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Myocardial ischemia tolerance in the newborn rat involving opioid receptors and mitochondrial k+ channels.

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Myocardial Ischemia Tolerance in the
Newborn Rat Involving Opioid
Receptors and Mitochondrial Kⴙ
Division of Electron Microscopy, Department of Anatomy, University of Göttingen,
Göttingen, Germany
Department of Thoracic and Cardiovascular Surgery, University of Göttingen,
Göttingen, Germany
Neonatal rat hearts are more tolerant to ischemia compared to adult rat hearts. We
hypothesized that opioid receptors and mitochondrial potassium channels are involved in the
elevated ischemia tolerance of neonatal rats. Newborn rats were treated by an intraperitoneal injection with sodium chloride (placebo, Pla; n ⫽ 7), naloxone (Nal; n ⫽ 8), or K⫹ (ATP)
channel blocker 5-hydroxydecanoate (HD; n ⫽ 8), or were left untreated (sham; n ⫽ 8). Thirty
minutes after injection, the rats were sacrificed and hearts were arrested cardioplegically and
fixed with aldehyde fixative 90 min after global ischemia at room temperature. For control,
newborn rat hearts were fixed immediately after sacrifice. Ventricular tissue blocks were
prepared for electron microscopy. Mitochondrial (volume-weighted mean volume of mitochondria) and cardiomyocyte volume (cellular edema index, CEI) were estimated to quantify the
ischemic injury. Compared to control myocardium, CEI was increased by 244% ⫾ 39% in
sham, 173% ⫾ 28% in Nal, 142% ⫾ 25% in HD, and 101% ⫾ 24% in Pla (P ⬍ 0.05 between
groups). Volume-weighted mean volume of mitochondria was increased by 514% ⫾ 235% in
sham, 341% ⫾ 110% in Nal, 458% ⫾ 149% in HD, and 175% ⫾ 70% in Pla. Differences
between Pla and other groups were significant (P ⬍ 0.01 for all). No significant difference was
observed between the other groups. Thus, ischemic injury was smallest with placebo, indicating a mechanism similar to preconditioning induced by the intraperitoneal injection. This
response was attenuated by blockade of opioid receptors and mitochondrial potassium channels, suggesting their involvement in the elevated ischemia tolerance of newborn rat hearts.
2006 Wiley-Liss, Inc.
Key words: stereology; ischemia tolerance; preconditioning; myocardium;
Neonatal mammals are known to possess a higher tolerance to hypoxia and ischemia than adult animals of the
same species (Fazekas et al., 1941; Mortola, 1999; Ostadal
et al., 1999). Physiological adaptations known to increase
hypoxia tolerance in newborn mammals include polcythaemia, a leftward shift of the oxygen dissociation curve,
reduction of body temperature and heart rate, and a deviation from body size allometry (Singer, 1999). Body size
allometry, also known as Kleiber’s rule (Kleiber, 1961),
refers to the fact that mass-specific basal metabolic rate
increases with decreasing body size, i.e., the smaller the
animal, the higher its specific metabolic rate. In a recent
study, we have demonstrated that deviation from body
size allometry in the newborn rat is not restricted to the
whole organism but also occurs in ischemic isolated hearts
of newborn rats, whereas in older hearts, no such deviation was present (Mühlfeld et al., 2005).
*Correspondence to: Christian Mühlfeld, Division of Electron
Microscopy, Department of Anatomy, University of Göttingen,
Kreuzbergring 36, 37075 Göttingen, Germany. Fax: 49-551397004. E-mail:
Received 27 September 2005; Accepted 14 November 2005
DOI 10.1002/ar.a.20315
Published online 3 February 2006 in Wiley InterScience
TABLE 1. Experimental groups
Number of
Injection 30 minutes before
isotonic sodium chloride solution
10ng/g body weight
50ng/g body weight
10␮l/g body weight
Nal, naloxone group; HD, 5-hydroxydecanoate group; Pla, placebo group; Con, control group.
To approach the mechanisms that are involved in the
increased neonatal tolerance to ischemia, we hypothesized
that neonatal mammals are capable of using strategies
known from other physiological states of increased tolerance to ischemia. Ischemic preconditioning (Schultz et al.,
1997) and natural hibernation (Su, 2000) are related to
the effects of endogenous opioid peptides and the opening
of mitochondrial ATP-regulated potassium [K⫹ (ATP)]
channels. Most authors have provided evidence for a cardioprotective effect of opioid receptor stimulation and
opening of K⫹ (ATP) channels during ischemia of the
adult mammalian heart (Bolling et al., 1997; Huh et al.,
2001), although differences exist between species and opioid receptor subtypes (Romano et al., 2004).
