Download Phosphodiesterase 5 inhibitors: are they cardioprotective?

Review
Cardiovascular Research (2009) 83, 204–212
doi:10.1093/cvr/cvp170
Phosphodiesterase 5 inhibitors: are they
cardioprotective?
Thorsten Reffelmann1,2* and Robert A. Kloner2
1
Klinik und Poliklinik für Innere Medizin B, Universitätsklinikum der Ernst-Moritz-Arndt-Universität Greifswald, FriedrichLöffler-Str. 23 a, 17475 Greifswald, Germany; and 2The Heart Institute, Good Samaritan Hospital, Division of Cardiology, Keck
School of Medicine, University of Southern California, 1225 Wilshire Boulevard, Los Angeles, CA 90017-2395, USA
Received 7 April 2009; revised 20 May 2009; accepted 25 May 2009; online publish-ahead-of-print 27 May 2009
Time for primary review: 16 days
KEYWORDS
Phosphodiesterase 5;
Sildenafil;
Vardenafil;
Tadalafil;
Preconditioning
A growing body of animal studies provides evidence for potential cardioprotective effects of inhibitors of
the enzyme phosphodiesterase isoform 5. Infarct size reduction by administration of phosphodiesterase
5 inhibitors was described in various experimental models of ischaemia and reperfusion. Furthermore,
potential beneficial effects were demonstrated in experimental models of congestive heart failure and
left ventricular hypertrophy. Some of the observed effects resemble the basic mechanisms of ischaemic
pre-conditioning, mimicking both acute and delayed effects. Other effects may be due to action on systemic and cardiac haemodynamics. Mechanisms and signalling pathways, characterized in some of the
experimental models, appear to be complex: for instance, the rate of cyclic guanosine monophosphate
(cGMP) synthesis and the functional compartmentalization of intracellular cGMP metabolism as well as
interaction with ß-adrenergic and nitric oxide signalling may influence effects in different experimental
settings. In this review, we discuss mechanisms, signalling pathways, and experimental limitations and
touch on considerations for translation into potentially useful applications in the clinical arena.
1. Introduction
To date, three pharmacological inhibitors of the enzyme phosphodiesterase (PDE) isoform 5 (PDE 5) have been approved for
the treatment of erectile dysfunction: sildenafil, vardenafil,
and tadalafil. While sildenafil was initially developed in an
effort to find a novel pharmacological class with anti-anginal
properties, the side effect of enhancing penile erections, as
reported by a substantial number of volunteers in the first
clinical investigations, soon became the new focus of
research. Anti-anginal effects, however, turned out to be
limited in these clinical studies.1 Shortly after the approval
of sildenafil for the treatment of erectile dysfunction in
1998, potential cardiovascular effects of sildenafil again
attracted increasing attention. Both the popular press and
scientific journals published reports about adverse cardiovascular events, such as myocardial infarction, in temporal
relation to sildenafil intake,2–4 which initiated a vigorous
debate about the cardiovascular safety of sildenafil. Investigations of potential side effects of sildenafil2,5–9 were followed by systematic statistical analyses of cardiovascular
events related to sildenafil, vardenafil, and tadalafil intake,
in comparison to control populations. These studies did not
support a cardiovascular risk specifically attributable to the
* Corresponding author. Tel: þ49 3834 86 7574; fax: þ49 3834 86 6657.
E-mail address: [email protected]
pharmacological action of PDE 5 inhibitors.10–15 It became
rather obvious that many of the patients suffering from erectile dysfunction are characterized by a high cardiovascular risk
profile which may predispose them to an increased rate of
adverse cardiovascular events.16–20 Following this extensive
debate on potential cardiovascular side effects, research
focus again shifted to the field of cardioprotection.21 A
growing body of experimental animal studies provided evidence for infarct size reducing effects in myocardial ischaemia
and reperfusion, mimicking in part mechanisms of ischaemic
pre-conditioning. Furthermore, potential beneficial effects
were also demonstrated in experimental models of congestive
heart failure and left ventricular hypertrophy.
The following review summarizes recent studies, predominantly from animal research, investigating a potential
cardioprotective profile of PDE 5 inhibitors, discusses mechanisms, perspectives, and experimental limitations of these
effects, and touches on considerations for translation into
clinically meaningful indications.
