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Division of Cardiology, Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, Maryland 21287, USA; and
* Department of Pathology, New York Medical College, Valhalla, New York, USA
2Correspondence: Halsted 500, Division of Cardiology, Johns Hopkins Medical Institutions, 600 N. Wolfe St., Baltimore, MD 21287, USA. E-mail: dkass{at}bme.jhu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Key Words: tissue activity • protein expression • coronary blood flow • PDE5
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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2
3
4)
. Another
mechanism gaining attention is augmented cGMP synthesis. CGMP, which is
produced by two isoforms of guanylyl cyclase—a membrane-bound form
(ANP/BNP receptors) and a cytosolic form activated by nitric oxide
(NO)—enhances or opposes the action of cAMP in a
concentration-dependent manner (5
, 6)
. Evidence has
accumulated that myocardial cGMP signaling increases in heart failure
via both ANP/BNP and NO pathways, (7
8
9
10
11)
, and likely
contributes to reduced ß-adrenergic responsiveness (9
, 12)
.
cGMP content is also regulated by catabolic enzymes, yet little is
known about their role in the heart. Various phosphodiesterases
regulate cGMP catabolism, including PDE5A, PDE6, PDE9A, PDE10A, and
PDE11A (13
14
15
16
17
18
19
20
21)
, and their expression is often selective
to specific tissues. Among these, PDE5A is the most widely studied, and
its inhibition is a primary target for the treatment of erectile
dysfunction (22)
and pulmonary hypertension
(23)
. PDE5A plays a modulatory role on coronary tone, as
its inhibition enhances coronary blood flow to hypoperfused myocardium
during exercise-induced ischemia (24)
. However, unlike its
vascular effects, the functionality of PDE5A to cardiac contraction and
relaxation is far less clear (25)
despite evidence of
robust gene expression in normal human and canine hearts (26
, 27)
. Furthermore, whether or not this enzyme is altered by
cardiac failure to potentially contribute to altered adrenergic
signaling is unknown.
Accordingly, the present study tested the hypothesis PDE5A inhibition blunts ß-adrenergic-stimulated cardiac function in conscious normal dogs and that this inhibitor effect is diminished by dilated cardiac failure. We further sought to determine whether differences in enzyme expression, activity, and/or cellular localization might account for altered regulation in the failure state. Using a canine model of rapid pacing-induced cardiomyopathy, we show for the first time that PDE5A inhibition markedly blunts dobutamine-stimulated systolic and diastolic function in normal hearts and that this pathway is profoundly diminished in heart failure in association with altered protein regulation. These data support a new biochemical pathway that can contribute to ß-adrenergic hyporesponsiveness in cardiac failure.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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. Catheters and leads were
externalized at the mid-scapulae and protected by an external jacket.
Control data were obtained in 22 dogs and cardiac failure data in 12
animals. Hemodynamics were measured in conscious animals under
steady-state conditions and during transient load reduction by IVC
occlusion at a constant atrial paced rate of 
160
min-1.
PDE5A was inhibited by the pyrazolinone EMD82639
(4-(4-[2-ethyl-phenylamino)-methylene]-3-methyl-5-oxo-4,5-di-hydro-pyrazol-1-y1)-benzoic
acid, Merck KGaA, Darmstadt, Germany), a highly selective novel PDE5A
inhibitor, with an IC50 of 0.01 µM for purified
PDE5A (similar to sildenafil) vs. 10–20 µM for PDE1 or PDE3 (higher
than sildenafil). EMD82639 was dissolved in 0.003N NaOH and infused at
0.08 mg/kg/min intravenous (i.v.) for 10 min, achieving 0.25–0.6 µM
plasma concentrations. In addition to basal responses, effects of PDE5A
inhibition on dobutamine-stimulated function (2.5–15 µg/kg/min i.v.)
were determined (n=14 normal, n=6 CHF dogs). For
these studies, an initial DOB dose response was measured, DOB was
discontinued, EMD82639 was administered after re-establishing basal
state, and a second DOB response was determined during continued
EMD82639 infusion. The effects of infusion vehicle (NaOH) alone on both
basal and DOB dose response were tested (n=13) and found to
be negligible. We previously reported full reproducibility of
sequential DOB responses in the absence of any intervening changes
(28)
.
