Full text of DOI:10.1038/sj.bjp.0704740

British Journal of Pharmacology (2002) 136, 415 ± 420
ã
2002 Nature Publishing Group All rights reserved 0007 ± 1188/02 $25.00
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E€ects of nitric oxide donors on cardiac contractility in wild-type
and myoglobin-de®cient mice
2
2
1J.W. Wegener, A. Godecke, J. Schrader & *,1H. Nawrath
2
1Pharmakologisches Institut, Universitat Mainz, Obere Zahlbacher Str. 67, 55101 Mainz, Germany and Institut fur Herz- und
Kreislaufphysiologie, Heinrich-Heine-Universitat, Universitatsstr. 1, 40225 Dusseldorf, Germany
1
The e€ects of the nitric oxide (NO) donors S-nitroso-N-acetylpenicillamine (SNAP), sodium(Z)-
1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate), and (Z)-1-[N-(2-Aminoethyl)-N-(2-
ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate) on force of contraction (Fc) were
studied in atrial and ventricular muscle strips obtained from wild-type (WT) and myoglobin-de®cient
(myo7/7) mice.
2
SNAP slightly reduced Fc in preparations from WT mice at concentrations above 100 mM; this
e€ect was more pronounced in myo7/7 mice.
DEA-NONOate reduced Fc in preparations from myo7/7 mice to a larger extent than those from
WT mice.
3
4
DETA-NONOate reduced Fc in preparations from myo7/7 but not from WT mice.
5
Pre-incubation with an inhibitor of the soluble guanylyl cyclase (1H-[1,2,4]oxadiazolo[4,3-
a]quinoxalin-1-one; 100 mM) prevented the e€ects of SNAP, DEA-NONOate and DETA-NONOate
on Fc in myo7/7 mice.
6
It is suggested that, in physiological conditions, myoglobin acts as intracellular scavenger
preventing NO from reaching its intracellular receptors in cardiomyocytes, whereas, in myoglobin-
de®cient conditions, NO is able to reduce contractility via activation of the soluble guanylyl cyclase/
cyclic GMP pathway.
British Journal of Pharmacology (2002) 136, 415 ± 420
Keywords: Nitric oxide; force of contraction; myoglobin; heart muscle; guanylyl cyclase
Abbreviations: DEA-NONOate, sodium(Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate; DETA-NONOate, (Z)-1-[N-(2-Ami-
noethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2diolate; DMSO, dimethylsulphoxide; eNOS, endothelial
NO synthase; Fc, force of contraction; myo7/7, myoglobin-de®cient; NO, nitric oxide; NS, not statistically
signi®cant; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; sGC, soluble guanylyl cyclase; SNAP, S-nitroso-
N-acetylpenicillamine; WT, wild-type
Introduction
It is well established that nitric oxide (NO) represents an
important signal molecule involved in several physiological
processes including regulation of vascular tone and platelet
aggregation (Moncada et al., 1991). However, the e€ects of
NO on myocardial contractility are still discussed controver-
sially. For instance, NO or related substances have been
reported either to reduce (Flesch et al., 1997; Smith et al.,
1991), to increase (Kojda et al., 1996) or not to a€ect cardiac
contractility (Nawrath et al., 1995; Weyrich et al., 1994).
Several possibilities have been suggested to account for this
discrepancy. Developmental changes in the expression of the
NO/cGMP signalling pathway may induce di€erent responses
to NO at di€erent ages of the preparations used (Ji et al.,
1999; Vulcu et al., 2000). Alternatively, the myoglobin
content in cardiac muscle, which is di€erent among species
(O'Brien et al., 1992) and even between cardiac muscle
preparations of one species (Ishibashi et al., 1993) may
account for the di€erences in NO actions on the heart since
myoglobin acts as an NO scavenger (Flogel et al., 2001;
Mittal et al., 1978). In the present study, the latter hypothesis
was tested by investigating the e€ects of di€erent NO donors
on force of contraction (Fc) in cardiac muscle preparations
from wild-type mice (WT) and myoglobin-de®cient (myo7/7
mice.
)
Methods
Myo7/7 mice were obtained from the Dept. of Cardiovas-
cular Physiology, Dusseldorf, at which they were generated as
described (Godecke et al., 1999). Brie¯y, myoglobin-speci®c
genomic clones were isolated from a genomic library of
mouse strain 129Sv by using a 266-bp, PCR-ampli®ed cDNA
fragment of the murine myoglobin gene spanning exon 2. A
targeting vector containing the neomycin resistance gene
instead of exon 2 was used to transfect the embryonic stem
cell line R1. Transfected stem cells were aggregated with
murine embryos isolated from NMRI mice.