Northern blot analysis and in situ hybridization studies
revealed the expression of opioid peptides in the developing and adult rat heart in both cell culture and myocardial
tissue (Springhorn and Claycomb, 1992; McLaughlin and
Wu, 1998; McLaughlin and Allar, 1998). Binding assay
studies proved the existence of ␬- and ␦-opioid receptors in
different stages of postnatal development of rats and mice
(Zimlichman et al., 1996). The existence of mitochondrial
K⫹ (ATP) channels in neonatal cardiomyocytes has also
been confirmed (Kicinska and Szewczyk, 2003).
We hypothesized that both the endogenous opioid system and mitochondrial potassium channels influence the
neonatal rat heart’s response to ischemia and that blockade of these systems by naloxone (nonselective opioid receptor antagonist) or 5-hydroxydecanoate [blockade of mitochondrial K⫹ (ATP) channels], respectively, reduces the
ischemia tolerance of newborn rat hearts. Support for our
hypothesis came from two studies on simulated ischemia
of neonatal cardiomyocyte cell culture. Diazoxide, an
opener of mitochondrial K⫹ (ATP) channels, protected isolated neonatal cardiomyocytes against ischemia and this
protection was blocked by 5-hydroxydecanoate (Kicinska
and Szewczyk, 2003). A metabolic downregulation of noncontracting neonatal mammalian myocytes subjected to
hypoxia was demonstrated (Casey and Arthur, 2000).
The present study, therefore, investigated ischemic
myocardium in untreated, sodium chloride-, naloxone-,
and 5-hydroxydecanoate-treated neonatal rats to evaluate
the degree of ischemic injury by electron microscopy. Special emphasis was placed on a sound quantification of the
ischemic swelling of mitochondria and cardiomyocytes using design-based stereological methods.
All experiments performed in this study comply with
the current German laws. The investigation conforms
with the Guide for the Care and Use of Laboratory Ani-
mals published by the U.S. National Institutes of Health
(NIH publications 85-23, revised 1996). The experiments
were approved by the Bioethical Committee of the District
of Braunschweig, Germany.
Experimental Protocol
A total of 36 newborn rats (body mass, 6.85 ⫾ 1.02 g;
heart mass, 48.43 ⫾ 11.39 mg) less than 24 hr old were
assigned to a control (Con; n ⫽ 5), a sham (sham; n ⫽ 8),
a placebo- (Pla; n ⫽ 7), a naloxone- (Nal; n ⫽ 8), or a
5-hydroxydecanoate-treated (HD; n ⫽ 8) group. Thirty
minutes prior to sacrifice, Pla animals received an isotonic
NaCl injection (10 ␮l/g body weight, i.p.); animals of Nal
and HD were injected naloxone (10 ng/g body weight, i.p.)
or 5-hydroxydecanoate (50 ng/g body weight, i.p.), respectively (needle size, 25 G). Thus, Pla, Nal, and HD animals
received the same volume of injection. Animals of sham
and Con did not receive any treatment but were also taken
from their littermates and kept alone for 30 min. All
animals were sacrificed by cervical dislocation. After a
parasternal thoracotomy, cardiac arrest was instituted by
perfusion of HTK solution (25 ␮l/min for 4 min at room
temperature; Custodiol, Köhler Chemie, Germany) via the
left ventricle in sham, Pla, Nal, and HD. The hearts were
kept in situ and were subjected to global ischemia at room
temperature. Drying was prevented by regular drops of
NaCl solution. The hearts were fixed by perfusion with
aldehyde fixative (1.5% glutaraldehyde, 1.5% paraformaldehyde in 0.15 M HEPES buffer) 90 min after cardiac
arrest. Control hearts were immediately fixed by perfusion with aldehyde fixative after sacrifice (Table 1).