2. The cyclic guanosine monophosphate –
phosphodiesterase 5 pathway
2.1 Basic mechanisms and effects
Phosphodiesterase is a family of enzymes catalyzing the
breakdown of 30 ,50 -cyclic nucleotide monophosphates,
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Phosphodiesterase 5 inhibitors
205
Figure 1 Schematic illustrating regulation of cGMP-synthesis and breakdown, the action of phosphodiesterases, and potential external factors, such as heart
failure, diabetes, atherosclerosis, or adrenergic drive, that could have significant influence on the complex interaction of various signal pathways.
such as cyclic adenosine monophosphate (cAMP) and cyclic
guanosine monophosphate (cGMP), which serve as important intracellular second messengers. Isoforms 5 and 6 are
specific for the hydrolysis of cGMP, whereas PDE 3 and
4 catalyze the breakdown of cAMP specifically. Phosphodiesterase 1 and 2 hydrolyze both cAMP and cGMP. As a
consequence, in tissue containing significant activity of
PDE 5, inhibition of PDE 5 should result in slowed breakdown of the second messenger cGMP.1 Therefore, the rate
of cGMP production will determine the final functional
effects of PDE 5 inhibitors. Cyclic GMP is synthesized by
guanylyl cyclases in response to endo- and exogenous
stimuli: nitric oxide (NO), a product of the enzyme NO
synthase, activates the soluble guanylyl cyclase. Natriuretic peptides activate the particulate guanylyl cyclase22
(Figure 1). cGMP activates a pattern of specific protein
kinases, in particular protein kinase G, which serve as
main effectors of cGMP, for example via reducing intracellular calcium.23 Interestingly, a functional compartmentalization of cGMP synthesized by the soluble and
particulate guanylyl cyclase was suggested in cardiomyocytes with differential effects on ß-adrenergic modulation
of cardiac contractility and differential control of
cGMP-breakdown by PDE 5 inhibitors.24 These findings
need further investigation as they might explain some of
the unresolved issues in understanding the cardiovascular
effects of PDE 5 inhibitors.
2.2 Tissue distribution of PDE 5: the basis
for effects in vivo
PDE 5 is found in high concentration in vascular smooth
muscle cells of the corpora cavernosa of the penis, in
smooth muscle cells of the peripheral arterial and venous
vessels as well as coronary and pulmonary circulation, and
in platelets.25 During sexual stimulation, NO is released
from non-adrenergic, non-cholinergic nerve endings and
endothelial cells in the corpora cavernosa. As a consequence, arterial smooth muscle cells in penile arteries
relax due to increased cGMP-synthesis, which results in
increased blood within the tunica albuginea, and hence a
penile erection. This may be intensified and prolonged by
PDE 5 inhibitors, slowing the breakdown of cGMP and enhancing smooth muscle relaxation and vasodilatation of penile
blood vessels. Similarly, PDE 5 inhibitors may reduce vascular tone in the peripheral, pulmonary, or coronary circulation. In healthy volunteers, oral administration of sildenafil
resulted in lowering of systolic and diastolic blood pressure
by 7–10 mmHg in a dose-independent manner.26 Similar
effects were described after vardenafil or tadalafil.11,27
Thus, under baseline conditions, vasodilatory effects of
PDE 5 inhibitors are modest; however, when PDE 5 inhibitors
and NO donors are administered simultaneously, which is
absolutely contra-indicated, life-threatening hypotension
may occur. Again, the degree of cGMP-production
206
determines functional effects of PDE 5 inhibitors. In the pulmonary circulation, vasodilatory effects were found to be
beneficial in the treatment of primary arterial pulmonary
hypertension, which finally led to the approval of oral sildenafil for this indication.28,29 Using aortic rings as an
experimental model for the peripheral and—with limitations—also the coronary circulation, vasodilatory effects,
accompanied by cGMP-accumulation, were demonstrated.25
According to measurements by Herrman et al.,7 using intracoronary Doppler-wire technique in patients during coronary
angiography, coronary flow after sildenafil intake increased
by 13% in stenosed coronary arteries as well as angiographically normal arteries. In most studies, PDE 5 inhibitors did
not exacerbate myocardial ischaemia during exercise
stress testing, but appeared to be neutral or slightly beneficial.30,31 Also, some anti-aggregatory effects on platelets,
which also contain PDE 5, were described in these studies.31
2.3 PDE 5 activity in cardiomyocytes
When discussing potential direct cardioprotective effects of
PDE 5 inhibitors, a crucial issue is the question whether PDE
5 is expressed in ventricular cardiomyocytes. This issue has
remained controversial. Initial investigations did not
provide evidence for the presence of significant PDE 5
activity in ventricular cardiomyocytes. The cGMPhydrolyzing activity found in ventricular tissue appeared to
be mainly due to other isoforms of PDE.25 In the study by
Wallis et al.,25 no modulating effect of sildenafil on contractility of isolated trabeculae carneae was observed, which
also argued against a functional role of PDE 5 in ventricular
cardiomyocytes. However, in contrast to the investigations
by Wallis et al., several publications suggested significant
PDE 5 activity in canine, and murine ventricular cardiomyocytes32,33 and also expression of PDE 5 mRNA in human
cardiomyocytes.34 In human atrial tissue, treatment with
sildenafil resulted in increased levels of cGMP,35 which
might also indicate significant activity of PDE 5 in atrial
cardiomyocytes.