Mice of either sex were killed by cervical dislocation at the
age of 4 ± 12 weeks. The heart was quickly removed and
immersed in warmed (378C) and oxygenated bu€er solution
(containing in mM: NaCl 137, KCl 5.4, CaCl2 3.6, MgCl2 1,
NaHCO3 12, NaH2PO4 0.42, glucose 5.6; aerated with 95%
*Author for correspondence; E-mail: Nawrath@mail.uni-mainz.de

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Figure 1 E€ects of SNAP on FC in cardiac muscle from WT and myo7/7 mice. (a, b) Original recordings of FC from atrial
preparations of WT (a) and myo7/7 mice (b). Arrows indicate the times at which SNAP (100 mM) or the solvent DMSO (0.5%,
v v71) was added. (c) Bars represent means+s.e.mean (n=6 ± 19). Data were obtained 5 min after the addition of SNAP (100 mM)
or DMSO. NS indicates the absence of a statistically signi®cant di€erence. (d) Concentration-response curves of SNAP in atria from
WT mice and from myo7/7 mice in the absence and presence of ODQ (100 mM). Symbols represent means+s.e.mean (n=3 ± 4).
Data are presented as original recordings or expressed as
means+s.e.means. The magnitude of Fc was measured as the
di€erence between resting and peak tension. Statistical
analysis was performed using either paired or unpaired
Student's t-test. The present study conforms with the Guide
for the Care and Use of Laboratory Animals (U.S. National
Institute of Health, publication 8523, revised 1985).
O2+5% CO2; pH 7.4). The left and right atria were supplied
at either end with silk ligatures as well as ventricular muscle
strips as described (Wegener & Nawrath, 1997).
The cardiac preparations were mounted vertically in organ
baths (5 ml) containing oxygenated bu€er solution at
36+18C. One end was ®xed to a hook of a muscle holder
while the other end was connected to an inductive force-
displacement transducer whose output was fed to a carrier
frequency preampli®er (TA2000, Gould, www.gouldis.de).
The preparations were stretched to the apex of the preload
active tension curve, mounted next to two platinum electrodes
built in a muscle holder, and electrically stimulated by square-
wave voltage pulses at 3 Hz (1 ms duration, voltage 20%
above threshold; Grass S4, www.astro-med.com). Drugs were
applied as single dose or cumulatively to achieve the
concentrations indicated.
Results
The e€ects of three NO donors on myocardial contractility
were investigated in WT and myo7/7 mice. The NO donor
SNAP, at a concentration of 100 mM, fully e€ective in smooth
muscle (Henry et al., 1989), did not change signi®cantly force
of contraction (Fc) in atrial and ventricular preparations from
both groups of animals (Figure 1a, b). The minor decrease of
Fc after addition of the drug was not statistically di€erent
from that observed if the solvent (DMSO) was applied
without drug (Figure 1c). However, concentration of SNAP
above 100 mM slightly reduced Fc in atrial preparations from
WT mice; this e€ect was larger in preparations from myo7/7
mice (Figure 1d). In the presence of ODQ (100 mM), the
e€ects of SNAP in myo7/7 mice were not statistically
di€erent from those obtained in WT mice.
S-nitroso-N-acetylpenicillamine (SNAP), sodium(Z)-1-
(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONO-
ate),
(Z)-1-[N-(2-Aminoethyl)-N-(2-ammonioethyl)amino]-
diazen-1-ium-1,2-diolate (DETA-NONOate), and 1H-[1,2,4]
oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) were purchased
from Alexis (www.alexis-corp.com). Stock solutions of SNAP
and ODQ were prepared in dimethylsulphoxide (DMSO) and
further diluted to the concentrations indicated. The DMSO
content in the test solutions did not exceed 0.5% v v71. All
other chemicals used were as pure as commercially available
and purchased from Sigma (www.sigma-aldrich.com).
The NO donor DEA-NONOate (100 mM) transiently
reduced Fc in atrial and ventricular preparations from both
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J.W. Wegener et al
Cardiac effects of NO in myo
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417
Figure 2 E€ects of DEA-NONOate on FC in cardiac muscle from WT and myo7/7 mice. (a) Original recordings of FC from atrial
preparations of WT (upper panel) and myo7/7 mice (middle and lower panel). Arrows indicate the times at which DEA-NONOate
(100 mM) was added. The bar indicates the presence of ODQ (100 mM). (b) Bars represent means+s.e.mean (n=3 ± 14). Data
represent the maximal e€ects of DEA-NONOate (100 mM) which occurred after 2 to 5 min. ODQ (100 mM) was applied 10 min
before DEA-NONOate. NS and asterisks indicate the absence and presence of statistically signi®cant di€erences, respectively. (c)
Concentration-response curves of DEA-NONOate in atria from WT and myo7/7 mice in the absence and presence of ODQ
(100 mM). Symbols represent means+s.e.mean (n=3 ± 6).