Tissue Processing
After perfusion fixation, the hearts were stored in aldehyde fixative for 12–24 hr at 4°C. The atria were removed,
the ventricles weighed. Afterward, the ventricles were cut
into 7–9 slices. Slices for electron microscopy were chosen
by systematic random sampling and cut according to the
orientator principle (Mattfeldt et al., 1990) to obtain isotropic uniform random (IUR-) tissue blocks. These were
subsequently rinsed in 0.15 M HEPES buffer and 0.1 M
sodium cacodylate buffer, osmicated, washed repeatedly
in distilled water, and dehydrated in an ascending acetone
series. Finally, the specimens were embedded in araldite
(SERVA Electrophoresis, Heidelberg, Germany).
Electron Microscopy
Three araldite blocks of each animal were chosen randomly and cut according to a systematic random method
(Weibel, 1979). Semithin sections were stained with methylene blue for qualitative light microscopic analysis. Ul-
trathin sections for quantitative investigations were
mounted on copper grids and stained with lead citrate and
uranyl acetate.
EM analysis was performed with an EM 900 (Zeiss,
Oberkochen, Germany) equipped with a digital camera
and a computer image analysis system (Analysis威 3.1, Soft
Imaging System, Münster, Germany) at a final magnification of 36,000⫻. Stereological test fields were gained
systematically randomly. A test system with 18 points
was projected onto each test field. Volume densities of
cardiomyocytic compartments were estimated by point
counting (Weibel, 1979; Schmiedl et al., 1990). The volume
densities were calculated according to Equation 1 and
referred to the cardiomyocyte (fi) as the reference volume:
VV(C/R) ⫽ P(C)/P(R)
where VV(C/R) is the volume density of a specific compartment within the reference volume, P(C) is the number of
points hitting the particular compartment, and P(R) is the
number of points in the reference volume. Thus, volume
densities of myofibrils, VV(mf/fi), sarcoplasm, VV(sp/fi),
mitochondria, VV(mi/fi), and the myocyte nucleus, VV(nuc/
fi), were assessed.
From the volume densities of mitochondria, sarcoplasm,
and myofibrils, the cellular edema index (CEI) was calculated according to Equation 2:
CEI ⫽ VV(mi/fi) ⫹ VV(sp/fi)/VV(mf/fi)
The volume-weighted mean volume of mitochondria,␯៮ ៮V
(mi), was estimated using the point sampled intercepts
method (Gundersen and Jensen, 1985). The formula for
this parameter is given by
␯៮ V(mi) ⫽ ␲/3 ⫻ ៮l03 ⫽ ␲/3 ⫻ 1/P(mi) ⫻ ⌺l03
where l0 is the edge-to-edge chord length of a mitochondrial profile along a line intercept passing through a sampling point, and P(mi) is the number of points of a rectangular point grid hitting mitochondrial profiles.
Body and ventricle mass are given as mean ⫾ standard
deviation. Stereological results are presented as mean
(CV), with CV being the coefficient of variation, calculated
by standard deviation divided by the mean. For evaluation
of the ischemic injury, the results of each animal were
normalized by referring them to the corresponding mean
of control hearts, e.g., CEI (Pla)Norm (%) ⫽ CEI (Pla)/
CEIMean(Con) ⫻ 100. Data were analyzed with the twosided nonparametric Mann-Whitney U-test. Differences
between corresponding data were considered significant
at P ⬍ 0.05 and highly significant at P ⬍ 0.01.
Qualitative Analysis
Qualitative light and electron microscopy confirmed
that all hearts except controls were arrested in diastole by
HTK solution (relaxed myofibrils) and perfused properly
(wide-open capillaries, only a small amount of erythrocytes within blood vessels). In control group, myocardium
was well preserved with electron-dense mitochondria,
high sarcoplasmic glycogen content, evenly dispersed nuclear chromatin, and well-preserved mitochondrial and
sarcolemmal membranes. As a typical feature of neonatal
myocardium, the content of free sarcoplasm was relatively
high. The contraction state in control hearts was slightly
higher than in the experimental groups due to the lack of
Most of the ischemic hearts showed typical signs of
ischemic damage such as large, less electron-dense mitochondria with loss of cristae, increase in sarcoplasm, loss
of sarcoplasmic glycogen granules, clumping and margination of chromatin. The myofibrillar contraction state usually increased in ischemic myocardium, was widely relaxed due to the cardiac arrest. Small areas of increased
contraction state were observed in all experimental
The most pronounced alterations indicating ischemic
injury were observed in sham animals and less pronounced in the naloxone and 5-hydroxydecanoate groups.
In the placebo group, however, only small ischemic
changes were found. Mitochondrial volume was highly
increased in sham, HD, and Nal. Mitochondria of placebo
group were only slightly swollen (Fig. 1).