Hemodynamics in conscious animals
Pressure-dimension signals were digitized at 250 Hz.
Signal-averaged data from 5–10 consecutive beats were used to derive
steady-state parameters and data measured during transient IVC
occlusion were used to assess pressure-dimension relations. Systolic
function was indexed by peak rate of pressure rise
(dP/dtmax) adjusted for preload, end-systolic
pressure-dimension relation, diastolic function by end-diastolic
pressure and time constant of pressure relaxation, and arterial load by
effective arterial elastance index (EaI) equal to the ratio of ESP to
stroke dimension. Methods for index calculations have been described
(28)
.
PDE5A protein expression and tissue localization
PDE5A protein expression and tissue localization were determined
using a polyclonal rabbit antibody to bovine lung PDE5A (developed and
generously provided by Drs. Jackie D. Corbin and Sharon H. Francis; ref
22
). Whole left ventricular myocardial tissue and
cardiomyocytes were analyzed separately. For myocyte isolation, the
region of the heart perfused by the left anterior descending artery was
excised, cannulated, and perfused at 15 ml/min with nominally
Ca2+-free modified Tyrode’s solution [in
mmol/l: NaCl 138, KCl 4, MgCl2 1,
NaH2PO4 0.33, glucose 10
and HEPES 10 (pH 7.3 with NaOH)] at 37°C and oxygenated with 100%
O2 for 30 min; the same solution with added
collagenase (type I, 178 U/ml, Worthington Biochemical Corp., Freehold,
NJ) and protease (type XIV, 0.12 mg/ml, Sigma) for 40 min; and washout
solution (with 200 mmol/l CaCl2) for 15 min
(29)
. Myocardial cells were mechanically disaggregated and
filtered through a nylon mesh; cell pellets were frozen for protein
assay or cell suspensions were plated onto coverslips and frozen for
immunohistochemistry. Chunk tissue was either frozen in liquid nitrogen
for immunoblot/enzyme activity analysis or preserved in OCT and
paraffin-embedded for immunohistochemistry.
Cytosolic LV chunk or isolated myocyte (20 µg) proteins
(30
31
32)
were prepared by differential centrifugation and
quantified by the Bradford method (Bio-Rad microassay). Aortic
microsomal fractions were used as a negative control. Protein (20 µg
per lane) was concentrated by precipitation with an equal volume of
10% trichloroacetic acid that was denatured in sample buffer,
electrophoresed through 1.5 mm-thick 0.1% SDS-8% polyacrylamide
reducing mini-gels (10–15 wells), and electroblotted onto PVDF
membranes (0.45 µm, MSI) as described (31
, 33)
.
Membranes were incubated overnight at 4°C using a final anti-bovine
PDE5A dilution of 1:20,000. After washing, blots were incubated with
secondary antibody (1:20,000 anti-rabbit alkaline phosphatase
conjugate), washed, and processed for detection of chemifluorescent
signals with the Amersham Vistra kit. Blots were scanned and quantified
with ImageQuant software using a Molecular Dynamics Storm Imager.
Results are summarized from four different blots in which three or four
different animals in each group were included.