It appears doubtful whether contamination with PDE 5
from vascular cells, as initially surmised to be the reason
for the detection of PDE 5 activity in cardiac ventricular
tissue preparations, is the sole explanation for the described
discrepancies regarding the presence of PDE 5 in ventricular
cardiomyocytes. An increasing number of studies reported
functional effects of PDE 5 inhibition on cardiac contractility. In the study by Senzaki et al.,32 the presence of PDE 5
in canine cardiomyocytes was demonstrated by immunohistochemistry, and in parallel experiments, a modulatory
effect of PDE 5 inhibition on ß-adrenergic modification of
cardiac inotropy was observed. In murine hearts and isolated
cardiomyocytes, contractility was reduced by sildenafil only
when these hearts were stimulated with isoproterenol, but
substantially less under baseline conditions.24,36 Interestingly, in mice with pharmacological inhibition of the
enzyme NO synthase or transgenic mice lacking NO synthase
3, sildenafil did not exhibit any modulatory effects on
ß-adrenergic modulation of cardiac contractility.36 In
humans, contractility indices as assessed by echocardiography and blood pressure measurements during dobutamine
infusion were significantly reduced after sildenafil intake,
pointing to modulatory effects of PDE 5 inhibition also in
the human heart.37 In investigations on functional
T. Reffelmann and R.A. Kloner
compartmentalization of cGMP derived from soluble and
particulate guanylyl cyclase, Takimoto et al.24 found suppression of isoproterenol-stimulated increase in contractility by sildenafil which was prevented by pre-treatment
with an inhibitor of protein kinase G, and also by inhibition
of the NO synthase. However, atrial natriuretic peptide, an
activator of the particulate guanylyl cyclase, did not alter
isoproterenol-induced changes of contractility, and unlike
sildenafil treatment it was accompanied by pronounced
increase in myocardial cGMP. Other experimental models
suggested that the ‘particulate cGMP-pool’ is under exclusive control of PDE 2 and, in contrast to the soluble
cGMP-pool, not under control of PDE 5.38 In transgenic
mice lacking NO synthase 3 activity or mice with chronic
pharmacological inhibition of NO synthase, the typical
co-localization of PDE 5A and z-band striations of the sarcomere, as visualized by immunostaining in normal wild-type
mice, disappeared, and a diffuse PDE 5A staining without
preference to the z-band was apparent.36 Similar translocation of PDE 5A activity away from z-bands during the development of heart failure in a canine tachycardia-induced
heart failure model was observed by Senzaki et al.,32
which was accompanied by diminished modulatory effect
of sildenafil on ß-adrenergic effects. These findings suggest
a complex interaction of various pathways, including
ß-adrenergic signalling, NO, and natriuretic peptide, and
separate functional compartments of cGMP with differential
functionality under regulation of distinct enzymatic patterns, which might in part explain some of the apparently
contradictory results with respect to cardioprotective properties of PDE 5 inhibitors.
3. PDE 5 inhibitors in experimental myocardial
ischaemia and reperfusion
3.1 PDE 5 inhibitors prior to ischaemia
3.1.1 Infarct size
The landmark study, suggesting powerful infarct size reducing effects of sildenafil in an experimental model of coronary artery occlusion and reperfusion in rabbits, was
published by Ockaili R et al.39 in 2002. In this study,
0.7 mg/kg sildenafil, administered as an intravenous bolus,
30 min prior to 30 min of coronary artery occlusion followed
by 180 min of reperfusion, reduced infarct size by 68%
(10.8 + 0.9% of the risk area vs. 33.8 + 1.7% in the
control group). Administration of sildenafil 24 h prior to
ischaemia resulted in 41% reduction of infarct size. 5Hydroxydecanoate, a blocker of the mitochondrial ATPsensitive potassium channel, abolished these infarct-size
reducing effects. As opening of ATP-sensitive potassium
channels is regarded as a key step in signalling of ischaemic
pre-conditioning, sildenafil might be termed as a preconditioning mimetic agent. In this study, intravenous
administration of sildenafil resulted in a substantial drop
of arterial blood pressure prior to ischaemia (238.8%
decline in mean arterial pressure within 2 min), which
then returned to baseline within 5 min. During coronary
occlusion, haemodynamics were not significantly different
between groups in this experimental study.
In 2001, Das S et al.40 had published a study, investigating
the effect of sildenafil on infarct size in an isolated
working-heart preparation in rats. In these investigations,
Phosphodiesterase 5 inhibitors
0.05 mg/kg sildenafil resulted in significant infarct size
reduction, but with higher doses of sildenafil substantial
exacerbation of myocardial necrosis was observed over a
very narrow dosing range (0.1–0.5 mg/kg sildenafil). Dosedependent effects of sildenafil on infarct size and ventricular function during reperfusion in working rat hearts were
also described in a study by du Toit et al.41 In that study,
higher doses of sildenafil were associated with decreased
contractility during reperfusion.