WT and myo7/7 mice. The e€ect was more pronounced in
preparations from myo7/7 than in those from WT mice
(Figure 2). Fc was maximally reduced after 2 ± 5 min and
reached control values within 10 min. Pre-incubation of the
preparations with ODQ (100 mM) for 10 min prevented the
e€ects of DEA-NONOate on Fc in myo7/7 mice (Figure 2).
The concentration-dependence of the e€ects was studied in
atrial preparations: the EC50 of DEA-NONOate was
calculated to 0.3 and 0.1 mM in preparations from WT
and myo7/7 mice, respectively (Figure 2d). However, a
major part of the preparations developed a contracture at
concentrations higher than 1 mM and was rejected from the
analysis.
The NO donor DETA-NONOate was without e€ect in
atrial and ventricular preparations from WT mice but
reduced Fc in those from myo7/7 mice (Figure 3). The e€ect
of 100 mM DETA-NONOate developed slowly and reached a
steady state within 30 min (Figure 3a). The e€ects of DETA-
NONOate were absent, if the preparations were pre-
incubated with ODQ (100 mM) for 10 min (Figure 3). The
concentration-dependence of the e€ects were studied in atrial
preparations from myo7/7 mice: the EC50 amounted to
0.1 mM (Figure 3c).
Discussion
The present study has shown that NO donors of the
NONOate group, DEA-NONOate and DETA-NONOate,
were more e€ective in reducing contractility in cardiac
preparations from myo7/7 than from WT mice. The S-
nitrosothiol-type NO donor SNAP, although e€ective in
smooth muscle preparations (Henry et al., 1989), did barely
change cardiac Fc in WT mice but reduced weakly Fc in
myo7/7 mice, albeit at concentrations greater than 100 mM.
In line with these ®ndings, DEA-NONOate but not SNAP
have been reported to reduce b-adrenergic-stimulated Ca2+
current in rat ventricular cardiomyocytes (Abi-Gerges et al.,
2001). A possible explanation for this di€erence may be the
way how the substances release NO. NONOates are
relatively stable as the pH is raised but release NO
spontaneously at physiological or low pH, albeit at di€erent
time constants (Keefer et al., 1996). S-nitrosothiols are
believed to decompose non-enzymatically to give NO but
also to transfer the nitroso group to other molecules
(Williams, 1996). In addition, the generation of NO from
S-nitrosothiols has been shown to depend on protein-bound
Cu2+ sources (Dicks & Williams, 1996). However, it remains
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Cardiac effects of NO in myo
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Figure 3 E€ects of DETA-NONOate on FC in cardiac muscle from WT and myo7/7 mice. (a) Original recordings of FC from
atrial preparations of WT (upper panel) and myo7/7 mice (middle and lower panel). Arrows indicate the times at which DETA-
NONOate (100 mM) was added. The bar indicates the presence of ODQ (100 mM). (b) Bars represent means+s.e.mean (n=3 ± 9).
Data were obtained 30 min after the addition of DETA-NONOate (100 mM). ODQ (100 mM) was applied 10 min before DETA-
NONOate. NS and asterisks indicate the absence and presence of statistically signi®cant di€erences, respectively. (c) Concentration-
response curves of DETA-NONOate in atria from myo7/7 mice in the absence and presence of ODQ (100 mM). Symbols represent
means+s.e.mean (n=3 ± 5).
open, whether these reactions have a bearing on the weak
cardiac e€ects of SNAP.
study. Similarly, fast and slow time courses were also shown
for the vasorelaxant e€ects of DEA-NONOate and SPER/
NO (t1/2=39 min) and related to the di€erent time courses of
NO release by each drug (Morley et al., 1993).
The e€ects of DEA-NONOate and DETA-NONOate on
Fc had completely di€erent time courses. The e€ect of DEA-
NONOate reached a maximum within 2 min and returned to
control values within 10 min. In contrast, the e€ect of
DETA-NONOate reached its maximum within 30 min. An
explanation for this di€erence may be the di€erent time
course of the NO release by each drug. The decomposition of
DEA-NONOate and DETA-NONOate to release NO shows
a half-life (at 378C) of about 2 min and 20 h, respectively
(Keefer et al., 1996). Using these values, the concentration-
time pro®le of the NO release from DEA-NONOate and
from DETA-NONOate was calculated taking into account
NO auto-oxidation as described (Schmidt et al., 1997).