Immunohistochemistry was performed on mid-LV myocardium and isolated
myocytes from four control and four failing hearts (10–15 myocytes
examined per heart). Tissue in OCT was sectioned and fixed in 4%
paraformaldehyde/0.5% Triton X-100. Primary incubation was performed
overnight with a sequence-specific PDE5A antibody (26)
(generously provided by J. Kotera and K. Omori) at 1:10,000 dilution
and either mouse monoclonal 
-actinin (1:100 dilution) or caveolin-3
(1:400) antibody (Chemicon International, Temecula, CA). Costaining
with -actinin identified localization of PDE5A relative to the
z-band sarcomere structure and costaining with caveolin-3 identified
the outer myocyte membrane. Secondary incubation was performed at room
temperature for 1 h using anti-rabbit Alexa 488 (Molecular Probes,
Eugene, OR) and anti-mouse rhodamine Red-X (Jackson Immunoresearch,
West Grove, PA). Imaging was performed on a Nikon Diaphot 300 inverted
epifluorescence microscope attached to a PCM-2000 laser confocal
scanning microscope system (Nikon, Inc., Melville, NY). The Alexa 488
was excited by the 488 nm line of a Spectra-Physic argon laser,
followed by imaging of the fluorescence from 505 to 535 nm. The
rhodamine Red-X was excited by a Uniphase helium-neon laser at 543 nm
with imaging of the emission from 589 to 621 nm.
PDE5 tissue activity
Total low Km cGMP phosphodiesterase activity
was assayed in duplicate at 1 µmol/l substrate by the two-step method
under linear conditions (30)
in the presence and absence
of EMD82639 (0.1–0.3 µM) and 0.1 mg/ml BSA with 0.1 mM EGTA.
EMD82639 stock solution was prepared in 100% dimethyl sulfoxide or
0.003N NaOH (results were similar with either) as vehicle. Vehicle was
included at the same (<0.1%) dilution for noninhibited cGMP PDE
assays as a control. Phosphodiesterase assays at 1 µM cGMP detected
several high-affinity cGMP PDEs (PDE5A, PDE9A) or dual-specificity PDE
activities (e.g., PDE1C, PDE3A, PDE10A and PDE11A). The 0.3 µM
EMD82639 dose was similar to that achieved in vivo and was sufficient
to inhibit PDE5A but well below the IC50 values
for PDE1 or PDE3. Effects were comparable to those of 3–6 µM
zaprinast (PDE5/6 selective at submicromolar; PDE1C/PDE11A1
IC50 values at 5 to 12 µM).
Isobutylmethylxanthine (50 µM, a nonselective inhibitor of
PDE1C/3/5A/10A/11A) reduced LV chunk cytosolic total cGMP PDE activity
by 66–71%, a concentration with little to no inhibitory effect on
PDE9A1 or PDE11A1 (17
, 19)
Statistical analysis
Data are presented as mean ± SE. Within-group
comparisons were made by repeated measures ANOVA, with post hoc testing
with a Bonferroni correction. Between-group comparisons for baseline
response and DOB dose response relations were analyzed by unpaired
t test and a multivariate linear regression model,
respectively.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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displays left ventricular pressure-dimension loops and
relations before (left) and after (right) EMD82639 in a control heart
at rest. Group data are provided in Table 1
. EMD82639 induced modest changes, with a slight decline in
contractility (right downward shift of the systolic pressure-dimension
relation), and improved diastolic distensibility (downward shift of
diastolic pressure-dimension relation). Modest venous and arterial
dilation reflected by lower right atrial pressure and EaI,
respectively, was also observed and was consistent with earlier reports
using zaprinast or sildenafil (34
, 35)
.
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Cardiodepressive effects of EMD82639 were markedly enhanced when
combined with ß-adrenergic stimulation (Fig. 1B
).
Dose-dependent increases of systolic and diastolic (relaxation)
function from dobutamine were reduced 30–70% by EMD82639 (Fig. 1B
). To test whether this interaction occurred upstream or
distal to adenylate cyclase stimulation, forskolin (5 nM/kg/min) was
substituted for dobutamine in four animals (Fig. 1C
). There
was similar depression of forskolin-mediated effects by PDE5A
inhibition, supporting downstream signaling.