A similar experimental protocol, as used by Ockailli et al.,
was tested in another set of experiments from our group:
1.45 mg/kg sildenafil, administered intravenously over
5 min 30 min prior to 30 min of coronary artery occlusion
in open chest rabbits, followed by 3 h of reperfusion did
not result in significant infarct size reduction nor in exacerbation of myocardial necrosis.42 After a small blood pressure
drop prior to ischaemia (217 to 219 mmHg for systolic and
diastolic blood pressure, respectively), haemodynamics
during ischaemia were not significantly different between
groups in this study. Similarly, zones of no-reflow, regional
myocardial blood flow in ischaemic and non-ischaemic
tissue, and incident arrhythmias were not significantly
different. The reasons for these discrepancies have not
been well understood, as very parallel experimental protocols were used.
Vardenafil was also tested in a similar open-chest rabbit
model of regional myocardial ischaemia, followed by reperfusion: 0.014 mg/kg vardenafil, given 30 min prior to coronary artery occlusion resulted in 58% of infarct size reduction
(14.3 + 2.2% of the risk area in comparison to 33.8 + 1.3% in
control animals).43 This effect was blocked by an inhibitor of
the mitochondrial ATP-sensitive potassium channel, but not
by an inhibitor of the sarcolemmal ATP-sensitive potassium
channel. Vardenafil administration caused a rapid decrease
in blood pressure (215% in mean arterial blood pressure)
within 5 min along with increasing heart rate. Over the
next 10 min blood pressure increased and heart rate
decreased, but did not return to baseline. Again, these
experiments were interpreted as a pre-conditioningmimetic action of the PDE 5 inhibitor vardenafil, resulting
in marked attenuation of myocardial necrosis.44
Tadalafil, a long-acting inhibitor of PDE 5, was tested in
rats. Treatment with tadalafil (10 mg/kg by gastric gavage)
2 h prior to coronary artery occlusion for 30 min followed
by 3 h of reperfusion significantly reduced infarct size
(41.7 + 2.4 vs. 54.0 + 3.4% of the risk area).45 In these
investigations, mean arterial blood pressure was significantly lower in tadalafil-treated animals in comparison
with controls, which may provide an explanation for
infarct size reduction as a consequence of reduced cardiac
workload during ischaemia. No other potential mechanisms
were studied in this set of experiments.
Further investigations regarding the mechanism of cardioprotection induced by sildenafil suggested that protein
kinase C is a crucial step in cardioprotection induced by sildenafil.46 In these experiments, chelerythrine, an inhibitor
of protein kinase C, abolished infarct size reduction
induced by sildenafil after 30 min of ischaemia and 3 h of
reperfusion. In isolated mouse ventricular myocytes, sildenafil reduced necrosis and apoptosis after simulated ischaemia (replacing the cell medium with a modified ‘ischaemic
buffer’, incubated at 1–2% O2).33 This protective effect of
sildenafil was absent in inducible NO-synthase knock-out
207
mice and attenuated in endothelial NO-synthase knock-out
mouse cardiomyocytes. These results point to NO signalling
as an essential step in cardioprotection induced by PDE 5
inhibitors. Importantly, these isolated cell experiments
were the first to demonstrate cardioprotective effects in
ventricular cardiomyocytes completely independent from
vascular responses or haemodynamic effects. Further experiments using isolated cardiomyocytes suggested that cardioprotection induced by sildenafil was dependent upon protein
kinase G activated by cGMP, and involves phosphorylation of
Erk 1/2 and glycogen synthase kinase 3ß.47 In Langendorffperfused mouse hearts, the same group investigated
whether infarct size reduction induced by sildenafil administered prior to global ischaemia and reperfusion could be
abolished by an Erk inhibitor. In this study, infarct size in
control hearts (29.4 + 2.4%) was similar as in hearts after
co-treatment with sildenafil and the Erk-inhibitor (31.8 +
4.4%), while sildenafil alone reduced infarct size to 15.9 +
3.0%, which provided strong evidence for direct involvement
of Erk in sildenafil-induced cardioprotection in this model.48
These results regarding various signal pathways, in particular involvement of the NO system, may be important
when considering potential clinical transferability of
results. Diseases with altered NO signalling may result in
different responses to sildenafil treatment.49 In dogs with
alloxan- and streptozotocin-induced diabetes, sildenafil did
not reduce infarct size after 60 min of ischaemia and 3 h
of reperfusion, while pronounced attenuation of necrosis
was found in non-diabetic dogs (16 + 2 vs. 31 + 2% of risk
area).50 In contrast, another study, investigating a very
low dose of sildenafil (0.06 mg/kg sildenafil) given 5 min
prior to reperfusion after 30 min of coronary occlusion in
wild-type mice, and inducible and endothelial NO-synthase
knock-out mice demonstrated marked infarct size reduction
in all three strains, which may suggest that additional mechanisms are involved in the cardioprotective effects of
sildenafil.51
The mentioned studies highlight the need for detailed
characterization of the experimental protocol, when investigating cardioprotective effects induced by phosphodiesterase 5 inhibitors. Concomitant diseases, such as diabetes or
heart failure, might significantly alter the observed effects.