Stereological Analysis
Swelling of cardiomyocytes was evaluated using the
cellular edema index. In sham, Nal, and HD, a significant
increase in CEI was observed in comparison to control
myocardium, whereas CEI was not increased in placebo
group (Table 2). Compared with control, CEI was increased by 244% ⫾ 39% in sham, 173% ⫾ 28% in Nal,
142% ⫾ 25% in HD, and 101% ⫾ 24% in Pla (P ⬍ 0.05
between groups; Fig. 2).
Mitochondrial volume and size distribution were evaluated using the volume-weighted mean volume of mitochondria. In all experimental groups, a significant increase in mitochondrial volume was observed in
comparison to control myocardium (Table 2). Volumeweighted mean mitochondrial volume was increased by
514% ⫾ 235% in sham, 341% ⫾ 110% in Nal, 458% ⫾
149% in HD, and 175% ⫾ 70% in Pla (Fig. 3). In placebo
group, this parameter was remarkably smaller than in the
other groups (P ⬍ 0.01). No differences were found between sham, Nal, or HD.
The major results of the present study are as follows.
One, the ultrastructure of arrested newborn myocardium
(sham) is highly sensitive to ischemia. The observed features of ischemic newborn myocardial damage are similar
to alterations known from adult ischemic myocardium
(Jennings and Reimer, 1991). Two, the ischemic swelling
of mitochondria was comparably high in sham and in
naloxone- and 5-hydroxydecanoate-treated animals and
was very small in placebo group. Three, ischemic swelling
of cardiomyocytes was most pronounced in sham animals,
whereas in naloxone- and 5-hydroxydecanoate-treated
groups, lower CEI values were observed. In placebo animals, hardly any cardiomyocyte swelling was present.
In neonatal rats, the intraperitoneal injection itself induced a cardioprotective effect that could partly be
blocked by naloxone or 5-hydroxydecanoate, suggesting a
mechanism similar to that of preconditioning. Naloxone
Fig. 1. Representative electron micrographs of ischemic myocardium from all experimental groups.
Mitochondria in sham (a), naloxone (b), and 5-hydroxydecanoate (c) groups are equally large with an electron
lucent matrix space, whereas mitochondria in placebo (d) group have normal size and appearance. Primary
magnification ⫽ 30,000. Scale bar ⫽ 500 nm.
TABLE 2. Summary of stereological data
VV (mf/fi)
VV (sp/fi)
VV (mi/fi)
VV (nuc/fi)
v៮ V(Mi) [␮m3]
0.34 (0.09)
0.34 (0.12)b,c,d,e
0.22 (0.08)c
0.10 (0.21)
1.24 (0.16)b,c,d,e
0.391 (0.46)d,e
0.41 (0.12)
0.26 (0.05)a,c,d,e
0.23 (0.15)c
0.09 (0.21)
0.88 (0.16)a,c,d,e
0.259 (0.32)d,e
0.44 (0.09)
0.21 (0.17)a,b,e
0.25 (0.02)a,b
0.10 (0.17)
0.72 (0.71)a,b,d,e
0.348 (0.33)d,e
0.50 (0.08)
0.15 (0.41)a,b
0.20 (0.14)
0.10 (0.22)
0.51 (0.24)a,b,c
0.133 (0.40)a,b,c,e
0.53 (0.04)a,b,c,d
0.14 (0.21)a,b,c
0.24 (0.09)
0.09 (0.19)
0.51 (0.11)a,b,c
0.076 (0.55)a,b,c,d
Results are given as mean (CV) with CV being the coefficient of variation.
Nal, naloxone group; HD, 5-hydroxydecanoate group; Pla, placebo group; Con, control group; VV, volume density; mf,
myofibrils; sp, sarcoplasm; mi, mitochondria; nuc, nucleus; fi, cardiomyocyte; CEI, cellular edema index; v៮ V(mi), volumeweighted mean volume of mitochondria.
Statistically significant results (p ⬍ 0.05) are indicated in the rows below the values: a, vs. sham; b, vs. Nal, c, vs. HD; d, vs.
Pla; e, vs. Con.
had a similarly high effect on mitochondrial and cardiomyocyte swelling, whereas 5-hydroxydecanoate mainly affected mitochondrial volume regulation.