PDE5A inhibition in failing heart
Figure 2
displays effects of EMD82639 on basal and dobutamine-stimulated cardiac
function in failing hearts. In contrast to controls, PDE5A inhibition
had no significant cardiac effects under basal conditions whereas
peripheral arterial and venous dilation was still observed. Arterial
dilation was even slightly enhanced in failure animals (Table 1)
. As
expected, dobutamine responsiveness was diminished at baseline in
failing hearts (Fig. 2B
; compare with Fig. 1B
).
However, unlike controls, neither DOB-mediated inotropic nor relaxation
effects were further modified by coinfusion of EMD82639.
|
Plasma cGMP levels (by radioimmunoassay) were measured before and after
EMD82639 in four animals (two normal, two failure). In all instances,
cGMP increased in coronary sinus and systemic arterial blood (mean
4.2±1.2 to 8.6±2.1 pmol/ml, P<0.05 by Wilcoxan).
Furthermore, the rise in cGMP across the cardiac circulation
(arterial-venous difference) in both control animals was nearly twofold
greater than that in the systemic circulation, but was 75% smaller
than systemic change in both failure animals.
Cyclic GMP-phosphodiesterase activity is reduced in failing
heart
To probe potential mechanisms underlying different responses to
PDE5A inhibition in normal and failing hearts, we examined
cGMP-specific PDE activity (Fig. 3A
). Activity was reduced by 30% (P<0.05 vs.
controls) in whole LV myocardium from failing hearts. Enzyme activity
was also found in isolated myocytes, but this was similar in cells from
control or failing hearts. To better test for disparities in PDE5A
activity, LV tissue and myocytes were preincubated with 0.1 or 0.3 µM
EMD82639. Analogous to that achieved in vivo, EMD82639-inhibitable
cGMP-PDE activity in whole LV was 31% lower in failing heart at the
higher concentration (120±28 vs. 83±22 pmol/min/mg protein). The
percent of total activity inhibited by EMD82639 in myocytes was also
diminished in failing cells (19 vs. 33%, P=0.02, Fig. 3B
). Thus, EMD82639-inhibitable cGMP-PDE activity was
diminished in both whole tissue and myocytes.
|
PDE5 protein expression
Figure 4A
shows Western immunoblots of cytosolic LV and myocyte
PDE5A. A 90 kDa band was present in whole LV tissue and isolated
myocytes, which is consistent with the molecular size reported for
PDE5A (14
, 22
, 26
, 27
, 36)
. Two splice variants of canine
PDE5A (90–100 kDa) have been reported; given its migration slightly
below phosphorylase (94 kDa), the single cardiac form we observed might
represent PDE5A1. LV cytosolic PDE5A expression was 50% lower in
failing hearts (763±111.3 vs. 377.8±57 arbitrary units,
P<0.05). In contrast, there was no significant difference
in PDE5A expression between normal and failing LV myocytes. As a
negative control, we probed aortic membrane fractions that should be
free of PDE5A. The results (Fig. 4B
) confirmed this
prediction and support assay specificity. However, the same size
protein was detectable in rat and mouse ventricular tissue (data not
shown) and in rat gastrocnemius skeletal muscle (Fig. 4B
),
further supporting antibody binding specificity.
|
Immunohistochemistry
Figure 5
a
, b
, c
shows confocal images of LV mid-myocardium stained for
PDE5A and the myocyte-specific caveolin-3. The latter localized
predominantly to the myocyte membrane with negligible vascular
staining. PDE5A was present in both myocardial and vascular tissue,
with more intense staining in smooth muscle and perivascular region
(arrow). Figure 5d
shows positive (left) vs. negative
(right) controls, with the latter using solely the secondary antibody.
Figure 5e
shows a normal isolated myocyte costained for
PDE5A and -actinin. Both proteins colocalized to the z-bands (lower
panel in 5E); unlike -actinin, however, PDE5A also had somewhat
diffuse particulate staining between z-bands within the cytosol.
|
The z-band localization of PDE5A found in normal cells was not observed
when cells were isolated from failing hearts. Figure 6
displays control and failing cells with caveolin-3 and PDE5A
costaining. In the failing cell, there is loss of z-band localization
of PDE5A, leaving a ground-glass cytosolic pattern with some areas of
focal intensity. This was not due to loss of sarcomere structures, as
-actin staining was unaltered in failing cells (data not shown).