3.1.2 Cardiac pre- and afterload
Administration of sildenafil reduced arterial blood pressure
in most of the studies presented above. Depending on the
velocity of intravenous application, a marked transient
blood pressure drop may occur. For example, in the study
by Ockaili et al.,39 0.7 mg/kg sildenafil reduced systolic,
diastolic, and mean arterial blood pressure by 24.5, 47.3,
and 38.8% respectively. Haemodynamics returned to nearly
baseline levels within 5 min thereafter. In another study,
when sildenafil was given slowly over 5 min, no acute
blood pressure drop occurred, but blood pressure was
reduced by 217 to 219 mmHg over 30 min.42 Regional myocardial blood flow was neither significantly increased nor
reduced by sildenafil in this study, both in the ischaemic
myocardial region and the non-ischaemic tissue. Taking
reduction of blood pressure by sildenafil into account, PDE
5 inhibition significantly reduced specific coronary vascular
resistance within the risk area, meaning that myocardial
perfusion was not compromised in this experimental
setting despite reducing coronary perfusion pressure, due
208
to coronary vasodilatation. Therefore, the reduction of
arterial blood pressure, as an important determinant of
cardiac afterload, may also contribute to cardioprotective
effects during ischaemia, as it may reduce left ventricular
work-load and energy expenditures during ischaemia
without altering coronary blood supply.45
In addition, transient reduction of arterial blood pressure
prior to coronary artery occlusion could serve as a preconditioning stimulus, similar to brief, non-lethal coronary
artery occlusions, used to induce ischaemic pre-conditioning.
Accordingly, in various experimental settings, protective
effects by PDE 5 inhibitors may involve different mechanisms
also including inadvertent pre-conditioning.
Furthermore, PDE 5 inhibitors were shown to attenuate
inotropic responses to catecholaminergic agents in several
models.32,34,36,37,52 Therefore, reduction of cardiac inotropy
during ischaemia should also be considered as a potential
mechanism of infarct size reduction. Functional effects of
PDE 5 inhibitors may not only be influenced by the degree
of pre-activation of the NO-guanylyl cyclase pathway, but
also by the degree of activation of the catecholaminergic
system. Implicitly, this could mean that the depth of anaesthesia in experimental conditions could modify functional
effects of sildenafil.
It is important to keep in mind that PDE 5 inhibitors, at
least in theory, could also increase cardiac contractility: as
a mechanism of cross-talk between cGMP and cAMP, cGMP
at high concentrations is known to partially inhibit PDE
3. This could in turn result in increased cAMP concentrations.53,54 Therefore, PDE 5 inhibition in cardiomyocytes
and subsequently increasing cGMP levels could lead to secondary increase in cAMP-levels that may result in enhanced
cardiac contractility. However, it is not clear whether this is
of any relevance in clinical circumstances.
As vasodilating effects of PDE 5 inhibitors occur both in
the arterial, pulmonary, and venous system, cardiac
preload might also be reduced under the influence of PDE
5 inhibitors. In a rabbit model, sildenafil during coronary
occlusion resulted in normalization of end-diastolic left ventricular pressures, yet another potential cardioprotective
mechanism. Similarly, sildenafil reduced left ventricular
end-diastolic pressure without coronary occlusion.42
3.2 PDE 5 inhibitors at reperfusion
Potential cardioprotective effects of vardenafil and sildenafil were also studied when given at reperfusion. Parallel to
the phenomenon of ischaemic pre-conditioning and to
pharmacological interventions mimicking ischaemic preconditioning, pharmacological interventions initiated at
reperfusion may be compared with the experimental protocol of post-conditioning, where brief episodes of coronary
re-occlusion within the very early phase of reperfusion are
performed in an intention to reduce myocardial reperfusion
injury.55 As the majority of cardiovascular events in the
clinical arena are unpredictable, recent research has
focused on interventions effective in reducing infarct size
when initiated at reperfusion, which might be more realistic
when transferring it to the clinical realm.