Evaluation of myocardial ultrastructure by quantifying
different degrees of ischemic injury was applied by several
authors (DiBona and Powell, 1980; Schmiedl et al., 1995).
It was shown that the swelling of mitochondria and cardiomyocytes, respectively, is closely correlated with the
functional status of the ischemic heart (Murry et al., 1990;
Gorge et al., 1991). Although the present study does not
present data on the functional properties of the tissues
investigated, it presents strong parameters indicative of
the functional status of the myocardium. The volumeweighted mean volume of mitochondria is an unbiased
parameter determined by using the point-sampled intercepts method (Gundersen and Jensen, 1985). It combines
information on mitochondrial size and size distribution
and gives an estimation of mean mitochondrial volume in
terms of ␮m3 obtained from single sections. In contrast to
the previously used mitochondrial volume-to-surface ratio, it does not depend on changes in mitochondrial surface
area. However, two limitations of the point-sampled intercepts method have to be taken into account with respect to
the application to mitochondria. First, it is impossible to
say to what extent the variation in mitochondrial size
contributes to the volume-weighted mean volume unless
the number-weighted volume is also estimated. The latter
requires the use of the physical disector (Sterio, 1984) at
the EM level, which was considered to be not efficient in
the context of the present study. Second, it cannot be
excluded from a single ultrathin section that different
mitochondrial profiles belong to the same mitochondrion.
In each group, a few physical disector sections were analyzed in a pilot study. Less than 2% of mitochondrial
profiles were observed that did not represent discrete mitochondria but represented different profiles that belong
Fig. 2. The cellular edema index was significantly increased in sham,
naloxone, and 5-hydroxydecanoate groups compared to placebo group.
In sham group, CEI increase was most pronounced; in naloxone and
5-hydroxydecanoate groups, increases in CEI were smaller than in sham
but still significantly higher than in placebo.
Fig. 3. The volume-weighted mean volume of mitochondria was
highly increased in sham, naloxone, and 5-hydroxydecanoate groups
compared to placebo group.
to the same mitochondrion. Therefore, we assumed that
each mitochondrial profile represents a single mitochondrion accepting an error of about 2%.
The CEI as a parameter for myocyte swelling has been
introduced by DiBona and Powell (1980). It is based on the
understanding that the total volume of myofibrils is not
altered during ischemia and therefore volume increases in
free sarcoplasm and mitochondria will increase the index
In the present study, all hearts (except controls) were
arrested cardioplegically using HTK solution as a cardioplegic solution widely used in cardiac surgery. This
approach was used because of two reasons. First, ischemia
in the newborn human mainly occurs when newborns
undergo surgical treatment of congenital heart diseases
and often hearts are arrested during surgery. Second,
naloxone was shown to decrease the frequency of arrhythmias during cardiac ischemia (Hung et al., 1998). Since
arrhythmias such as fibrillation may significantly influence myocardial metabolism and thus the susceptibility of
the heart to ischemia, we excluded this factor by arresting
the hearts. Our data are therefore based on cardiomyocyte
energy-consuming processes apart from contraction.
Controversial reports exist about the effect of ischemic
preconditioning on neonatal hearts and the importance of
mitochondrial K⫹ (ATP) channel in this event. In neonatal
rats, less than 7-day-old ischemic preconditioning failed to
protect the functional status (contractility, left ventricular
pressure) of the isolated heart (Langendorff mode) during
subsequent ischemia (Awad et al., 1998; Ostadalova et al.,
2002). No evidence was found for an involvement of mitochondrial K⫹ (ATP) channels in ischemic preconditioning
in neonatal rats (Ostadalova et al., 2002). However, immature rabbit hearts could be preconditioned and evidence was provided for a close correlation of ischemic
preconditioning and activation of mitochondrial K⫹ (ATP)
channels in neonatal rabbit hearts since the preconditioning-induced cardioprotection was completely abolished by
5-hydroxydecanoate (Baker et al., 1999). It has been speculated that ischemic pre-condtioning may be a reactivation of newborn ischemia tolerance in adult hearts (Doenst
et al., 2003; Mühlfeld et al., 2005). The present study
reports a reaction of the newborn rat to mild stress induced by an intraperitoneal injection that clearly shows
characteristics of preconditioning. It has not been possible
yet to speculate about the adequate stimulus for this reaction, but it may involve mild pain, stress, or inflammation. The large difference between placebo and sham myocardium suggests that the elevated ischemia tolerance of
newborn rats depends partly on a humoral or paracrine/
autocrine signaling, which in its broadest sense can be
described as a response mechanism (as in this case an
intraperitoneal injection) and may be related to the phenomenon of ischemic preconditioning. This interesting
phenomenon was confirmed by a very recent study on mice
hearts: in isolated adult mice hearts, a delayed preconditioning-like effect in terms of reduced infarct size and
better ventricular function was obtained by an intraperitoneal injection of Ringer solution (Labruto et al., 2005).