These localization patterns were quite consistent among the animals
studied. Thus, in addition to alterations in the expression of protein
and EMD82639-inhibitable enzyme activity, cardiac failure was
associated with altered PDE5A distribution within myocytes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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The data obtained in intact control hearts are consistent with the
reported negative influence of cGMP in vitro on cardiac ß-adrenergic
responses (5
, 6)
. Only recently has degradation of cGMP
also been considered a mechanism whereby this signaling could be
affected. For example, the PDE5A inhibitor zaprinast reduced myocardial
contractility in adrenergically stimulated cardiac papillary muscle
(37)
. However, other studies reported no influence of
PDE5A inhibition on isolated muscle function nor found evidence of
myocardial protein expression (25)
. The disparity with the
present results may relate to the use of intact conscious animals and
concomitant ß-adrenergic stimulation. Recent reports have found
robust PDE5A mRNA expression in human and canine heart (14
, 26
, 27)
, supporting a more potent role. The current study provides
several lines of evidence for myocardial and myocyte enzyme expression
and functionality, supporting both the presence and physiological
significance of PDE5A in normal canine heart.
The mechanisms by which PDE5A inhibition diminished dobutamine
responsiveness are likely similar to those reported with enhanced cGMP
production via nitric oxide (10
, 11)
or 8-bromo cGMP
(5)
. In nonstimulated hearts, cGMP can augment contractile
function at low concentrations, likely via cross-talk with
cAMP-dependent signaling (6)
. At higher concentrations,
cGMP has a negative inotropic effect by antagonizing cAMP via PKG
(primarily in mammals) or PDE2 stimulation (primarily in amphibians).
ß-Adrenergic activation increases both cAMP and cGMP synthesis, with
the net effect of cGMP being negative (a brake) on inotropic and
lusitropic responses. Reducing cGMP concentrations in this setting,
such as by NOS inhibition, enhances ß-adrenergic responsiveness,
whereas PDE5A inhibition produces the opposite effect. cAMP levels were
not directly measured in our study, so it is possible that PDE5A
inhibition also altered this signaling. As the net effect was a
decline in ß-adrenergic stimulation, it seems more likely that cAMP
levels declined, perhaps by an influence on PDE2. Further study is
needed to test the role of this PDE in normal and failing hearts.
PDE5 activity in heart failure
Reduced ß-adrenergic responsiveness is a major hallmark of
cardiac failure. Its mechanisms are related to reduced cAMP generation
from diminished ß-receptor density, altered G-protein coupling,
increased ß-receptor kinase activity, and reduced adenylate cyclase
activity (1
2
3
4)
. Enhancement of competing pathways
involving cGMP synthesis due to atrial and/or brain natriuretic peptide
(7
, 8)
and NO signaling (10
, 12)
are also
observed. The latter is mediated both by induction of high-output NOS2
(38
, 39)
as well as enhancement of pathways regulating
NOS3 activity (e.g., caveolae, ß3 receptors)
(12
, 40)
.
The finding of down-regulated cGMP catabolic enzymes (PDE5A) in cardiac
failure may help explain recent observations of enhanced cGMP-dependent
inhibition of adrenergic signaling in these hearts. Such reduced
expression would be consistent with lower PDE3A also observed in heart
failure (31
, 32)
, suggesting the possibility of shared
upstream stimuli for such changes. NOS inhibition augments dobutamine
responsiveness in failing hearts more so than in controls in humans
(9)
and animals (12)
. In the canine pacing
tachycardia model specifically, this occurs without a concomitant
increase in NOS-3 expression or calcium-dependent or -independent NOS
activity (12)
. Our recent study suggesting enhanced
ß-receptor coupling with caveolin-3 and NOS may in part explain this
phenomenon. Still other studies suggest enhanced
ß3-receptor NOS signaling (40
, 41)
as another contributor. The present data add reduced cGMP catabolism as
another factor.