In an open-chest rabbit model, infarct size after 30 min of
coronary occlusion and 3 h of reperfusion was significantly
reduced when sildenafil or vardenafil was started 5 min
prior to reperfusion as an intravenous infusion and continued
T. Reffelmann and R.A. Kloner
for the first hour of reperfusion (19.2 + 1.3% with sildenafil,
17.0 + 2.0 with vardenafil vs. 33.8 + 1.7% of the risk area in
controls).56 Cardioprotective effects were abolished by
5-hydroxydecanoate, again suggesting a mechanism involving opening of mitochondrial KATP-channels. Nitroglycerine,
given at reperfusion, did not reduce infarct size in this
set of experiments. The authors speculated that ‘rapid
up-regulation of cGMP by PDE 5 inhibition rather than
increasing its synthesis through NO’ is an effective approach
to limit reperfusion injury in this model. A presumably more
swift increase in intracellular cGMP after PDE 5 inhibition in
comparison with guanylyl cyclase activation by nitroglycerine was discussed to be the pathophysiological basis for
the observed differences. Additional direct effects of NO,
other than increasing intracellular cGMP, might be an
alternative explanation.
With regard to the downstream cascade of signalling, they
also discussed whether cGMP activation of the RISK-pathway
via phosphatidyl-inositol-3 hydroxy-kinase-Akt and p44/p42
extracellular signal regulated kinases Erk 1/2 could be
involved in mediating the observed cardioprotective
effects, although there were no further data to support
this hypothesis in this publication.
Another study, investigating the effect of different doses
of vardenafil, administered during 120 min of reperfusion
after 30 min of ischaemia in isolated rat hearts, demonstrated infarct size reduction with 10 mM, but not with
higher doses of vardenafil.57 Specific inhibitors of guanylyl
cyclase and of protein kinase G prevented infarct size
reduction in this study, suggesting that both guanylyl
cyclase and protein kinase G are required for mediating
vardenafil-induced cardioprotection.58 The authors also
demonstrated restoration of the mitochondrial membrane
potential under the influence of vardenafil despite challenge
with calcimycin (calcium ionophore to induce mitochondrial
permeability transition pores).57 This might indicate that
vardenafil could exhibit its cardioprotective properties via
inhibition of mitochondrial permeability transition pore formation which is regarded to be crucial for the determination
of final infarct size under various cardioprotective interventions. These studies support other experimental work, in
which various substances administered at reperfusion were
shown to limit infarct size, presumably by reducing reperfusion injury, a potential attractive mechanism as an adjunct
to reperfusion therapy for acute myocardial infarction in
the clinical arena.
3.3 Cardioprotection late after application
of PDE 5 inhibitors
A substantial number of scientific publications on ischaemic
pre-conditioning described a second, delayed phase of protection, a term used for infarct size reduction and attenuation of myocardial stunning even more than 24 h after a
short period of sublethal ischaemia (the ‘pre-conditioning’
stimulus). Salloum et al.59 investigated whether similar
effects late after exposure to sildenafil could be demonstrated. In these experiments, mice received an intraperitoneal injection of 0.7 mg/kg sildenafil or placebo 24 h prior to
excision of the hearts. Immediately, the excised hearts were
mounted on a Langendorff apparatus, and then subjected to
20 min of ischaemia followed by 30 min of reperfusion.
Infarct size reduction by sildenafil was striking in this
Phosphodiesterase 5 inhibitors
study (6.9 + 1.2 vs. 27.6 + 3.3%). Furthermore, intraperitoneal injection of sildenafil was followed by a transient
increase in inducible and endothelial NO synthase in the
hearts between 45 and 120 min (mRNA level) and after
24 h (protein level). Inhibition of inducible NO synthase
abolished cardioprotection by sildenafil. The involvement
of increased expression of inducible NO synthase in the
delayed cardioprotective response of sildenafil parallels
other studies on delayed cardioprotection by ischaemic
stimuli.60 A parallel set of experiments with a nearly identical protocol demonstrated that sildenafil-induced delayed
cardioprotection could be blocked by inhibitors of the mitochondrial ATP-sensitive as well as the mitochondrial Caþþ activated potassium channel.59 In other experiments, using
30 min of ischaemia followed by 1 h of reperfusion, infarct
size reduction by sildenafil was abolished both in adenosine-1-receptor knock-out mice and under the influence of
a specific antagonist of the adenosine 1 receptor,61
suggesting involvement of the adenosine-1 receptor in signalling of sildenafil-induced delayed protection. In a Langendorff model, involvement of the survival kinase Erk in
late protective effects of sildenafil, similar to early cardioprotective effects (see above), could also be demonstrated.48 Interestingly, delayed cardioprotective effects
may be amplified by oral pre-treatment with a statin, as
suggested in a study investigating the effects of 30 min
ischaemia and 240 min of reperfusion in rats.62
4. Effects of phosphodiesterase 5 inhibitors
on heart failure and the development
of cardiac hypertrophy
In a chronic murine model of myocardial infarction-induced
heart failure, the effect of sildenafil treatment over 4 weeks
was tested.63 After coronary artery ligation, 0.71 mg/kg sildenafil was given (intraperitoneal injection bid) or placebo.