Our study demonstrates this effect not only in neonatal
rats but also provides information about the nature of this
In the present study, naloxone, a nonselective opioid
receptor antagonist, reduced the cardioprotective effect
present in placebo group as indicated by pronounced sarcoplasmic and mitochondrial swelling. Although information exists about protective effects of naloxone on different
organs subjected to ischemia (Elkadi et al., 1987; Machuganska et al., 1989; Chen et al., 2001), a large amount of
recent studies confirmed that opioids, either endogenously
secreted during ischemic preconditioning (Schultz et al.,
1997) or pharmacologically administered (Bell et al., 2000;
Romano et al., 2004) before ischemia display protective
effects on adult cardiomyocytes in vivo and in vitro. These
effects are characterized by limitation of infarct size and
improved postischemic functional recovery during ischemia (Bolling et al., 1997; Okubo et al., 2004). Although it
is yet not possible to say whether this effect is related to
opioid receptor antagonism or to a distinct effect of naloxone, the similarities to other physiological states of increased ischemia tolerance are noticeable.
If administered in micromolar doses, 5-hydroxydecanoic
acid selectively blocks a certain type of mitochondrial potassium channel (Jarburek et al., 1998). This K⫹ (ATP)
channel is physiologically regulated, among other features, by the cellular ATP concentration. Opening of the
channel allows potassium ions to enter the mitochondrial
matrix space, thus helping mitochondria to maintain functional and structural integrity (Carreira et al., 2005).
Opening of these channels by pharmacological influences
(e.g., diazoxide) has proved to be a powerful tool to protect
the heart from ischemic injury (Garlid et al., 1997). Studies on the signaling pathways of ischemic preconditioning
have shown that 5-hydrxoydecanoate, at least in part,
blocks the beneficial effects of ischemic preconditioning,
indicating that opening of K⫹ (ATP) channels is an important feature of cardioprotection induced by ischemic preconditioning (Kevelaitis et al., 1999). Furthermore, the
opioid-induced cardioprotection known from adult myocardium and cardiomyocyte culture can be blocked by
5-hydroxydecanoate (Fryer et al., 1999; Huh et al., 2001).
Diazoxide had a protective effect on cultured neonatal rat
cardiomyocytes (Kicinska and Szewczyk, 2003). In accordance with these data and our hypothesis, 5-hydroxydecanoate induced a larger ischemic injury in newborn rat
hearts than that observed in placebo group. Interestingly,
this effect was much stronger with respect to mitochondrial volume regulation than to cardiomyocyte swelling.
Both naloxone and 5-hydroxydecanoate were not able to
abolish the preconditioning-like effect observed in the placebo group completely. In particular, cardiomyocyte swelling was more pronounced in sham than in the other
groups. This may indicate an immediate response and
effect of opioids in response to the intraperitoneal injection so that the resorption of the experimental substances
could only in part block the beneficial effects.
In summary, the present study provides evidence for a
preconditioning-like response mechanism in the newborn
rat that increases the ischemia tolerance of the heart and
helps to maintain better control of cardiomyocyte and
mitochondrial volume regulation. This endogenous cardioprotection is independent of myocardial contraction since
only arrested hearts were used in the present study. Although more work is needed on this topic, our data indicate that the endogenous opioid system and mitochondrial
ATP-regulated potassium channels are highly important
in the signaling of the increased tolerance of the newborn
rat heart to ischemia.
The authors thank Ms. S. Freese, Ms. H. Hühn, and Ms.
S. Wienstroth for their reliable and expert technical assistance and Ms. C. Maelicke for proofreading our English.
Supported by a research grant from the Deutsche Stiftung
für Herzforschung (F/01/03; to H.D., J.R., and M.M.) and
by a grant from the Boehringer Ingelheim Fonds (to C.M.).
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channel, tolerance, ischemia, opioids, involving, myocardial, receptors, rat, newborn, mitochondria
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