We observed minimal to no functional effect of EMD82639 in failing
hearts. Protein expression and total cGMP PDE activity was diminished
in whole tissue but not in myocytes. This might suggest a role of
vascular myocardial signaling, since PDE5A is expressed and can mediate
vascular tone in resistance coronary vessels (24)
.
However, the component of cGMP PDE activity inhibited by EMD82639 (more
likely PDE5A selective) was lower in both whole tissue and myocytes,
and protein distribution was markedly altered in failing myocytes. This
latter observation is intriguing, since modulation of cGMP signaling
with ß-adrenergic stimulation might be anticipated to involve
proteins localized near the receptor complex, i.e., at z-band
structures. Loss of this distribution in failing cells might explain a
functional decline exceeding that measured by total protein or activity
in in vitro assays. This may be better clarified by future analysis of
enzyme activity in specific subcellular fractions. Other potential
mechanisms that remain to be tested are secondary changes in PDE5A
function related to phosphorylation (42
, 43)
or the
decline in other PDEs involved with cGMP catabolism (e.g., PDE9A,
PDE10A, and PDE11). Human PDE5A has cAMP response elements and levels
of some PDE5A transcripts (such as PDE5A2) can increase by cAMP
stimulation in cultured smooth muscle cells (44)
. It is
therefore possible that alterations in cAMP signaling in cardiac
failure could play a role in modifying gene expression of PDE5A, and
thereby cGMP catabolism.
Given the current lack of means to selectively up-regulate PDE5A or
prevent changes with cardiac failure, the present data could not
definitively link altered regulation with blunted adrenergic signaling
in this disorder. However, is notable that the magnitude of adrenergic
suppression in normal hearts treated with EMD82639 was marked,
indicating that even partial reduction of PDE5A activity could
contribute to ß-adrenergic hyporesponsiveness in heart failure. Last,
it is possible that reduced expression and functionality of PDE5A in
heart failure may play a beneficial role. Whereas the negative
inotropic action of cGMP can contribute to decreased basal and
adrenergic stimulated cardiac function in heart failure, it also
reduces oxygen consumption (45)
and offsets the
development of cardiac hypertrophy (46)
, which can be
cardioprotective. Furthermore, PDE5A inhibition can enhance blood flow
in ischemic myocardium during exercise in hearts with fixed coronary
artery stenoses (24)
. Thus, whether altered PDE5A
enzymatic activity in heart failure plays a primary role in its
evolution or is an adaptive change limiting progressive toxicity
remains to be clarified. Our data warrant such studies, as they could
lead to the development of a new mode of heart failure therapy.
Alternative cGMP-selective PDE-phosphodiesterases
The present study focused on PDE5A; however, several other members
of the PDE family have recently been described, some having even
greater selectivity for cGMP. For example, PDE9A1, an IBMX-insensitive
PDE highly expressed in kidney, spleen, brain, and small intestine
(17)
, is currently one of the highest affinity PDEs for
cGMP known (i.e., a 20–40 lower Km for cGMP than
PDE5A). However, PDE9A1 expression in heart so far has appeared
minimal, and to date its role in regulating cardiac cGMP catabolism is
unknown. PDE10A is a recently described dual cAMP and cGMP
phosphodiesterase expressed most strongly in testes and brain, but
cardiac expression remains unclear (18
, 47)
. In
preliminary studies, we found that 3–6 µM zaprinast
(IC50 for PDE5A is 0.2 µM; (26)
inhibited 25–53% of total LV chunk cGMP-esterase activity, comparable
to EMD82639. Since recombinant forms of human PDE9A, murine PDE10A, and
human PDE11A have zaprinast IC50 values of 11–35
µM, it is unlikely that these enzymes would be relevant at the
zaprinast dose used (17
18
19
20
21)
.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication February 1, 2001.
Revision received April 9, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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