Infarct size, measured 24 h post-infarction, was significantly
smaller in sildenafil-treated animals (40.0 + 4.6 vs. 69.6 +
4.1%), a protective effect that could be blocked by an inhibitor of NO synthase. Western blot analysis demonstrated a
significant increase of inducible and endothelial NO synthase
after 24 h. Apoptosis was significantly reduced by sildenafil.
Furthermore, parameters of left ventricular remodelling,
such as end-diastolic left ventricular dimensions, fractional
shortening, and cardiac hypertrophy, were favourably influenced by sildenafil treatment which resulted in a pronounced difference in survival over 4 weeks (36 vs. 93%).
In a model of doxorubicin-induced cardiotoxicity in mice,
sildenafil or saline was administered prior to doxorubicin
treatment (total dose of doxorubicin 15 mg/kg).64 Sildenafil
(0.7 mg/kg intraperitoneally prior to doxorubicin) appeared
to protect the hearts from doxorubicin toxicity, namely
attenuating doxorubicin-induced cardiomyocyte apoptosis,
and improving left ventricular developed pressure, as
assessed by a Langendorff preparation (for example after
8 weeks: 92 + 1 mmHg for co-treatment with sildenafil and
doxorubicin vs. 62 + 3 mmHg for co-treatment with saline
and doxorubicin). Importantly, in these experiments, an
inhibitor of NO synthase and an inhibitor of the mitochondrial
ATP-sensitive
potassium
channel
abrogated
sildenafil-induced protection. In the tachycardia-induced
heart failure model in dogs, described by Senzaki et al.,32
209
PDE 5 inhibition had little effect on dobutamine-induced
increase in contractility. In animals without heart failure,
however, PDE 5 inhibition markedly blunted inotropic
responses to dobutamine. Similarly, PDE 5 expression was
reduced in failing hearts and the co-localization of PDE 5
with z-bands of the sarcomere, which appeared to be
characteristic in normal hearts, was not preserved in the
failing hearts. The results suggest different effects of PDE
5 inhibition when heart failure has already developed, a
fact that could be important when transferring the results
to the clinical arena. A study by Chen et al.65 also suggested
altered PDE 5 expression in the coronary vasculature in
models of heart failure.
PDE 5 inhibition was also tested in models of hypertrophic
heart failure and ventricular remodelling, induced by
increasing after-load. Sildenafil was shown to prevent or
even reverse cardiac hypertrophy and exhibit beneficial
effects on parameters of ventricular remodelling.66,67 In a
mouse model of transverse aortic constriction, sildenafil
was demonstrated to arrest progression of hypertrophy, to
increase protein kinase G activity, and to enhance calcium
transients at a time point, when hypertrophy had already
developed.67 In this study, an increase in cardiac left ventricular mass of þ135% and increase in left ventricular enddiastolic dimensions of þ10% occurred within three weeks
of aortic constriction. While left ventricular mass further
increased in mice treated with placebo, starting sildenafil
treatment after the first 3 weeks almost completely
arrested the development of hypertrophy at this level.67 In
a similar model, PDE 5 inhibition attenuated the development of left ventricular hypertrophy and left ventricular
dilation.68 These potential beneficial effects were not exclusively dependent on the calcineurin pathway, which is
known to be a target of protein kinase G. Hypertrophy and
depressed heart function in mice lacking a subunit of calcineurin was favourably influenced by sildenafil in these
experiments. Alternative pathways, such as the
phosphoinositol-3 kinase Akt and Erk 1/2 signalling
pathways, may also contribute to the cGMP signalling of
hypertrophic responses.66 Other chronic animal models
investigated whether cardiac morphological and functional
changes induced by treatment with inhibitors of the NO
pathway could be attenuated by sildenafil.69 After 8 weeks
of treatment with L-nitro-arginine-methyl-ester, an inhibitor
of NO synthesis, in rats, arterial hypertension developed,
which was accompanied by a reduction of cardiac output
and morphological changes of the left ventricle.
Co-treatment with sildenafil significantly reduced myocardial lesions and attenuated the reduction in cardiac output
compared with NO inhibition alone in these experiments.
However, from most of the protocols, it is not absolutely
clear which differential effects of PDE 5 inhibition are
crucial: its effect on the cardiomyocyte, vasodilatatory
effects on the systemic vasculature, or both.
