cGMP is preferentially shifted to inhibition of PDE-3 (15). This leads to increase in cAMP, protein kinase A activation, increased intracellular calcium and increased contractility. The increased PDE-5 expression predisposed mice to adverse LV remodeling after myocardial infarction (MI). LV systolic and diastolic dysfunction were more marked in PDE5-TG than in wild-type mice, associated with enhanced hypertrophy and reduced contractile function in isolated cardiomyocytes from remote myocardium (13). Chronic treatment with sildenafil immediately following MI or beginning 3 days post MI attenuated ischemic cardiomyopathy (53) suggesting that PDE-5 inhibition may be a promising therapeutic tool for advanced HF in patients. Interestingly, PKG activation with sildenafil was associated with the inhibition of Rho kinase (54), which is known to suppress LV remodeling post MI in mice (55).

Cardiac Dysfunction in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a progressive and fatal genetic disorder of muscle degeneration. Patients with DMD lack dystrophin as a result of mutations in the X-linked dystrophin gene. The loss of dystrophin leads to severe skeletal muscle pathologies and cardiomyopathy, a delayed symptom of the disease that usually develops by the second decade of life, with more than 90% of patients presenting clinical symptoms by 18 y of age (56). Reduced NO-cGMP signaling is a key contributor to DMD cardiac pathogenesis. Dystrophin-deficient mice (mdx mice) show cardiac dysfunction with decrease in diastolic function followed by systolic dysfunction later in life. Loss of dystrophin prevents normal nNOS expression and/or signaling in all (skeletal, smooth and cardiac) muscle systems (57). The stimulation of cGMP synthesis by overexpression of cardiac-specific nNOS reduced impulse-conduction defects in dystrophin-deficient (mdx) mice (58;59). Similarly, increased pGC activity in young mdx mice decreased susceptibility to cardiac damage during sympathetic stress (60). Chronic treatment with sildenafil reduced functional deficits in cardiac performance of aged mdx mice without any effect on normal cardiac function in wild type controls (57). When sildenafil treatment was started after cardiomyopathy had developed, the established symptoms were rapidly reversed within a few days. These results suggest that PDE-5 inhibitors may be useful in treatment of cardiomyopathy in DMD patients.

cGMP and Doxorubicin-Induced Cardiotoxicity

Doxorubicin (DOX) is one of the most powerful and widely used anti-cancer drugs in clinics. Specially, the cumulative doses over 550 mg/m² increase the risk of developing cardiac side effects, including congestive heart failure and dilated cardiomyopathy (61). DOX cardiotoxicity involves increased oxidative stress, inhibition of nucleic acid and protein synthesis, release of vasoactive amines, alteration of mitochondrial energetics and altered adrenergic function. Reduction in fractional shortening and abnormalities in the nonspecific T wave and ST segment of EKG are typically observed in DOX-induced ventricular dysfunction (62). Treatment with sildenafil prior to DOX inhibited cardiomyocyte apoptosis, preserved mitochondrial membrane potential (Δψm), myofibrillar integrity and prevented left ventricular dysfunction as well as ST segment prolongation (63). Similarly, Tadalafil also improved left ventricular function and prevented cardiomyocyte apoptosis in DOX-induced cardiomyopathy through mechanisms involving up-regulation of cGMP, PKG activity, and MnSOD level without interfering with the chemotherapeutic benefits of DOX (64). Thus, prophylactic treatment with PDE-5 inhibitors might become a promising therapeutic intervention for managing the clinical concern of DOX-induced cardiotoxicity in patients.

cGMP in Cancer Chemotherapy

Sildenafil and vardenafil induce caspase-dependent apoptosis and antiproliferation effect in B-cell chronic lymphatic leukemia (65). Vardenafil and DOX combination significantly improved survival and reduced the tumor size in brain-tumor-bearing rats (66). Oral administration of vardenafil and sildenafil increased the rate of transport of compounds across the blood-tumor-brain and improved the efficacy of DOX in treatment of brain tumors. We recently reported that sildenafil is both a powerful sensitizer of DOX-induced killing of prostate cancer and provides concurrent cardioprotective benefit (67). Co-treatment with sildenafil and DOX enhanced apoptosis in PC-3 and DU145 prostate cancer cells through enhancing ROS generation as compared to normal prostate cells where such combination attenuated DOX-induced ROS generation. The basic difference in mitochondrial respiration between normal and cancer cells (which produce large amount of lactate regardless of the availability of oxygen) appears to make cancer cells more sensitive to oxidative stress (68). DOX-induced apoptosis is mainly initiated by oxidative DNA damage although this apoptosis may involve topoisomerase II inhibition as well. The increased apoptosis by sildenafil and DOX was associated with enhanced expression of proapoptotic proteins Bad and Bax and suppression of antiapoptotic protein, Bcl-2 and Bcl-xL. Moreover, treatment with sildenafil and DOX in mice bearing prostate tumor xenografts resulted in significant inhibition of tumor growth (67).