According to this calculation, NO released by 100 mM
DEA-NONOate produced a peak concentration of 10 mM
within 80 s and returned to about 2 mM after 5 min. NO
released by 100 mM DETA-NONOate produced a steady-
state concentration of 0.6 mM within 1000 s during the
calculated time interval of 40 min. These calculated pro®les
of [NO] ®t well to and are, therefore, assumed to determine
the time courses of the cardiac e€ects of DEA-NONOate and
DETA-NONOate in myo7/7 mice observed in the present
Recently, myoglobin has been suggested to be involved in
the inactivation of NO and to substantially determine the NO
e€ects on coronary blood ¯ow (Flogel et al., 2001). In the
latter study, NO decreased dp/dt in a Langendor€-type
preparation, and this e€ect was slightly pronounced in hearts
from myo7/7 mice. The present study shows a more complete
dissociation of the e€ects of NO donors in normal and
myoglobin-de®cient hearts. Most probably, the di€erent
techniques to assess contractility (whole heart vs isolated
strips in isometric conditions) account for these di€erences. It
seems likely, therefore, that the level of myoglobin (around
100 ± 200 mM; (Wittenberg, 1970)) being present in the
cardiomyocytes from WT mice acts as an intracellular
scavenger for NO, as has been shown for extracellular
haemoglobin (Martin et al., 1985), reducing the e€ective NO
concentration in the cardiomyocytes. As a consequence, only
high levels of exogenously applied NO may reach intracel-
lular target molecules to induce physiological e€ects, as seen
for 100 mM DEA-NONOate (corresponding to about 10 mM
NO) in the preparations from WT mice (Figure 2). On the
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Cardiac effects of NO in myo
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419
other hand, low levels of myoglobin may make it easier for
exogenously derived NO to reach intracellular receptors in
cardiomyocytes. Indeed, di€erent levels of myoglobin were
taken as evidence for the di€erences in NO donor-induced
increases in cGMP levels in atrial and ventricular prepara-
tions from the rabbit (Ishibashi et al., 1993) and the
di€erences of NO donor-dependent e€ects in cardiac muscle
from neonatal and adult rats (Vulcu et al., 2000).
of Fc, as for example inhibition of mitochondrial energy
supply (Stumpe et al., 2001).
In summary, the present study has shown the absence of
myocardial e€ects of exogenously applied NO, at least at
concentrations below 10 mM. This has been explained by the
presence of intracellular myoglobin acting as scavenger for
NO. However, the in vitro ®ndings do not unequivocally deny
a physiological role for NO in the myocardium, if liberated
from the cardiovascular endothelium or cardiomyocytes
(Shah & Maccarthy, 2000). An intra-myocardial source for
NO may be able to induce physiological functions in spite of
the presence of myoglobin, if NO is generated in close vicinity
to its target molecules. Indeed, an isoform of the NO synthase
(endothelial NO synthase; eNOS) has been found closely
associated to membrane caveolae in cardiomyocytes (Feron et
al., 1999) and reported to mediate the stretch dependence of
Ca2+ release (Petro€ et al., 2001). In addition, it has been
shown that eNOS co-puri®es with ryanodine receptor
channel-containing sarcoplasmatic reticulum fractions from
the myocardium (Zahradnikova et al., 1997). In cerebellar
neurones, an NO synthase is co-localized with the NMDA
receptor via an anchoring protein (Christopherson et al.,
1999). A compartimentalization would escape the scavenge of
NO by myoglobin in cardiomyocytes and may lead to local
NO concentrations sucient to exert e€ects on b-adrenergic
signalling, Ca2+ handling, or oxygen consumption.
The e€ects of the NO donors DEA-NONOate and DETA-
NONOate were absent in the presence of ODQ, an inhibitor
of soluble guanylyl cyclase (sGC) (Garthwaite et al., 1995).
These results suggest that the cGMP/sGC signalling pathway
is involved in mediating the e€ects of NO in the murine
myocardium. Intracellular application of cGMP has also
been shown to induce similar e€ects on L-type Ca2+ current
(ICa) in rat cardiomyocytes (Vandecasteele et al., 2001) as did
the NO donor SIN-1 (Mery et al., 1993). DEA-NONOate
(100 mM) has been reported to reduce contractility in rat
cardiomyocytes which was, however, not inhibited by 25 mM
ODQ (Sandirasegarane & Diamond, 1999). Since ODQ is
able to interact with other heme containing proteins besides
sGC (Feelisch et al., 1999; Zhao et al., 2000), e.g. with
intracellular myoglobin (100 ± 200 mM) as suggested (Wegener
et al., 1999), the ODQ concentration used in the former study
may have been too low to result in a sucient inhibition of
sGC. However, concentrations of SNAP and DEA-NON-
Oate above 100 mM reduced Fc in preparations from myo7/7
mice in the presence of 100 mM ODQ and even from WT
mice. This ®nding indicates that, at these concentrations,
e€ects besides activation of sGC are involved in the reduction
This study was supported by a grant from the Deutsche
Forschungsgemeinschaft (SFB 553).
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(Received January 4, 2002
Accepted March 26, 2002)
British Journal of Pharmacology vol 136 (3)