It is of particular interest that protective effects in these
chronic experiments were closely related to the NO
pathway. In humans, for example, treatment with tadalafil
for 4 weeks resulted in significantly improved endotheliumdependent vasodilation, assessed as flow-mediated dilation
of the brachial artery.70 As endothelium-dependent vasodilation is regarded as a crucial step in the pathophysiology
of congestive heart failure, one might suggest that PDE 5
inhibition could also be beneficial via this mechanism.71
210
Some clinical studies examined effects of PDE 5 inhibition in
heart failure.72,73 Hirate et al.,72 for example, found
increased cardiac indices and decreased peripheral resistance 60 min after 50 mg sildenafil in patients with systolic
heart failure. Therefore, PDE 5 inhibition may be an attractive pharmacological principle in heart failure patients with
direct effect on the myocardium and also additive vascular
effects.
5. Other cardiac effects of phosphodiesterase
5 inhibitors
Other cardiac effects of PDE 5 inhibitors have been less well
investigated. High doses of sildenafil were reported to influence membrane ion currents. The rapid component of the
inward rectifier potassium channel was blocked by high concentrations of sildenafil in a study by Geelen et al.8
However, it is not clear whether this is of any relevance in
clinical circumstances. In an animal model of right ventricular pacing, the threshold for arrhythmia under co-treatment
with sildenafil and NO donors was lowered.9 According to the
prescribing information, vardenafil slightly prolongs electrocardiographic QTc (heart rate corrected QT interval).74 This
does not seem to be the case for sildenafil and tadalafil. In
patients with heart failure, no promotion of arrhythmia
was reported; however, a slight increase in sympathetic
heart rate modulation occurred in one study.75 Sympathetic
activation by a central nervous system-dependent effect
was described in patients in another study, but its clinical
relevance is not clear.76
6. Perspective and summary
The foregoing discussion depicts a complex puzzle of
studies, mainly from animal research, suggesting cardioprotective effects of phosphodiesterase 5 inhibitors including
different models and mechanisms. Some of the observed
effects resemble the basic mechanisms of ischaemic preconditioning, mimicking both acute and delayed effects.
Other effects may be due to action on systemic and
cardiac haemodynamics. As several investigations reported
controversial results, close attention to the specific experimental protocol should be paid when estimating potential
clinical applications of PDE 5 inhibitors in cardiovascular
disease.
The following aspects appear to be of outstanding importance and should be clarified before the value of the
observed effects can be estimated for clinical circumstances
(compare Figure 1):
First, some of the studies investigating infarct size
reduction after PDE 5 inhibition reported a very narrow
dose-effect relationship.40,41,57 As both theoretical concepts53,54 as well as the study by Das et al.40 suggested,
the possibility of exacerbation of myocardial necrosis
under certain experimental conditions, the exact dosing,
being safe and effective, must be defined very accurately.
Second, many of the observed effects appeared to be
dependent on adequate functioning of the NO–guanylyl
cyclase pathway. Therefore, conditions, which potentially
could lead to altered NO signalling,49 such as diabetes mellitus50 or heart failure,32 must be separately investigated,
also with respect to potentially unforeseen effects. Third,
T. Reffelmann and R.A. Kloner
pre-activation of the nitric oxide–guanylyl cyclase pathway
may determine the functional and potential protective
effects of PDE 5 inhibition. This should be carefully
accounted for both in experimental and in clinical situation.
Co-administration of NO donors and PDE 5 inhibitors, for
example, is absolutely contra-indicated. Fourth, in experimental conditions of ischaemia and reperfusion, inadvertent pre-conditioning due to blood pressure lowering
effects of PDE 5 inhibitors prior to occlusion should be differentiated from direct cardioprotective effects, also mimicking ischaemic pre-conditioning. Fifth, pre-activation of the
ß-adrenergic pathway in cardiomyocytes also appears to
determine functional and potentially protective effects of
PDE 5 inhibition.32 In experimental animal research, this
should be carefully standardized, including the depth of
anaesthesia. Also in clinical circumstances, this modulatory
effect should be accounted for, in particular as conditions
such as congestive heart failure might significantly alter
this interaction.32 Sixth, the compartmentalization of
cGMP, its functional effects, and enzymatic control should
be further investigated in order to understand to what
extent effects of cGMP derived from particulate guanylyl
cyclase, for example in response to natriuretic peptides,
differs from cGMP derived from soluble guanylyl cyclase,
and whether this transfers into different cardioprotective
action.
In summary, further research on cardioprotective effects
should be consequently pursued as the discussed investigations provide promising results with the perspective of
clinical therapeutic strategies in coronary artery disease,
as an adjunct to reperfusion therapy for myocardial infarction or as an additional strategy to improve symptoms and
presumably improve prognosis in congestive heart failure.
Conflict of interest: R.A.K. is a speaker, consultant, and
researcher for Pfizer, Lilly ICOS, and Bayer (T.R.: no conflict
of interest).
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