Concluding Comments

It is clear from the above summarized studies that NO-cGMP-PKG pathway plays a key role in protection against MI, pre- and post-conditioning, hypertrophy, heart failure and DOX-induced cardiotoxicity. A number of clinically relevant therapeutic modalities including GC activators and PDE-5 inhibitors are promising agents in modulating the cGMP pathway in these disease states. Research over the past 9 years on the cardiac uses of PDE-5 inhibitors have helped initiate human trials including NIH multicenter trial (RELAX: Evaluating the Effectiveness of sildenafil at Improving Health Outcomes and Exercise Ability in People With Diastolic Heart Failure; NCT00763867) in patients with heart failure; clinical trial of sildenafil (Revatio) (REVERSE-DMD, NCT01168908) to treat Duchenne Muscular Dystrophy and Becker Muscular Dystrophy patients with cardiac disease, which is currently recruiting patients at the Johns Hopkins Medical Institutions. The role of sildenafil in enhancing the chemotherapeutic efficacy of DOX in prostate and other cancer cell lines, while alleviating the cardiotoxic effects of DOX, suggests that a new paradigm may be emerging for a safer use of this agent in the treatment of various types of cancer.

Table 1.

Pharmacological agents that target the cGMP signaling pathways

Drug/Agent	Target	Model/species	Results	Reference
Atrial natriuretic peptide (ANP)	pGC	Rabbit and rat heart, adult rat ventricular myocytes	Protects against I/R injury and RO-induced hypercontracture by cGMP-dependent nuclear accumulation of zyxin and Akt.	(32–35)
Bradykinin	B2 receptor	Rabbit heart and cardiomyocytes	Mimics ischemic preconditioning by NO-cGMP-PKG dependent opening of mitoKATP channels	(25)
B-type natriuretic peptide (BNP)	pGC	Rat heart	Protects against I/R injury through cGMP-PKG dependent modulation of mitoKATP channels	(31)
8-Br-cGMP	cGMP analog	Neonatal rat ventricular myocytes	Protects against SI-RO via cGMP-dependent protein kinase (PKG)	(36)
Cinaciguat (BAY 58-2667)	sGC	Rabbit, rat, mouse and dog heart	Induces pre- or post-conditioning like effect against I/R by PKG mediated opening of mitoKATP channels	(38–40)
Sildenafil	PDE-5	Rabbit and mice heart, and adult mouse cardiomyocytes Mice, adult mouse cardiomyocytes Mdx mice (dystrophin-deficient mice) Mice prostate cancer xenograft	Protects against I/R and SI-RO injury through eNOS/iNOS, activation of PKC, increased accumulation of cGMP, activation of PKG, phosphorylation of ERK and opening of mitoKATP channels Inhibits DOX-induced cardiomyocytes apoptosis, preserved mitochondrial membrane potential (Δψm), myofibrillar integrity and prevents DOX-induced left ventricular dysfunction Reduced functional deficits in the cardiac performance of aged mdx mice Sensitizes DOX-induced tumor reduction and provides concurrent cardioprotective benefits.	(2;11;42;47–49;53) (57;63) (57) (67)
S-nitroso-N-acetyl-L,L-penicillamine (SNAP)	NO donor	Neonatal rat ventricular myocytes	Mimics preconditioning effect against SI-RO, which is cGMP dependent but independent of PKC or KATP channels	(22)
Vardenafil	PDE-5	Rabbit heart Brain-tumor-bearing rat	Protects against I/R injury via opening of mitoKATP channels Improved survival and reduced DOX-induced tumor size	(3;45) (66)
Tadalafil	PDE-5	Mice heart	Limit I/R injury by hydrogen sulfide (H2S) signaling in a PKG-dependent fashion. Prevents DOX-induced cardiomyopathy and improved left ventricular function through up-regulation of cGMP, PKG activity and MnSOD level without interfering with chemotherapeutic benefits of doxorubicin.	(3) (57;64)

Cyclic guanosine 3’,5’-monophosphate, cGMP; cGMP-dependent protein kinases, PKG; Doxorubicin, DOX; Ischemia-reperfusion, I/R; Nitric oxide, NO; Nitric oxide synthase, NOS; Mitochondrial K_ATP channel, MitoK_ATP channel; Manganese superoxide dismutase, MnSOD; Phosphodiesterase, PDE-5; Simulated ischemia-reoxygenation, SI-RO.

List of Abbreviations

cGMP

Cyclic guanosine 3’, 5’-monophosphate

GC

Guanylyl cyclase

PKG

cGMP-dependent protein kinases

DOX

Doxorubicin

DMD

Duchenne muscular dystrophy

I/R

Ischemia-reperfusion

NO

Nitric oxide

NOS

Nitric oxide synthase

MitoK_ATP

Mitochondrial K_ATP

PDE

Phosphodiesterase